Z
THE SKAGGS INSTITUTE FOR
CHEMICAL BIOLOGY
S c i e n t i f i c
R e p o r t
2 0 0 8
O n t h e c o v e r : A key structure of Ebola virus. Scientists at The Scripps Research
Institute, led by Erica Ollmann Saphire, have determined the structure of a critical
protein from Ebola virus. This image shows the virus spike protein (blue and white),
which is necessary for entry of Ebola virus into human cells, bound to an immune system antibody (yellow) acting to neutralize the virus. The structure provides a major
step forward in understanding how the deadly virus works and may be useful in developing vaccines against or treatments for Ebola virus infections. Image created by
Christina Corbaci, administrative assistant, and Jeff Lee, Ph.D., senior research associate,
in the laboratories of Erica Ollmann Saphire, Ph.D., associate professor, and Dennis
Burton, Ph.D., professor. X-ray images courtesy of Dr. Lyle Conrad, Cynthia Goldsmith,
and Dr. Fred Murphy, Centers For Disease Control and Prevention.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
TA B L E O F C O N T E N T S
Engineering Eukaryotic Algal Chloroplasts for Production of
Human Therapeutic Proteins and of Biofuels
Skaggs Members
4
Skaggs Scholars
5
Board Members
6
President’s Introduction
7
Director’s Overview
9
Stephen P. Mayfield, Ph.D.
Auditory Perception and Hearing Impairment: From
Mouse Models to Human Genetic Disease
Ulrich Müller, Ph.D.
37
Chemical Synthesis and Chemical Biology
K.C. Nicolaou, Ph.D.
I N V E S T I G AT O R S ’ R E P O R T S
36
38
Filling Space at the Molecular Level
Julius Rebek, Jr., Ph.D.
Catalysis, Cancer, and the Regulation of Genes: Inventing
Molecules With Defined Functions
Carlos F. Barbas III, Ph.D.
11
Role of Mistranslation in Disease
Paul R. Schimmel, Ph.D.
Cytokine Inflammatory Signaling in Obesity and
Neurodegeneration
Tamas Bartfai, Ph.D.
13
14
42
K. Barry Sharpless, Ph.D.
43
Molecular Biology of Olfaction
15
Structure and Biology of Multidrug Transporters
Geoffrey Chang, Ph.D.
Peter Schultz, Ph.D.
Click Chemistry and Biological Activity
Synthetic, Medicinal, and Bioorganic Chemistry
Dale L. Boger, Ph.D.
41
New Amino Acid Building Blocks
Training in Molecular and Experimental Medicine
Ernest Beutler, M.D.
40
17
Lisa S. Stowers, Ph.D.
45
Macromolecular Master Keys for Genome integrity, Reactive
Oxygen Control, and Pathogenesis
John A. Tainer, Ph.D.
46
Chemical Physiology
Benjamin F. Cravatt, Ph.D.
17
Chemical Approaches to Disease
Paul Wentworth, Jr., Ph.D.
48
Fundamental Processes in Neural Development
Gerald M. Edelman, M.D., Ph.D.
19
James R. Williamson, Ph.D.
Chemical Etiology of Nucleic Acid Structure
Albert Eschenmoser, Ph.D.
20
Intracellular RNA Assembly
Martha J. Fedor, Ph.D.
Pathway Engineering for Enzymatic Synthesis
50
X-ray Crystallographic Studies of Therapeutically Important
Macromolecular Targets
Ian A. Wilson, D.Phil.
51
21
Bioorganic and Synthetic Chemistry
Click and Virus-Based Chemistry for Biological
Discovery
M.G. Finn, Ph.D.
Chi-Huey Wong, Ph.D.
22
Insights Into Protein Chemistry and Biology From
Protein Structure
Elizabeth D. Getzoff, Ph.D.
Structure-Based Design of Bioactive Agents
M. Reza Ghadiri, Ph.D.
30
31
Understanding and Ameliorating Protein Misfolding
Diseases
Jeffery W. Kelly, Ph.D.
Published by TSRI Press®.
All rights reserved.
©
Copyright 2008, The Scripps Research Institute.
59
Staff Awards and Activities
61
Author Index
63
Subject Index
65
27
Synthetic Enzymes, Catalytic Antibodies, Biomolecular
Computing, and Synthetic Capsids
Ehud Keinan, Ph.D.
Automation of Nuclear Magnetic Resonance Structure
Determination of Proteins in Solution
Kurt Wüthrich, Ph.D.
Continuous In Vitro Evolution of RNA Enzymes
Gerald F. Joyce, M.D., Ph.D.
56
26
Bacterial Quorum Sensing
Kim D. Janda, Ph.D.
Studies of Macromolecular Recognition by Multidimensional
Nuclear Magnetic Resonance
Peter E. Wright, Ph.D.
24
55
33
3
4
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
THE SKAGGS INSTITUTE
FOR CHEMICAL BIOLOGY
Elizabeth D. Getzoff, Ph.D.††
Professor
MEMBERS
M. Reza Ghadiri, Ph.D.*
Professor
Julius Rebek, Jr., Ph.D.*
Professor and Director
Carlos F. Barbas III, Ph.D.**
Professor
Janet and W. Keith Kellogg II
Chair in Molecular Biology
Tamas Bartfai, Ph.D.***
Professor
Chairman, Molecular and
Integrative Neurosciences
Department, Scripps
Research
Director, Harold L. Dorris
Neurological Research
Institute
Ernest Beutler, M.D. †
Professor
Chairman, Department of
Molecular and Experimental
Medicine, Scripps Research
Dale L. Boger, Ph.D.*
Richard and Alice Cramer
Professor of Chemistry
Geoffrey Chang, Ph.D.**
Associate Professor
Benjamin F. Cravatt,
Ph.D.****
Professor
Chairman, Department of
Chemical Physiology
Director, Helen L. Dorris
Child and Adolescent
Neuro-Psychiatric Disorder
Institute
Gerald M. Edelman, M.D.,
Ph.D.*****
Professor
Chairman, Department of
Neurobiology, Scripps
Research
Albert Eschenmoser, Ph.D.*
Professor
Martha J. Fedor, Ph.D.**
Associate Professor
M.G. Finn, Ph.D.*
Professor
Kim D. Janda, Ph.D.*
Professor
Ely R. Callaway, Jr., Chair in
Chemistry
Director, Worm Institute for
Research and Medicine
Gerald F. Joyce, M.D.,
Ph.D. †††
Professor
Dean, Faculty
Ehud Keinan, Ph.D.**
Adjunct Professor
Jeffery W. Kelly, Ph.D.*
Lita Annenberg Hazen
Professor of Chemistry
Chairman, Department of
Molecular and Experimental
Medicine, Scripps Research
Richard A. Lerner, M.D. †††
President, Scripps Research
Lita Annenberg Hazen
Professor of
Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
Stephen P. Mayfield,
Ph.D. ††††
Professor
Associate Dean, Kellogg
School of Science and
Technology
Peter Schultz, Ph.D.*
Professor
Scripps Family Chair
K. Barry Sharpless, Ph.D.*
W.M. Keck Professor of
Chemistry
Lisa T. Stowers, Ph.D. ††††
Assistant Professor
John A. Tainer, Ph.D.**
Professor
Paul Wentworth, Jr., Ph.D.*
Professor
Henry Dube, Ph.D.
Richard J. Hooley, Ph.D. ‡‡
University of California
Riverside, California
Jun-Li Hou, Ph.D.
Seiji Kamioka, Ph.D.
Lionel Moisan, Ph.D. ‡‡
CEA
Gif-Sur-Yvette, France
Severin Odermatt, Ph.D. ‡
Agustí Lledó Ponsati, Ph.D.
James R. Williamson,
Ph.D. †††
Professor
Dean, Graduate and Post
Graduate Studies
Per Restorp, Ph.D.
Ian A. Wilson, D.Phil.**
Professor
Siddhartha Shenoy, Ph.D.
Chi-Huey Wong, Ph.D.*
Ernest W. Hahn Professor
and Chair in Chemistry
Peter E. Wright, Ph.D.**
Professor
Cecil H. and Ida M. Green
Investigator in Biomedical
Research
Chairman, Department of
Molecular Biology, Scripps
Research
Ph.D. †††
Kurt Wüthrich,
Cecil H. and Ida M. Green
Professor of Structural
Biology
Michael Schramm, Ph.D. ‡‡
California State University
Long Beach, California
Craig Turner, Ph.D.
Shengxiong Xiao, Ph.D.
* Joint appointment in the
Department of Chemistry
** Joint appointment in the
Department of Molecular
Biology
*** Joint appointment in the
Molecular and Integrative
Neurosciences Department
**** Joint appointment in the
Departments of Chemical
Physiology and Chemistry
***** Joint appointment in the
Department of Neurobiology
†
††
Ulrich Müller ††††
Professor
R E B E K L A B O R A T O R Y †††††
†††
K.C. Nicolaou, Ph.D.*
Aline W. and L.S. Skaggs
Professor of Chemical
Biology
Darlene Shiley Chair in
Chemistry
Chairman, Department of
Chemistry, Scripps
Research
Dariush Ajami, Ph.D.
Assistant Professor of
Molecular Assembly
Paul R. Schimmel, Ph.D. ††
Ernest and Jean Hahn
Professor and Chair in
Molecular Biology and
Chemistry
Fernando R. Pinacho
Crisotomo, Ph.D. ‡‡
Burnham Institute for
Medical Research
La Jolla, California
††††
†††††
R E S E A R C H A S S O C I AT E S
Mark Ams, Ph.D.
Elizabeth Barrett, Ph.D ‡
‡
‡‡
Deceased
Joint appointments in the
Departments of Molecular
Biology and Immunology
Joint appointments in the
Departments of Chemistry and
Molecular Biology
Joint appointment in the
Department of Cell Biology
Rebek laboratory staff. Staff of
the other members are listed in
their respective departments
Appointment completed
Appointment completed; new
location shown
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
THE SKAGGS SCHOLARS
With a contribution from the
Skaggs family, Scripps Research
established the Skaggs Clinical
Scholars Program in an effort to
more closely integrate clinical
and basic research within the
Scripps organization. The program identifies research-oriented
clinicians and funds meritorious
collaborative research projects
between the clinical scholar and
appropriate scientists at Scripps
Research. A broader goal is to
expand the body of knowledge
related to human disease and to
develop effective therapeutic
interventions.
Faith Barnett, M.D., Ph.D.
Division of Neurosurgery,
Scripps Clinic
Rajesh Belani, M.D.
Division of Hematology and
Oncology, Scripps Clinic
Clifford W. Colwell, Jr., M.D.
Shiley Center for Orthopaedic
Research and Education at
Scripps Clinic (SCORE)
John W. Cronin, M.D.
Division of Chest and Critical
Care Medicine, Scripps
Clinic
George E. Dailey, M.D.
Division of Diabetes and
Endocrinology, Scripps
Clinic
Arthur Dawson, M.D.
Division of Chest and Critical
Care Medicine, Scripps
Clinic
J. Thomas Heywood, M.D.
Heart Failure Clinic, Division
of Cardiology, Scripps
Clinic
Ronald A. Simon, M.D.
Division of Allergy, Asthma,
and Immunology, Scripps
Clinic
Mary A. Kalafut, M.D.
Division of Neurology,
Scripps Clinic
Richard A. Smith, M.D.
Center for Neurologic Study,
Scripps Memorial Hospital
Andrew J. King, M.D.
Division of Nephrology,
Scripps Clinic
Donald D. Stevenson, M.D.
Division of Allergy, Asthma,
and Immunology, Scripps
Clinic
Michael P. Kosty, M.D.
Division of Hematology and
Oncology, Scripps Clinic
Daniel A. Nachtsheim, M.D.
Division of Urology, Scripps
Clinic
Shirley M. Otis, M.D.
Division of Neurology,
Scripps Clinic
Athena Philis-Tsimikas, M.D.
Whittier Institute for
Diabetes
Paul S. Phillips, M.D.
Interventional Cardiology,
Scripps Mercy Hospital
Paul J. Pockros, M.D.
Division of Gastroenterology
and Hepatology, Scripps
Clinic
Mathew J. Price, M.D.
Division of Interventional
Cardiology, Scripps Clinic
Robert J. Russo, M.D., Ph.D.
Division of Cardiovascular
Diseases, Scripps Clinic
Darlene J. Elias, M.D.
Division of Chest and Critical
Care Medicine, Scripps
Clinic
Farhad F. Shadan, M.D.,
Ph.D.
Division of Chest and Critical
Care Medicine, Scripps
Clinic
Sheila F. Friedlander, M.D.
Pediatric and Adolescent
Dermatology, Rady
Children’s Hospital
Alexander R. Shikhman,
M.D., Ph.D.
Division of Rheumatology,
Scripps Clinic
Williamson B. Strum, M.D.
Division of Gastroenterology
and Hepatology, Scripps
Clinic
Gary W. Williams, M.D.,
Ph.D.
Department of Medicine,
Scripps Clinic
Katharine M. Woessner, M.D.
Division of Allergy, Asthma,
and Immunology, Scripps
Clinic
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
THE SKAGGS INSTITUTE
THE SCRIPPS
FOR RESEARCH
RESEARCH INSTITUTE
BOARD OF TRUSTEES
BOARD OF TRUSTEES
Claudia S. Luttrell
President
Suzie Balukoff
Thomas C. Beecher
Douglas A. Bingham, Esq.
The Most Reverend Robert H.
Brom
Ron L. Cutshall
Kim D. Janda, Ph.D.
Jeffery W. Kelly, Ph.D.
Richard A. Lerner, M.D.
George L. Moosman
Don L. Skaggs
Mark S. Skaggs
Donna J. Weston
John J. Moores
Chair of the Board, Scripps
Research
Chairman, JMI and San Diego
Padres
Chair, Board of Trustees, the
Carter Center
Warren Beatty
President, Mulholland
Productions Incorporated
A. Brent Eastman, M.D.
Medical Director, Scripps Health
Richard J. Elkus, Jr.
Director, KLA-Tencor, Lam
Research, Virage Logic
Member, Board of Trustees, Palo
Alto Medical Foundation
Marjorie Fink
Philanthropist
Phillip Frost, M.D.
Chairman and Chief Executive
Officer, OPKO Health, Inc.
President, The Frost Group
Mrs. William McCormick Blair,
Jr.
Vice President, Albert and Mary
Lasker Foundation
Louis L. Gonda
Chairman and Chief Executive
Officer, Lexington Commercial
Holdings
Chairman, Lexington Ventures,
L.L.C.
Chairman, Lexington Realty,
L.L.C.
J. Gary Burkhead
Retired, Vice-Chairman, Fidelity
Investments
Paul L. Herrling, Ph.D.
Head, Corporate Research,
Novartis International AG
Gary N. Coburn
Retired Senior Managing
Director, Putnam Investments
Lawrence C. Horowitz, M.D.
President and Managing General
Partner, Selby Lane Enterprises
II, L.L.C.
Vincent E. Benstead
Former Partner,
PricewaterhouseCoopers
Gerald Cohn
Retired Executive, Private
Investor
George H. Conrades
Chairman and Chief Executive
Officer, Akamai Technologies,
Inc.
J. Michael Cook
Retired Chairman and Chief
Executive Officer, Deloitte +
Touche
Rod Dammeyer
President, CAC, L.L.C.
John G. Davies, Esq.
Of Counsel, Allen Matkins
Judicial Appointments Advisor
for Governor Arnold
Schwarzenegger
Thomas E. Dewey, Jr.
Member, McFarland Dewey &
Co., L.L.C.
Alexander W. Dreyfoos
Private Investor
Chairman, Raymond F. Kravis
Center for the Performing Arts
Thomas H. Insley
Vice President and Chief
Financial Officer, SkinMedica,
Inc.
Amin Khoury
Chairman and Chief Executive
Officer, B/E Aerospace, Inc.
Richard A. Lerner, M.D.
President, The Scripps Research
Institute
Claudia S. Luttrell
President, The Skaggs Institute
for Research
James R. Mellor
Former Chairman and Chief
Executive Officer, General
Dynamics Corporation
The Hon. Lynn Schenk
Former Congresswoman,
California
Ralph J. Shapiro
Chair, Avondale Investment
Company
Mark S. Skaggs
Board Member, the ALSAM
Foundation
The Hon. Alice D. Sullivan
(Ret.)
California Superior Court Judge,
Retired
Andrew Viterbi, Ph.D.
President, Viterbi Group, L.L.C.
OFFICERS
Richard A. Lerner, M.D.
President and Chief Executive
Officer
Douglas A. Bingham, Esq.
Executive Vice President and
Chief Operating Officer;
Secretary
Donna J. Weston
Senior Vice President and Chief
Financial Officer; Treasurer
Thomas E. Northrup, Esq.,
Ph.D.
Chief Business Counsel;
Assistant Secretary
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
President’s Introduction
am proud to report
on some of the many
accomplishments at
The Skaggs Institute for
Chemical Biology at The
Scripps Research Institute during the past year.
I
SCIENTIFIC
BREAKTHROUGHS
This past year’s scientific findings from scientists
Richard
at the Skaggs Institute are,
as in earlier years, extraordinary.
A. Lerner, M.D.
• Professor Chi-Huey Wong and colleagues developed
a new 2-punch strategy against HIV and successfully tested aspects of the strategy in the laboratory. The investigators created devices they call
glycodendrons that are designed to do 2 things at
once: (1) inhibit the transport of HIV from where
it traditionally enters the body, preventing the virus
from moving deeper inside where it can infect
immune cells, and (2) set up an immune antibody response to a unique carbohydrate structure
on the surface of the virus.
• Professor Kim Janda, Associate Professor Eric Zorrilla (Scripps Research), and colleagues discovered
a catalytic antibody that degrades a known appetite
stimulant. The antibody works against the gastric
hormone ghrelin, which has been linked to weight
gain and fat storage. These findings may lead to a
potentially novel treatment for obesity.
• Using samples from survivors of the 2005–2006
“bird flu” outbreak in Turkey, an international team,
including researchers at Sea Lane Biotechnologies,
L.L.C., Atherton, California, and me, created the
first comprehensive libraries of monoclonal antibodies against avian influenza virus (type H5N1).
These antibody libraries may be useful in developing a therapy that could stop an influenza pandemic
and provide treatment to the people infected and
in pointing the way to the development of a universal flu vaccine.
• Paul Schimmel, Ernest and Jean Hahn Professor
and Chair in Molecular Biology and Chemistry, and
colleagues uncovered 2 surprising new methods
for correcting mistakes in protein production. This
editing system is important because even small
mistakes in protein production can have profound
disease effects.
• Jeffery Kelly, chair of the Department of Molecular
and Experimental Medicine and Lita Annenberg
Hazen Professor of Chemistry, and colleagues discovered that 2 widely available prescription drugs
restore partial cellular folding, trafficking, and function to a variety of mutant enzymes responsible for
3 distinct lysosomal storage diseases, maladies
involving failure of multiple organ systems. The
team found that the calcium channel blockers diltiazem and verapamil, which are used to treat
hypertension, increased the overall function of
mutant lysosomal enzymes associated with Gaucher
disease, α-mannosidosis, and type IIIA mucopolysaccharidosis in cell lines derived from tissues from
patients with these diseases.
• Professor John Tainer and colleagues revealed how
tiny mutations in a single gene can produce 3
strikingly different childhood diseases. The scientists solved a crystal structure of the enzyme XPD
helicase, which unwinds DNA to fix damage that
regularly occurs. This research sheds light on 3
different inherited syndromes: xeroderma pigmentosum, which increases the risk for skin cancer by
several thousandfold, and Cockayne syndrome and
trichothiodystrophy, which are premature aging and
developmental disorders.
• Professor Elizabeth Getzoff and colleagues developed
a new method for chemically targeting a single
enzyme to block production of nitric oxide without
limiting the beneficial production of this oxide by
other closely related enzymes. The technique provides a general solution that should enable development of new drugs to treat medical problems
linked to nitric oxide overproduction, such as arthritis, and may aid in the discovery of treatments for
other conditions such as HIV disease and AIDS.
• Professor Gerald Joyce, dean of the faculty, and
colleagues demonstrated genetic adaptation to
selective pressure in real time. Under the control
of a computer, a population of billions of genes went
through 500 cycles of forced adaptation to emerge
as molecules that could grow faster and faster on
a continually dwindling source of chemical fuel.
• Professor Peter Schultz, who holds the Scripps
Family Chair, and colleagues produced a powerful
immune response in mice by incorporating an unnatural amino acid into a target protein. This novel
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
approach could be useful in developing new vaccines
for cancer, infectious diseases, and other disorders.
• Professor Benjamin Cravatt, chair of the Department
of Chemical Physiology and director of the Helen
L. Dorris Child and Adolescent Neuro-Psychiatric
Disorder Institute, and colleagues did a protein survey that nearly tripled the number of proteins known
to be involved in programmed cell death and refuted
a long-held idea about the life cycle of proteins.
The findings may open doors for the discovery of
new drugs.
PEOPLE NEWS
It is with great sadness that I report the death, on
October 5, 2008, of Professor Ernie Beutler, chair of
the Department of Molecular and Experimental Medicine
since 1978. His passing is a great loss to science, to
the Skaggs Institute and Scripps Research, and to all
who knew and worked with him over his long, brilliant
career. It is difficult to adequately acknowledge his host of
significant discoveries—among them X-inactivation and
novel treatments for Gaucher disease and several forms
of leukemia, including hairy cell leukemia—or to fully
recognize his authorship of more than 1000 scientific
articles in all the leading journals in his field, his numerous monographs and book chapters, and his editing of
the widely used textbook Williams Hematology. He was
an extraordinary man who led an exceptional life, and
I am most thankful that he crossed our path and stayed
with us for so long.
Filling the position of chair of the Department of
Molecular and Experimental Medicine is Jeffery Kelly,
who also recently became chair of the Board of Trustees
of the Skaggs Institute for Research.
Assuming the deanship of the Kellogg School from
Dr. Kelly is Professor Jamie Williamson. Dr. Williamson
will build on his 7 years as associate dean to lead this
top-ranked graduate program into the future.
FA C U LT Y H O N O R S
In 2008, members of the Skaggs Institute again
received many honors and awards.
• Professor Peter Wright, chair of the Department of
Molecular Biology and Cecil H. and Ida M. Green
Investigator in Biomedical Research, was acknowledged for his outstanding research achievements
by election to the National Academy of Sciences.
• Professor Albert Eschenmoser won the Benjamin
Franklin Medal in Chemistry. Franklin Institute
Awards are given for outstanding achievements
that have enhanced the quality of human life and
deepened our understanding of the universe. Dr.
Eschenmoser was recognized for his research on
the structure of nucleic acids, leading to the understanding of why RNA and DNA have the structures
they do.
• Professor Ian Wilson was showered with honors,
including an honorary degree from the University
of St. Andrews in Scotland in recognition of achievements “at the forefront of research to understand
the immune system and influenza”; election as
a Corresponding Fellow to the Royal Society of
Edinburgh, Scotland’s National Academy of Science
and Letters; and election to the Board of Directors
of the Keystone Symposia.
• Professor Carlos Barbas III received the 2009 Tetrahedron Young Investigator Award, Bioorganic and
Medicinal Chemistry, an award for scientists less
than 45 years old who have exhibited “exceptional
creativity and dedication” in their fields. In addition, Dr. Barbas was chosen for the American Chemical Society Arthur C. Cope Scholar Award, which
recognizes excellence in organic chemistry.
• Jeffery Kelly won the American Peptide Society’s
Vincent du Vigneaud Award, sponsored by Bachem,
Inc., Torrance, California, for “fundamental, visionary research on folding and aggregation processes
in peptides and proteins, and for courageous, insightful exploration of the biological and medical implications of his discoveries.”
I am delighted to take this moment to appreciate
the many, significant accomplishments that have brought
us this far, through the extraordinary generosity and continuing support of the Skaggs family. Thank you also to
the many members of the Scripps Research community,
including donors, trustees, friends, faculty, staff, postdoctoral fellows, and students, for your dedication, hard
work, and vision.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Director’s Overview
he Skaggs Institute
for Chemical Biology
was established in
1996 by a spectacular gift
from L.S. “Sam” Skaggs.
During the past 12 years,
more than 100 million
dollars has been awarded
in research support for
members of the Skaggs
Institute. Currently, the
funding supports 31 prinJulius Rebek, Jr., Ph.D.
cipal investigators, 99
postdoctoral fellows, and 61 graduate students. The
mission of the institute is to conduct science that leads to
new medicinal agents to relieve suffering. Here I describe
some of the progress made by members toward these
goals. More details can be found in the individual reports.
Stephen Mayfield has used his genetically modified
algae to produce carbon-neutral liquid biofuels, a splendid result at a time when fossil fuels reserves are dwindling. Algae can produce biomass at a rate higher than
terrestrial plants do and can be used to synthesize therapeutic proteins. In short, algae are a versatile and
renewable energy source.
M. Reza Ghadiri has developed new cyclic peptide
mimetics as scaffolds to present amino acid side chains
involved in protein-protein interactions. Using the triazole
as a peptide bond surrogate, he has developed useful
bioactive probe molecules that imitate the 3-dimensional
pharmacophore of naturally occurring tetrapeptides.
M.G. Finn continues to modify the surfaces of intact
viral capsids by using, among other methods, click chemistry. These modifications have been used to display carbohydrates on the exterior capsid surface as well as
polycations that efficiently inhibit the action of heparin.
Jeff Kelly, the new chairman of the Department of
Molecular and Experimental Medicine, is studying the
role of amyloidosis in diabetes. Deposits of amylin in
the pancreas are related to the compromised function
of these secretory cells that characterize the disease.
Jamie Williamson has developed a powerful enzymatic synthesis of nucleotides such as adenosine triphosphate. The process involves 28 enzymes but can be
carried out in 60% yield starting from glucose, carbon
dioxide, ammonia, and serine. The synthesis is ideal
for isotopically labeled products for use in nuclear
T
magnetic resonance analysis of the structure of proteins and nucleic acids.
Ullrich Müller is studying the hair cells of the inner
ear that are the principal mechanosensors for the detection of sound and head movement. He is unraveling
the molecular composition of the mechanotransduction machinery in these cells by identifying the genes
that control their functions.
Ehud Keinan has proposed a general synthetic strategy of using a simple pentagonal core to produce chemical capsids that are approximately the size of spherical
viruses. He has modeled the assembly and dissociation of
these systems under controlled environmental conditions
and has made progress in synthesizing the molecules
that have the proper shapes and recognition surfaces.
Dale Boger and his group work on inhibiting enzymes
that control natural painkillers such as anandamide. They
have developed synthetic molecules that are more efficient than ibuprofen and are similar to morphine in
potency as analgesics in neuropathic pain.
Carlos Barbas used a reductionist approach on catalytic antibodies to identify the key features of their catalytic abilities. He has shown that simple chiral amines
can be nearly as effective in asymmetric catalysis for many
reactions that make complex carbon-carbon bond arrays.
Geoffrey Chang has developed x-ray crystallography to characterize molecules involved in multidrug
resistance. These molecules transport small drug molecules from inside the cell to outside and are involved
in the efflux of antibiotic compounds. The goal is to
develop inhibitors of the process that can be used in
the treatment of infections.
Gerald Joyce, dean of the Scripps Research faculty,
has developed “evolution on a chip.” This method combines a large population of RNA molecules and computer
controlled microfluidic chips that allow adaptation to
occur through hundreds of cycles in a few days. He
has also developed small molecules that can trigger
RNA enzymes to catalyze their own formation: molecular replication.
Kim Janda is working to manipulate the chemical
biology of cell-to-cell signaling known as quorum sensing.
His findings have applications in controlling virulence
and infectivity of bacterial and other microbial agents.
Peter Schultz continues to add more amino acids
to the repertoire of synthetic biology. Proteins made
from amino acids with an expanded genetic code can
confer an evolutionary advantage and improved pharmacologic properties. These proteins are directed to
applications in biomedical technology.
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Ian Wilson continues to study those few potent but
broadly neutralizing antibodies that recognize HIV type 1.
The elusive goal is still to develop the structural information in these complexes for use in a vaccine.
Lisa Stowers studies neural circuits that underlie
innate behavior. She uses olfactory stimulus in rodents
to identify the neurons involved. Her studies suggest
that maternal-infant behavior in rodents is also triggered
by olfactory mechanisms.
In prebiotic chemistry, a debate continues on the relative importance of replication vs metabolism in the origins of life. Albert Eschenmoser is making progress on
both of these fronts. He and his group make use of eversimplified backbones derived from glyceric acid for
replication and explore the chemistry of glyoxylate for
metabolism.
Chi-Huey Wong has invented a new method for the
ligation of peptides in which attached sugars are used as
delivery vehicles. The intent is to optimize the methods
to achieve the total synthesis of therapeutic glycoproteins as single isomers.
In my own research group, we continue to explore
the behavior of molecules in small spaces. These
arrangements, known as encapsulation complexes, isolate molecules from the medium and expose unusual
behaviors, shapes, and reaction intermediates that
cannot be seen in solution.
Among the honors bestowed on the Skaggs investigators, 2 were particularly noteworthy. Peter Wright,
chairman of the Department of Molecular Biology, was
elected to the National Academy of Science, and Tamas
Bartfai, Chairman of the Molecular and Integrative Neurosciences Department, was elected to the Swedish Academy of Sciences. Members of the Skaggs Institute won
numerous national and international prizes and earned
many honorary degrees in the past year.
My colleagues and I are grateful for the continued
support of the Skaggs Institute for Research. They provide generous funding for basic science at the interface
of chemistry and biology.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
INVESTIGATORS’ R EPORTS
Catalysis, Cancer, and
the Regulation of Genes:
Inventing Molecules With
Defined Functions
C.F. Barbas III, K. Albertshofer, T. Bui, R.P. Fuller, T. Gaj,
J. Gavrilyuk, C. Gersbach, B. Gonzalez, R.M. Gordley, J. Guo,
S. Juraja, D.H. Kim, A. Mercer, S. Mizuta, A. Onoda,
S. Salahuddin, M. Santa Marta, L.J. Schwimmer, F. Tanaka,
H. Uehara, N. Utsumi, U. Wuellner, K.S. Yi, H. Zhang
e are concerned with
problems at the interface of molecular
biology, chemistry, and medicine. Many of our studies
involve learning or improving
on Nature’s strategies to prepare novel molecules that perform specific functional tasks,
Carlos F. Barbas III, Ph.D.
Professor
such as regulating a gene,
Molecular Biology
destroying cancer, or catalyzing
a reaction with small molecules in an enzymelike manner. We hope to apply these novel insights, methods,
and products to provide solutions to human diseases,
including cancer, HIV disease, and genetic diseases.
W
C ATA LY T I C A N T I B O D I E S
We are extending and refining approaches to catalytic
antibodies by using novel recombinant strategies coupled
with reactive immunization, chemical-event selections,
and the design of unique multiturnover selection chemistries. We are developing in vitro selection and evolutionary strategies as routes for obtaining antibodies of
defined biological and chemical activity. These strategies involve the directed evolution of human, rodent,
and synthetic antibodies. Essentially, we are evolving
proteins to function as efficient catalysts, a task that
is naturally performed over eons, and one that we aim
to complete in weeks. The approach is a blend of
chemistry, enzymology, and molecular biology.
A major focus of our research is the development
of strategies to produce antibodies that efficiently form
and break carbon-carbon bonds. In addition to fashioning new enzymatic function to study the chemistry
of imines and enamines, we hope to apply these cata-
11
lysts in novel therapies against cancer and HIV type 1
infection that couple catalytic antibody activity with
activation of designed prodrugs.
O R G A N O C ATA LY S I S
In studying how proteins catalyze reactions, we
often examine how the constituent components react.
These studies have led to a new green approach to
catalytic asymmetric synthesis that can be applied to
the synthesis of drugs and druglike molecules. Using
insights garnered from our studies of aldolase antibodies, we prepared simple chiral amino acids and amines
to catalyze aldol and related imine and enamine chemistries such as Michael and Mannich reactions. We
also studied small amine-bearing peptides that are
catalytic. Although aldolase antibodies are superior
catalysts, simple chiral amino acids and amines are
enabling us to determine the importance of pocket
sequestration in catalysis.
We showed that L-proline and other chiral amines
can be efficient asymmetric catalysts of a variety of
important imine- and enamine-based reactions. Studies
from our laboratory and the contributions of others
have produced advances toward one of the ultimate
goals in organic chemistry: the catalytic asymmetric
assembly of simple and readily available precursor
molecules into stereochemically complex products
under operationally simple and, in some instances,
environmentally friendly experimental protocols. An
important result of these studies is the development of
catalysts that allow aldehydes, for the first time, to be
used efficiently as nucleophiles in a wide variety of
catalytic asymmetric reactions. Previously, only naturally
occurring enzymes were thought capable of this chemical
feat. With future efforts, small organic catalysts may
match some of Nature’s other heretofore unmatched
synthetic prowess. These catalysts might help explain
the development of complex chemical systems in the
prebiotic world and provide hints toward yet-to-be discovered mechanisms in extant biological systems.
Using this method, we directly synthesized a wide
variety of α and β amino acids, carbohydrates, and lactams. Stereochemically complex molecules can now be
assembled by using small molecules in a manner analogous to that of natural enzymes. Novel catalyst designs
have enabled us to synthesize particular diastereoisomers previously not accessible with proline, and we
envision that this approach will largely replace the use
of aldolase enzymes in synthesis (Fig. 1). New and
exciting catalytic asymmetric reactions continue to
emerge from these studies.
12
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
F i g . 1 . Organocatalysis with natural and designed amino acids
leads to a variety of efficient asymmetric syntheses previously
approachable only via enzyme catalysis. A–C, Design considerations for a family of organoaldolases that allow large families of
carbohydrates to be readily synthesized.
C H E M I C A L LY P R O G R A M M E D A N T I B O D I E S
In targeting cancer, we take a multidisciplinary
approach that involves gene regulation, catalytic antibodies, drug design, and combinatorial antibody libraries.
Using a chemically programmed antibody strategy, we
recently showed the power of combining small-molecule
chemistry with immunochemistry. We designed smallmolecule integrin antagonists to self-assemble into a
covalent complex with antibody 38C2 (Fig. 2). The
resulting chemically programmed antibody had significant advantages compared with small molecules or antibody alone in studies of metastatic melanoma, colon
cancer, and breast cancer. We recently developed a
powerful new approach to a programmable vaccine
strategy based on a universal vaccination that elicits
programmable antibodies.
DESIGNER TRANSCRIPTION FACTORS AND ENZYMES
From the simplest to the most complex, proteins
that bind nucleic acids are involved in orchestrating
gene expression. DNA and RNA are the molecules that
store the recipes of all life forms. The fertilized egg of
a human contains the genetic recipe for the development
and differentiation of a single cell into 2 cells, 4 cells,
and so on, finally yielding a complete individual. The
coordinated expression or reading of the recipes for life
allows cells containing the same genetic information
to perform different functions and to have distinctly
different physical characteristics. Lack of coordination
F i g . 2 . Combining the power of small-molecule chemistry with the
power of protein chemistry and immunology, we have created a new
and effective class of therapeutic molecules, chemically programmed
antibodies. A covalently bound diketone is shown in the active site
of an aldolase antibody. This covalent chemistry allows rapid antibody programming.
due to genetic defects or to viral seizure of control of
the cell results in disease.
In one project, we are developing methods to produce proteins that bind to specific DNA sequences to
control specified genes. As we showed earlier, these proteins can be used as specific genetic switches to turn
on or turn off genes on demand, creating an operating
system for genomes. To this end, we selected and
designed specific zinc finger domains that will constitute an alphabet of 64 domains that will allow any DNA
sequence to be bound selectively. The prospects for
this “second genetic code” are fascinating and should
have a major impact on basic and applied biology.
Billions of transcription factors can now be prepared
by using our approach. Our goal is to develop a new
class of therapeutic proteins that inhibit or enhance
the synthesis of proteins, providing a new strategy for
fighting diseases of either somatic or viral origin.
Using a novel library of transcription factors, we
developed a strategy that effectively allows us to turn
on and turn off every gene in the genome. We recently
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
extended this approach to enable us to endow a variety of enzymes with sequence specificity of our own
design (Fig. 3). In the future, these new enzymes will
enable us to insert, delete, or otherwise modify genes
with surgical precision within any genome.
13
Zhang, H.L., Mitsumori, S., Utsumi, N., Imai, M., Garcia-Delgado, N., Mifsud,
M., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Catalysis of 3-pyrrolidinecarboxylic acid and related pyrrolidine derivatives in enantioselective anti-Mannichtype reactions: importance of the 3-acid group on pyrrolidine for stereocontrol. J.
Am. Chem. Soc. 130:875, 2008.
Cytokine Inflammatory
Signaling in Obesity and
Neurodegeneration
T. Bartfai, B. Conti, I. Tabarean, M. Sanchez-Alavez,
O. Osborn, I. Klein
he “endogenous pyrogen,” the proinflammatory cytokine IL-1, which
has been a major topic of our
studies, is a key cytotoxic agent
for the pancreatic beta cells
that produce insulin. In obesity,
increased insulin resistance
necessitates the production
Tamas Bartfai, Ph.D.
and release of more and more Professor and Chairman
Molecular and Integrative
insulin to control blood glucose
Neurosciences
levels. We wish to know if blocking IL-1 signaling in obese individuals can protect the
beta cells and thus postpone or block the development
of type 2 diabetes and the need for insulin injection as
the endogenous production of insulin decreases and
finally stops. We plan to use AS-1 and EM163, blockers
of signaling by Toll-like receptors that were developed
in collaboration with J. Rebek, the Skaggs Institute.
These low molecular weight blockers, which are mimetics of the adaptor protein myeloid differentiation factor
88, are ideal for oral administration and may be used
for chronic treatment of obesity/type 2 diabetes.
As proof of principle of the therapeutic value of
blocking IL-1 signaling, we used high-affinity mouse
monoclonal antibodies that neutralize IL-1. In obese
animals with stressed, enlarged, and/or dying beta
pancreatic cells, blockade of cytotoxic IL-1 signaling
protected beta cells and prevented enlargement of the
cells. These findings suggest that such blockade would
likely postpone the development of type 2 diabetes, for
which obesity, alongside age, is a major risk factor. This
study opens the way for the use of molecules such
AS-1 and EM 163 and their derivatives in treatment of
obesity to postpone development of type 2 diabetes.
IL-1 is also a major inflammatory and neurogenesispromoting signal in the developing brain. Blockade of
T
F i g . 3 . Through a combination of rational and evolutionary design,
we created a variety of zinc finger enzymes that function in human
cells. Novel enzymes such as recombinases, methylases, nucleases,
and integrases are under development. A designed zinc finger recombinase enzyme is shown above the sequence on which it acts.
PUBLICATIONS
Alonso, D., Kitagaki, S., Utsumi, N., Barbas, C.F. III. Towards organocatalytic
polyketide synthases with diverse electrophile scope: trifluoroethyl thioesters as
nucleophiles in organocatalytic Michael reactions and beyond. Angew. Chem. Int.
Ed. 47:4588, 2008.
Blancafort, P., Tschan, M.P., Bergquist, S., Guthy, D., Brachat, A., Sheeter, D.A.,
Torbett, B.E., Edrmann, D., Barbas, C.F. III. Modulation of drug resistance by artificial transcription factors. Mol. Cancer Ther. 7:688, 2008.
Gordley, R.M., Gersbach, C.A., Barbas, C.F. III. Synthesis of programmable integrases. Proc. Natl. Acad. Sci. U. S. A., in press.
Jiang, L., Althoff, E.A., Clemente, F.R, Doyle, L., Röthlisberger, D., Zanghellini,
A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F. III, Hilvert, D., Houk, K.N.,
Stoddard, B.L., Baker D. De novo computational design of retro-aldol enzymes.
Science 319:1387, 2008.
Magnenat, L., Schwimmer, L.J., Barbas, C.F. III. Drug-inducible and simultaneous
regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches [published correction appears in Gene Ther. 15:1246,
2008]. Gene Ther. 15:1223, 2008.
Massa, A., Utsumi, U., Barbas, C.F. III. N-Tosylimidates in highly enantioselective
organocatalytic Michael reactions. Tetrahedron Lett. 50:145, 2009.
Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Barbas, C.F. III. Water-compatible organocatalysts for direct asymmetric syn-aldol reactions of dihydroxyacetone
and aldehydes. Org. Lett. 10:1621, 2008.
Tanaka, F., Hu, Y., Sutton, J., Asawapornmongkol, L., Fuller, R., Olson, A.J., Barbas, C.F. III, Lerner, R.A. Selection of phage-displayed peptides that bind to a particular ligand-bound antibody. Bioorg. Med. Chem. 16:5926, 2008.
Utsumi, N., Kitagaki, S., Barbas, C.F. III. Organocatalytic Mannich-type reactions
of trifluoroethyl thioesters. Org. Lett. 10:3405, 2008.
Zhang, H., Ramasastry, S.S.V., Tanaka, F., Barbas, C.F. III. Organocatalytic antiMannich reactions with dihydroxyacetone and acyclic dihydroxyacetone derivatives:
a facile route to amino sugars. Adv. Synth. Catal. 350:791, 2008.
14
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
IL-1 signaling during development blunts the division
of nerve cells and thus produces cognitive deficits. This
effect is an important caveat for using blockers of IL-1
signaling in nonadults.
The protein transthyretin can produce plaques similar to the proteolytic degradation product of the amyloid
precusor protein, which forms amyloid plaques, the
hallmark of Alzheimer’s disease. It was assumed that
transthyretin acts as “seed” in the formation of amyloid
plaques and that it indirectly may influence the concentration and actions of the neurotoxic monomers amyloid
β-peptide1-40/42. However, collaborative studies with
J.N. Buxbaum, Scripps Research, in transgenic mice
that overproduce the amyloid peptide indicated the opposite. Transthyretin can protect the brain from the neurotoxic effects of amyloid β-peptides, probably by promoting
the clearance of the neurotoxic monomers, and thus
may be part of endogenous neuroprotection mechanisms. These findings open a previously unknown therapeutic possibility for treatment of Alzheimer’s disease.
PUBLICATIONS
Buxbaum, J.N., Ye, Z., Reixach, N., Friske, L., Levy, C., Das, P., Golde, T.,
Masliah, E., Roberts, A.R., Bartfai, T. Transthyretin protects Alzheimer’s mice
from the behavioral and biochemical effects of Aβ toxicity. Proc. Natl. Acad. Sci.
U. S. A. 105:2681, 2008.
Osborn, O., Brownell, S.E., Sanchez-Alavez, M., Salomon, D., Gram, H., Bartfai,
T. Treatment with an interleukin 1 beta antibody improves glycemic control in dietinduced obesity. Cytokine 44:141, 2008.
Spulber, S., Oprica, M., Bartfai, T., Winblad, B., Schultzberg, M. Blunted neurogenesis and gliosis due to transgenic overexpression of human soluble IL-1ra in the
mouse. Eur. J. Neurosci. 27:549, 2008.
Training in Molecular and
Experimental Medicine
E. Beutler
strong relationship
between the basic sciences of chemistry and
biology and clinical medicine
is essential for understanding
the basic biology of disease
and the directed development
of therapeutic interventions.
Ernest Beutler, M.D.
Professor and Chairman
This understanding requires
Molecular and
specific technical training that
Experimental Medicine
provides a perspective encompassing both sides. The Skaggs Institute for Chemical
A
Biology has attempted to provide such training by supporting young scientists in the Department of Molecular
and Experimental Medicine.
Christian Nievera, under the supervision of Xiaohua
Wu, associate professor, is studying molecular mechanisms involved in the maintenance of genome stability
and repair after DNA damage. Genome instability and
aberrant DNA repair lead to gross chromosomal rearrangement, a major underlying cause for tumorigenesis.
Dr. Nievera is determining the role of the Mre11/Rad50/
Nbs1 (MRN) complex and its interaction with replication protein A in modulating the S-phase checkpoint
after DNA damage. He is examining the mechanism
by which this interaction leads to the suppression of
replication origin firing in response to DNA damage.
Furthermore, he has found that MRN interacts directly
with the breast cancer suppressor protein BRCA1. He
is determining how BRCA1 works with MRN to repair
DNA. Because much of natural resistance to malignant
transformation due to mutational events appears to be
related to the capacity of cells to preserve the integrity
of DNA, an understanding of the mechanisms involved
in these responses is critical.
Jaroslav Truksa, a trainee in my laboratory, has been
studying transcriptional regulation of hepcidin, a critical
regulator of iron metabolism. Hepcidin appears to be
particularly important in anemia of chronic inflammation
and iron refractory anemia. Aberrant hepcidin expression is also associated with hemochromatosis. Dr. Truksa
has used innovative methods—a luciferase reporter and
in vivo bioluminescence—to study transcriptional regulation in intact animals. With this technology, he has
defined an upstream region of the hepcidin promoter
that is important in the response to ingested iron. Using
tissue culture methods, he has defined a second, distinct region in the hepcidin promoter that responds to
cytokine stimulation.
In addition, he examined the role of Tmprss6, a
novel protein associated with iron refractory anemia in
humans, and found that it suppresses the total level of
expression of hepcidin induced by inflammatory cytokines and bone morphogenic proteins. Dr. Truksa is
also examining the repression of hepcidin by growth
differentiation factor GDF15, which is highly expressed
in thalassemia. He is investigating the intracellular
mediators involved in signaling between the cell-surface modulators of iron (hemojuvelin, HFE, TfR2, and
Tmprss6) and the hepcidin gene. Understanding the
pathway of iron regulation by hepcidin will provide
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
insight into the future management both primary and
secondary iron storage diseases.
Each of the trainees has fulfilled the goals of the
Skaggs program by applying basic scientific knowledge
and techniques to disease-related biologic systems. They
have each published several articles in outstanding
journals and have made or are making contributions to
the understanding of clinical disorders.
15
cycles) that are active in vivo in all rodent models of
pain examined. The most extensively studied of the
inhibitors to date (e.g., OL-135 at 20 mg/kg) are as
efficacious as morphine (at 1–3 mg/kg), ibuprofen (at
100 mg/kg), and gabapentin (at 500 mg/kg) and, unlike
ibuprofen and gabapentin, maintain activity across all
rodent pain models (Fig. 1). This research constitutes
Synthetic, Medicinal, and
Bioorganic Chemistry
D.L. Boger, E. Anderson, Y. Ando, K. Boyle, C. Burke,
R. Clark, D. Colby, C. Crane, J. DeMartino, K. Duncan,
C. Ezzili, J. Garfunkle, H. Ge, D. Hochstatter, I. Hwang,
R. Jones, H. Kakei, D. Kato, F.S. Kimball, J. Lajiness,
S. Lee, K. MacMillan, K. Otrubova, P. Patel, W. Robertson,
R. Rodriguez, Y. Sasaki, M. Schnermann, S. Seto,
H. Shimamura, C. Slown, S. Stamm, J. Stover, D. Swingle,
S. Takizawa, A. Tam, P. Va, L. Whitby, A. Wolfe, J. Xie,
A. Zuhl
he research interests of
our group include the
total synthesis of biologically active natural products,
the development of new synthetic methods, heterocyclic
chemistry, bioorganic and medicinal chemistry, combinatorial
Dale L. Boger, Ph.D.
chemistry, the study of DNAProfessor
agent interactions, and the
Chemistry
chemistry of antitumor antibiotics. We place a special emphasis on investigations
to define the structure-function relationships of natural
or designed agents in efforts to understand the origin
of the biological properties.
T
FAT T Y A C I D A M I D E H Y D R O L A S E I N H I B I T O R S
Inhibiting fatty acid amide hydrolase (FAAH)
increases endogenous levels of anandamide, which in
turn leads to analgesia in models of neuropathic and
chronic pain. Extending studies conducted in collaboration with R.A. Lerner and B.F. Cravatt, the Skaggs
Institute, that led to the identification, isolation, characterization, and delineation of the functional role of
FAAH, we have developed a series of exceptionally
potent and selective inhibitors of FAAH (α-ketohetero-
F i g . 1 . Profile of a prototypical FAAH inhibitor.
a well-defined translation of basic science (target discovery, target structure) into useful tools (inhibitors of
enzyme for target validation) and their further development into potential therapeutic agents (optimization
for in vivo efficacy).
DNA-BINDING AGENTS
Our continuing examination of naturally occurring
antitumor agents that derive their biological properties
through sequence-selective DNA binding resulted in a
detailed study of yatakemycin. We defined the exceptional potency of the natural material; characterized
its DNA alkylation properties, consisting of an adenine
N3 alkylation central to a 5-bp adenine-thymine–rich
site; and conducted first- and second-generation syntheses of the natural product in efforts that provided
both the natural and the unnatural enantiomers. The
unnatural enantiomer was just as effective and potent
as the natural product itself.
16
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
These efforts not only provided a sufficient amount
of the scarce natural product for detailed studies of
its properties but also enabled the assignment of its
unknown absolute configuration. Unexpectedly, these
efforts also led to a reassignment of the structure of the
natural product and revealed a new group (thiomethyl
ester) in the molecule that contributes to the properties of yatakemycin. With the systematic preparation
of more than 70 analogs, we have defined key structural features responsible for the biological properties
of this antitumor agent.
VA N C O M Y C I N A N D R E L AT E D G LY C O P E P T I D E
ANTIBIOTICS
Vancomycin and its family of related naturally occurring glycopeptides, which include teicoplanin and ristocetin, are potent antibiotics used to treat infections
caused by microorganisms resistant to other antibiotics. Antibiotics in the vancomycin family inhibit the
synthesis of bacterial cell walls by binding to the terminal D -Ala-D -Ala of the precursor cell wall peptidoglycan, thereby inhibiting the action of transpeptidases
and transglycosylases required to complete the cell
wall synthesis.
As part of a program to define the structural features that contribute to the activity of members of the
vancomycin family and to explore approaches to improve
the biological properties of the drugs, we developed an
efficient de novo synthesis of the aglycons of these
antibiotics. The emergence of vancomycin resistance
is associated with an alteration of the bacterial cell wall
precursor to D -Ala-D -Lac, resulting in a 1000-fold loss
in vancomycin binding affinity and antimicrobial activity.
We showed experimentally that this loss in binding affinity is due primarily to destabilizing lone-pair
interactions (100-fold) rather than to the simple loss
of a single hydrogen bond (10-fold) in the bound complex (Fig. 2). This finding has consequences on the
reengineering of vancomycin to bind D -Ala- D -Lac so
that antimicrobial activity against vancomycin-resistant bacteria can be restored. We recently completed
the total synthesis of the [ψ[CH 2NH]Tpg4]vancomycin
aglycon in which the residue 4 carbonyl and its destabilizing lone pairs have been removed from the vancomycin structure. Examination of this aglycon revealed
that such a reengineering of vancomycin provides antibiotics active against vancomycin-sensitive and vancomycin-resistant bacteria.
Ramoplanin, a naturally occurring complex of 3
components, represents a new and potent antibiotic
F i g . 2 . Origin of vancomycin-destabilized binding to D-Ala-D-Lac.
effective against antibiotic-resistant bacteria, including
vancomycin-resistant strains. As a complement to our
studies on the reengineering of vancomycin, we are
examining this new class of antibiotics. To date, this
research has resulted in the total synthesis of the ramoplanin A2 aglycon (major component of the complex),
the total synthesis and structural reassignment of the
ramoplanin A1 and A3 aglycons (minor components),
and the preparation of a series of key analogs. One
analog, [Dap 2 ]ramoplanin A2 aglycon in which the
labile depsipeptide ester is replaced by a stable amide,
was slightly more potent and much more stable than
the natural product and provided a stable template
for detailed structure-function studies.
PUBLICATIONS
Eubanks, L.M., Hixon, M.S., Jin, W., Hong, S., Clancy, C.M., Tepp, W.H., Baldwin, M.R., Malizio, C.J., Goodnough, M.C., Barbieri, J.T., Johnson, E.A., Boger,
D.L., Dickerson, T.J., Janda, K.D. An in vitro and in vivo disconnect uncovered
through high-throughput identification of botulinum neurotoxin A antagonists [published correction appears in Proc. Natl. Acad. Sci. U. S. A. 104:6490, 2008].
Proc. Natl. Acad. Sci. U. S. A. 104:2602, 2007.
Hardouin, C., Kelso, M.J., Romero, F.A., Rayl, T.J., Leung, D., Hwang, I., Cravatt, B.F., Boger, D.L. Structure-activity relationships of the α-ketooxazole inhibitors of fatty acid amide hydrolase. J. Med. Chem. 50:3359, 2007.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Ishikawa, H., Boger, D.L. Total synthesis of (–)- and ent-(+)-4-desacetoxy-5desethylvindoline. Heterocycles 72:95, 2007.
Jin, W., Trzupek, J.D., Rayl, T.J., Broward, M.A., Vielhauer, G.A., Weir, S.J.,
Hwang, I., Boger, D.L. A unique class of duocarmycin and CC-1065 analogues
subject to reductive activation. J. Am. Chem. Soc. 129:15391, 2007.
Lee, S.Y., Clark, R.C., Boger, D.L. Total synthesis, stereochemical reassignment,
and absolute configuration of chlorofusin. J. Am. Chem. Soc. 129:9860, 2007.
Nam, J., Shin, D., Rew, Y., Boger, D.L. Alanine scan of [L-Dap2]ramoplanin A2
aglycon: assessment of the importance of each residue. J. Am. Chem. Soc.
129:8747, 2007.
Romero, F.A., Du, W., Hwang, I., Rayl, T.J., Kimball, F.S., Leung, D., Hoover,
H.S., Apodaca, R.L., Breitenbucher, J.G., Cravatt, B.F., Boger, D.L. Potent and
selective α-ketoheterocycle-based inhibitors of the anandamide and oleamide
catabolizing enzyme, fatty acid amide hydrolase. J. Med. Chem. 50:1058, 2007.
Tichenor, M.S., MacMillan, K.S., Stover, J.S., Wolkenberg, S.E., Pavani, M.G.,
Zanella, L., Zaid, A.N., Spalluto, G., Rayl, T.J., Hwang, I., Baraldi, P.G., Boger,
D.L. Rational design, synthesis, and evaluation, of key analogues of CC-1065 and
the duocarmycins. J. Am. Chem. Soc. 129:14092, 2007.
Tichenor, M.S., MacMillan, K.S., Trzupek, J.D., Rayl, T.J., Hwang, I., Boger, D.L.
Systematic exploration of the structural features of yatakemycin impacting DNA
alkylation and biological activity. J. Am. Chem. Soc. 129:10858, 2007.
Xu, L., Chong, Y., Hwang, I., D’Onofrio, A., Amore, K., Beardsley, G.P., Li, C.,
Olson, A.J., Boger, D.L., Wilson, I.A. Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine
monophosphate cyclohydrolase. J. Biol. Chem. 282:13033, 2007.
Structure and Biology of
Multidrug Transporters
17
member of the ATP-binding cassette transporter family.
We are also using electron cryomicroscopy, in collaboration with R. Milligan, Scripps Research, to study other
conformations. In addition, we have determined the
x-ray structures of proton-drug antiporters, including
EmrE from the small multidrug resistance transporter
family and EmrD from the major facilitator superfamily.
We recently determined the x-ray structure of P-glycoprotein in complexes with drugs to understand the
structural basis of polyspecificity.
With support from the Skaggs Institute, we have
focused on determining the structure of a bacterial MDR
transporter from the multiple antimicrobial toxin extrusion
family, which has important clinical relevance. This family is the last family of MDR transporters whose structure
has not yet been characterized, and these transporters
play an important role in the efflux of a variety of antibiotic compounds, including fluoroquinolones. MDR
transporters from this family also efflux antimicrobial
compounds in plants as part of the plants’ natural
defense mechanism against microbes. A structure of the
bacterial transporter would greatly aid the rational design
of inhibitors to reverse MDR in the treatment of infection.
Chemical Physiology
G. Chang, S. Aller, Y. Chen, X. He, A. Karyakin, S. Lieu,
T. Nguyen, M. Revin, P. Szewczyk, A. Ward, J. Yu
ultidrug resistance
(MDR) is a major clinical problem in the
chemotherapy of infection and
cancer. The structural characterization of MDR transporters
is important for the development of future compounds to
reverse drug resistance. We
Geoffrey Chang, Ph.D.
Associate Professor
are interested in the molecular
Molecular Biology
basis of the transport of drugs
and lipids across the cell membrane by bacterial and
mammalian MDR transporters. We combine structure,
function, and chemistry through collaborations with
M.G. Finn, the Skaggs Institute, and Q. Zhang, Scripps
Research. We use several techniques, including detergent/lipid protein chromatography, membrane protein
crystallization, and x-ray crystallography.
We have already solved the molecular structures
of several conformations of the lipid flippase MsbA, a
M
B.F. Cravatt, D. Bachovchin, J. Blankman, M. Dix, S. Ji,
F. Kopp, W. Li, J. Long, B. Martin, K. Masuda, M. McKinney,
D. Nomura, G. Simon, J. Thomas, S. Tully, E. Weerapana
e are interested in
understanding complex physiology and
behavior at the level of chemistry and molecules. At the
center of cross talk between
different physiologic processes
are endogenous compounds
that provide a molecular mode
Benjamin F. Cravatt, Ph.D.
Professor
for intersystem communicaCell Biology
tion. However, many of these
molecular messages remain unknown, and even in the
instances in which the participating molecules have
been defined, the mechanisms by which these compounds function and their modes of regulation are for
the most part still a mystery.
One family of chemical messengers we study is the
endogenous cannabinoids (“endocannabinoids”), a class
W
18
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
of lipid signaling molecules that activate cannabinoid
receptors in the nervous system and peripheral tissues.
The levels and signaling function of endocannabinoids
are tightly regulated by enzymes to maintain proper
control over the influence of the endocannabinoids on
brain and body physiology. One of our major goals is
to identify endocannabinoid biosynthetic and degradative enzymes and develop selective genetic and pharmacologic tools to perturb the function of these enzymes
in vivo. An example is fatty acid amide hydrolase (FAAH),
which terminates the signaling function of the endocannabinoid anandamide, as well as several other
amidated lipid transmitters.
We are using transgenic and synthetic chemistry
techniques to study the role of FAAH in regulating fatty
acid amide levels in vivo. We found that transgenic mice
that lack FAAH have highly elevated levels of fatty acid
amide in the brain that correlate with reduced pain
behavior, suggesting that FAAH may be a new therapeutic target for the treatment of pain and related neural
disorders. In collaboration with R.C. Stevens, Scripps
Research, we solved the first 3-dimensional structure
of FAAH. We are using this information to design potent
and selective inhibitors of the enzyme. In studies with
D.L. Boger, the Skaggs Institute, we have identified
potent FAAH inhibitors and using a functional proteomic screen developed by us, have shown that these
inhibitors are highly selective for this enzyme. We are
also interested in proteins responsible for the biosynthesis of endocannabinoids.
A second major focus in the laboratory is the design
and use of chemical probes for the global analysis of
protein function. The evolving field of proteomics, defined
as the simultaneous analysis of the complete protein
content of a given cell or tissue, encompasses considerable conceptual and technical challenges. We hope
to enhance the quality of information obtained from
proteomics experiments by using chemical probes that
indicate the collective catalytic activities of entire classes
of enzymes. Using activity-based probes that target
the serine and metallo hydrolases, we have identified
several enzymes with altered activities in human cancers. Using a combination of pharmacologic and molecular biology approaches, we are now testing the role
that these enzymes play in cancer pathogenesis.
Finally, we are developing proteomic and metabolomic platforms to map endogenous substrates of
enzymes in native biological systems. These large-scale
technologies are intended to provide a global, unbiased
portrait of the physiologic activities of enzymes, thereby
aiding in the functional annotation and assessment of
the enzymes as potential therapeutic targets for a range
of human diseases.
PUBLICATIONS
Ahn, K., Johnson, D.S., Fitzgerald, L.R., Liimatta, M., Arendse, A., Stevenson, T.,
Lund, E.T., Nugent, R.A., Nomanbhoy, T.K., Alexander, J.P., Cravatt, B.F. Novel
mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity. Biochemistry 46:13019, 2007.
Ahn, K., McKinney, M.K., Cravatt, B.F. Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem. Rev. 108:1687, 2008.
Blankman, J.L., Simon, G.M. Cravatt, B.F. A comprehensive profile of brain
enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem. Biol.
14:1347, 2007.
Carlson, E.E., Cravatt, B.F. Enrichment tags for enhanced-resolution profiling of
the polar metabolome. J. Am. Chem. Soc. 129:15780, 2007.
Cravatt, B.F., Simon, G.M., Yates, J.R. III. The biological impact of mass-spectrometry-based proteomics. Nature 450:991, 2007.
Cravatt, B.F., Wright, A.T., Kozarich, J.W. Activity-based protein profiling: from
enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77:383, 2008.
Dix, M.M., Simon, G.M., Cravatt, B.F. Global mapping of the topography and
magnitude of proteolytic events in apoptosis. Cell 134:679, 2008.
Salisbury, C.M., Cravatt, B.F. Optimization of activity-based probes for proteomic
profiling of histone deacetylase complexes. J. Am. Chem. Soc. 130:2184, 2008.
Simon, G.M., Cravatt, B.F. Anandamide biosynthesis catalyzed by the phosphodiesterase GDE1 and detection of glycerophospho-N-acyl ethanolamine precursors in
mouse brain. J. Biol. Chem. 283:9341, 2008.
Weerapana, E., Simon, G.M., Cravatt, B.F. Disparate proteome reactivity profiles
of carbon electrophiles. Nat. Chem. Biol. 4:405, 2008.
Fundamental Processes in
Neural Development
G.M. Edelman, A. Atkins, D.C.Y. Koh, D. Matsuda,
P. Panopoulos, J. Pilotte
cientists in the Department of Neurobiology
focus on the features of
primary cellular processes that
regulate the development of
the vertebrate nervous system.
In the past year, the emphasis
has been on factors that control the translation of mRNA
Gerald M. Edelman, M.D.,
Ph.D.
into protein, including the speProfessor and Chairman
cific regulation of local transNeurobiology
lation at synapses. Equally
important have been studies of RNA-binding proteins
such as the cold-induced RNA-binding motif protein 3
S
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
(RBM3) and its influence on the differentiation and
function of neurons at the level of both transcription
and translation.
Julie Pilotte has observed that RBM3 is highly
expressed in proliferative zones and plastic regions of
the brain in rats and is developmentally regulated. Within
mature neurons, RBM3 is present in nuclei and in dendrites. Dr. Pilotte’s studies in neurons and other cells
have shown that RBM3 localizes to leading edges of processes and to structures containing translation machinery that presage the formation of mature adhesion
plaques. Functional studies indicate that RBM3 has
a profound effect on cell motility and morphology, an
effect that appears to extend to neurite outgrowth.
These effects of RBM3 likely involve the potent regulatory influence of RBM3 on mRNA translation. Our
previous work established that RBM3 greatly enhances
mRNA translation rates, potentially by altering the production of microRNAs. Dr. Pilotte has conducted a
comprehensive analysis of the relationship between
RBM3 expression levels and the production of microRNAs. The data suggest several possible mechanisms
underlying the effects of RBM3 on cell morphology and
motility that involve microRNAs that target components
of the translation machinery and cytoskeleton. These
studies are ongoing. Overall, RBM3 appears to have a
variety of functions critical for cell morphology, migration, and maturation that may be involved in the brain
during neural development and in plasticity in adults.
In the cytoplasm, RBM3 plays important roles in
regulating mRNA transport and translation. In many
cells, however, the protein is more strongly expressed
in the nucleus, where its function is just beginning to
be defined. Annette Atkins used immunoprecipitation
of RBM3 from nuclear extracts and proteomic analyses
in collaboration with L. Liao and J.R. Yates, Scripps
Research, to show that the protein colocalizes with a
subset of proteins from the splicing machinery. Her
further studies have shown that the protein can influence
the splicing of specific mRNAs. Moreover, she obtained
convincing evidence that RBM3 regulates its own expression by splicing an exon with a premature termination
codon from its message, preventing degradation of the
mRNA via a process known as nonsense-mediated decay.
These results add an important new dimension to the
role of RBM3 and related RNA-binding proteins in regulating protein synthesis at multiple levels.
Vincent Mauro and his colleagues have been studying
basic mechanisms of translation initiation in eukaryotes.
19
Earlier studies by this group showed that a 9-nucleotide segment from the 5′ leader of the Gtx homeodomain mRNA facilitated translation initiation by base
pairing to 18S rRNA, the RNA component of 40S ribosomal subunits. Although the Gtx element was tested
in isolation in earlier studies, the results indicated that
eukaryotic mRNAs could initiate translation much as
the Shine-Dalgarno interaction does in bacteria. Studies by Panagiotis Panopoulos have now shown the
physiologic relevance of this element in the context
of 2 natural mRNAs that contain this sequence in their
5′ leaders: Gtx itself and fibroblast growth factor 2. For
these studies, he used modified RNA oligonucleotides to
block mRNA-rRNA base pairing by targeting complementary sequences in either the 18S rRNA or mRNAs and
by mutating the Gtx element in the context of the natural mRNA sequences.
Dora Koh has investigated the translation of the
β-site amyloid precursor protein–cleaving enzyme 1
(BACE1) mRNA. The increased translation of this mRNA
has been implicated in the etiology of Alzheimer’s disease. Her studies resolved an apparent discrepancy
between various published studies by showing that
the various results could be explained by the use of
different expression systems and differences in interpretation. She showed that the translation of the BACE1
mRNA was affected by the expression system and that
it occurred by a ribosomal shunting mechanism when
the mRNA was expressed in the nucleus via RNA polymerase II. In other studies, she has probed the RNA
conformation of 2 endogenous RNAs in living cells:
RNase P, which was probed as a proof of concept, and
BACE1. These studies revealed a strong correlation
between nucleotide accessibility and the site of translation initiation in the BACE1 mRNA, supporting the
tethering/clustering model of translation initiation that
was previously postulated by Dr. Mauro’s group. Daiki
Matsuda is using synthetic mRNAs with multiple potential initiation codons and various obstacles designed to
block individual codons to further investigate the notion
that the accessibility of the initiation codon is a key factor affecting its use.
All of these studies are designed to help define the
molecular and cellular events that regulate the development and function of the nervous system. Our focus
on fundamental processes is based on the belief that
understanding these processes can provide the necessary framework for defining the mechanisms underlying
not just one but many diseases. Indeed, our studies
20
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
have already provided insights into aspects of a number of diseases, including Alzheimer’s disease and
mental retardation syndromes.
Chemical Etiology of Nucleic
Acid Structure
A. Eschenmoser, R. Krishnamurthy, G.K. Mittapalli,
R.R. Kondreddi, Y. Osornio, V.S. Naidu
not considered a potentially prebiotic system, in contrast
to the oligomer system derived from glyceric acid and
tagged via amide bonds with 5-aminopyrimidines.
We have completed the synthesis of such a glyceric
acid–derived oligomer containing six 5-aminouracil
units (6-mer) and have studied its base-pairing properties with DNA, RNA, and α-L-threofuranosyl-(3′g2′)
nucleic acid. Base pairing was strong between the 6mer and poly-d(A) (Fig. 2), was somewhat weaker with
the corresponding poly-r(A), and even occurred with
α-L-threofuranosyl-3′g2′) nucleic acid.
n the general context of our
project to map the landscape of potentially primordial informational oligomer
systems, we have been working during the past year on
the following topics.
I
OLIGOMERS BASED ON
5 - A M I N O P Y R I M I D I N E – TA G G E D
2′g3′-PHOSPHODIESTER–
L I N K E D G LY C E R I C A C I D
Albert Eschenmoser, Ph.D.
Professor
Chemistry
BACKBONES
We have undertaken the synthesis and study of the
base-pairing properties of oligomers derived from a
2′g3′-phosphodiester–linked glyceric acid backbone that
bears 2,4-disubsituted 5-aminopyrimidines, attached to
the carboxyl group of glyceric acid via an amide bond
with the 5-amino group, as recognition elements (Fig. 1).
The structure of this oligomeric system is based on a
structural simplification of the oligonucleotides containing
lyxopyranosyl-(3′g4′)– and threofuranosyl-(2′g3′)–linked
phosphodiester backbones, which we have studied previously. Among the oligomer systems depicted in Figure 1,
the nucleic acid derived from the glycerol backbone is
F i g . 1 . Structural simplification of α- L -threofuranosyl-(3′g2′)
nucleic acid, which was inspired by studies on (3′g4′)-lyxopyranosyl nucleic acid, gives rise to acyclic informational oligomeric systems. Two examples are shown: glycerol nucleic acid and glyceric
acid nucleic acid.
F i g . 2 . UV (left) and circular dichroism (right) spectroscopic
data for base pairing between 5-aminouracil–tagged 2-phosphoglycerate hexamer and DNA, poly(dA); c = 5+5 µM. Measurements
were made in phosphate buffer.
E X P L O R I N G T H E C H E M I S T R Y O F G LY O X Y L AT E A N D
D I H Y D R O X Y F U M A R AT E
A research project such as mapping the landscape
of potentially primordial informational oligomer systems
eventually demands the conception of, and the commitment to, a detailed chemical scenario for the type of
organic chemistry that is supposed to have led to such
oligomers under primordial conditions. Figure 3 depicts
the chemical nature of the scenario we have decided
to study experimentally. In the reaction cycle, glyoxylate
would autocatalytically convert itself into its dimer
dihydroxyfumarate. Dihydroxyfumarate is a known compound that we postulate can act as a common starting material for a large variety of biomolecules, such as
sugars, α-amino acids, and pyrimidines, and for other
organics of etiologic interest by reactions that are essentially unexplored thus far but are deemed compatible with
the constraints of a primordial chemistry. We are conducting exploratory studies for assessing the chemistry
of selected intermediates postulated to be formed from
the chemistry of glyoxylate and dihydroxyfumarate.
Some of the promising preliminary results include the
formation of dihydroxyacetone from the reaction of dihydroxyfumarate with glyoxylate, conversion of 2,3-dioxobutanoic acid (one of the proposed products of the
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
F i g . 3 . Hypothetical autocatalytic cycle for the dimerization of
glyoxylate to dihydroxyfumarate and the biomolecules to be derived
from the constituents of that cycle.
reaction between glyoxylate and dihydroxyfumarate)
to alanine, and identification of hitherto undiscovered
reaction pathways and intermediates in the reaction of
dihydroxyfumarate with itself and with glyoxylate.
PUBLICATIONS
Eschenmoser, A. On a hypothetical generational relationship between HCN and
constituents of the reductive citric acid cycle. Chem. Biodivers. 4:554, 2007.
21
tures to perform these functions, these nucleic acids
tend to assemble into a mixture of properly folded and
misfolded structures during assembly reactions carried
out in vitro. We hope to learn how RNAs avoid misfolding and assemble into functional structures during
biogenesis in vivo.
We have developed a way to use self-cleaving ribozymes embedded in chimeric mRNAs and noncoding
RNAs to probe RNA assembly in living cells. Ribozyme
self-cleavage provides a sensitive, quantitative signal
that functional ribozyme structures have formed. We
began by engineering ribozyme variants with flanking
inserts that have the potential to form complementary
base pairs with parts of the ribozyme sequence. The
inserts are located either upstream or downstream of
the ribozyme sequence so they will be transcribed either
before or after the ribozyme. This arrangement allows
us to examine how the sequential nature of RNA synthesis affects folding outcomes (Fig. 1). Assembly of AltH1,
the alternative helix, is incompatible with assembly
of H1, the ribozyme helix, and prevents self-cleavage.
If RNA helices assemble sequentially during transcription, an upstream insert would inhibit ribozyme assem-
Eschenmoser, A. The search for the chemistry of life’s origin. Tetrahedron
63:12821, 2007.
Koch, K., Schweizer, B., Eschenmoser, A. Reactions of the HCN-tetramer with
aldehydes. Chem. Biodivers. 4:541, 2007.
Intracellular RNA Assembly
M.J. Fedor, J.W. Cottrell, L. Li, L. Liu, O. Tam, P. Watson,
S. Zimmerman
ur goal is to understand
how RNAs fold into the
correct 3-dimensional
structures inside cells. The
mRNAs have long been recognized as key intermediates in
the transmission of information from the DNA sequence
of genes to the amino acid
sequence of proteins, and nonMartha J. Fedor, Ph.D.
Associate Professor
coding RNAs are now known
Molecular Biology
to perform several essential
biological functions previously attributed to proteins.
Although RNAs must adopt precise 3-dimensional struc-
O
F i g . 1 . Chimeric RNAs contain sequences with the potential to
form self-cleaving ribozymes or nonfunctional structures, depending
on the relative contributions of kinetics and thermodynamics to RNAfolding outcomes. The secondary structure of this self-cleaving ribozyme contains an essential base-paired helix, called H1, with 14 bp.
A, A downstream insert (green) is complementary to the adjacent
ribozyme sequence and has the potential to form an alternative helix
with 12 bp, called 3′AltH1. During cotranscriptional RNA assembly
in vitro and in vivo, the H1 structure that forms first resists competition from the downstream 3′AltH1 and allows assembly of a functional, self-cleaving ribozyme. B, In a second ribozyme variant, an
upstream insert (red) is complementary to the ribozyme sequence
and can form a helix with 12 bp, called 5′AltH1, which is incompatible with H1 assembly. Although the 5′AltH1 helix has lower thermodynamic stability than does H1, 5′AltH1 can form first during
transcription to create a kinetic trap that blocks subsequent assembly of a functional ribozyme both in vitro and in vivo.
22
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
bly more than a downstream insert would. We also
designed AltH1 and H1 with different numbers of stabilizing base pairs to learn how thermodynamic stability
contributes to folding outcomes.
In the first set of ribozyme variants we examined,
upstream or downstream AltH1 sequences with 10 bp
were able to compete with assembly of H1 sequences
with 8 bp. This result suggested that thermodynamic
stability, and not the sequential nature of RNA synthesis,
was the major determinant of folding outcomes in vivo.
In vitro, an H1 helix with 8 bp dissociates slowly, with
a half-time on the order of days, so this evidence that
a downstream AltH1 can block assembly of an upstream
H1 implied that some feature of the intracellular environment accelerates exchange between upstream and
downstream structures.
In a second set of variants, H1 helices with 14 bp
were combined with AltH1 helices with 12 bp. These
changes slowed helix dissociation rates even further and
reversed the relative thermodynamic stability of H1 and
AltH1 so that H1 helices were now more stable. Strikingly, assembly of these variants did not always produce the most thermodynamically favored outcome. As
expected, significant cleavage did occur in the presence
of a downstream insert, evidence that stabilizing H1 by
adding base pairs can prevent competition from a shorter
downstream 3′AltH1 that has lower kinetic and thermodynamic stability. However, the upstream 5′AltH1 also
inhibits self-cleavage, even though H1 is longer and more
stable, in contrast to previous evidence that the most
thermodynamically stable structure predominates in vivo.
Thus, ribozymes with 8 bp in H1 and 10 bp in AltH1
seem to reach thermodynamic equilibrium during intracellular assembly, whereas ribozymes with 14 bp in H1
and 12 bp in 5′AltH1 seem to become trapped in an
upstream 5′AltH1 with lower thermodynamic stability.
Evidently, slow helix dissociation can trap RNAs in thermodynamically less favored structures in vivo, but
kinetic trapping requires much longer helices with
much slower dissociation kinetics than expected from
the behavior of similar RNA structures in vitro. Current
efforts focus on understanding the molecular basis of
accelerated exchange between adjacent helices during
RNA biogenesis in living cells.
PUBLICATIONS
Fedor, M.J. Comparative enzymology and structural biology of RNA self-cleavage.
Annu. Rev. Biophys., in press.
Click and Virus-Based Chemistry
for Biological Discovery
M.G. Finn, M. Baksh, D. Banerjee, J. Fiedler, V. Hong,
A. Kislukhin, A. Udit
irus capsids, the protein
shells of virus particles
that self-assemble in
host cells, are a readily available biological material that
we use for a variety of healthrelated applications. During
the past several years, with
M.G. Finn, Ph.D.
crucial support from the Skaggs
Professor
Institute, we have developed
Chemistry
methods for the chemical derivatization of these structures, enabling us to bring the full
power of both chemistry and molecular biology to bear
in the creation of biologically active particles. The chemical methods are derived from click chemistry, the field
of highly reliable synthetic methods developed and popularized by scientists in the laboratories of K.B. Sharpless, the Skaggs Institute, V.V. Fokin, Scripps Research,
and in our laboratory. The following applications were
pursued in the past year.
V
F U N D A M E N TA L C O N J U G AT I O N R E A C T I O N S O F
PROTEINS AND VIRUS PAR TICLES
We continue to develop new chemistry that allows
the facile connections of biologically active molecules
to capsid surfaces. Recently, we focused on connectors that become fluorescent when making bonds to the
desired compounds and that can break those bonds at
a predetermined rate (Fig. 1). This type of copper-free
click chemistry is useful for the synthesis of protein conjugates in vivo and for the construction of delivery agents
that release their cargoes at the desired site of action,
such as a tumor.
P O LY VA L E N T C A R B O H Y D R AT E C O N J U G AT E S
We have extended our investigation of the immune
response to carbohydrates displayed on the exterior surface of virus capsids. We found that the immunogenicity previously detected in chickens also occurs in mice
and that both IgM and IgG antibodies are produced that
are highly selective for the displayed glycans. This finding is important because many pathogens and disease
states are marked by the display of unusual surface glycans. If immune responses can be raised against these
glycans, the possibility exists to create novel vaccines.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
F i g . 1 . Top, Thiol-selective reagents that efficiently label proteins
and peptides, providing a fluorescent signal upon bond formation.
Bottom, Example of the generation of fluorescence upon the addition of a protein containing a free cysteine residue to a 100-µM
solution of the thiol-selective reagent.
C AT I O N I C PA R T I C L E S
Polycations are of interest as cell-penetrating agents
and as molecules that bind tightly to polyanions such
as DNA and RNA. The polyanion heparin is used widely
to inhibit blood clotting, but problems with heparin
overdose are widespread. In the operating room, overdoses are corrected by administering cationic molecules
that complex with heparin to reduce its effect, but the
cationic molecules themselves are anticoagulants when
used at too high a concentration. We created viruslike
particles with enhanced levels of positive charge on
their surfaces and found that these species efficiently
inhibit the action of heparin (Fig. 2). Furthermore, the
particles are not anticoagulants by themselves or in the
presence of heparin and so appear to circumvent the
major difficulty with current antiheparin agents in clinical use. Further testing of these particles in animal
models is under way.
PUBLICATIONS
Bourne, C., Lee, S., Venkataiah, B., Lee, A., Korba, B., Finn, M.G., Zlotnick, A.
Small-molecule effectors of hepatitis B virus capsid assembly give insight into virus
life cycle. J. Virol. 82:10262, 2008.
Hong, V., Udit, A.K., Evans, R.A., Finn, M.G. Electrochemically protected
copper(I)-catalyzed azide-alkyne cycloaddition. ChemBioChem 9:1481, 2008.
Kaltgrad, E., O’Reilly, M.K., Liao, L., Han, S., Paulson, J., Finn, M.G. On-virus
construction of polyvalent glycan ligands for cell-surface receptor. J. Am. Chem.
Soc. 130:4578, 2008.
23
F i g . 2 . Measurement of the time required for clotting of a standard
sample of normal human plasma upon the administration of heparin
and test antagonist. Control experiments indicated that clotting required
approximately 55 seconds in the absence of heparin and 2 minutes
in the presence of heparin. The active viruslike particles T18R and
D14R and the wild-type particle to which 95 copies of cationic peptide 1 (WT-(1)95) were attached by click chemistry completely inhibited
the anticoagulant activity of heparin without themselves inhibiting
coagulation at higher concentrations. This finding contrasts sharply
with the actions of peptide 1 and the clinical agent protamine. These
molecules inhibit heparin at low concentrations but then give rise
to strong anticoagulation when added at slightly higher concentrations,
making them difficult to use in a clinical setting. The data points
for protamine and peptide 1 at >400 seconds represent experiments
in which clotting was not observed within that time.
Miermont, A., Barnhill, H., Strable, E., Lu, X., Wall, K.A., Wang, Q., Finn, M.G.,
Huang, X. Cowpea mosaic virus capsid, a promising carrier for the development of
carbohydrate based antitumor vaccines. Chem. Eur. J. 14:4939, 2008.
Prasuhn, D.E., Jr., Singh, P., Strable, E., Brown, S., Manchester, M., Finn, M.G.
Plasma clearance of bacteriophage Qβ particles as a function of surface charge. J.
Am. Chem. Soc. 130:1328, 2008.
Strable, E., Prasuhn, D.E., Jr., Udit, A.K., Brown, S., Link, A.J., Ngo, J.T., Lander,
G., Quispe, J., Potter, C.S., Carragher, B., Tirrell, D.A., Finn, M.G. Unnatural
amino acid incorporation into virus-like particles. Bioconjug. Chem. 19:866, 2008.
Udit, A.K., Brown, S., Baksh, M.M., Finn, M.G. Immobilization of bacteriophage
Qβ on metal-derivatized surfaces via polyvalent display of hexahistidine tags. J.
Inorg. Biochem. 102:2142, 2008.
Udit, A.K., Everett, C., Gale, A.J., Kyle, J.R., Ozkan, M., Finn, M.G. Heparin
antagonism by polyvalent display of cationic motifs on virus-like particle. ChemBioChem, in press.
Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W.-X., Finn, M.G., Stevens, R.C.,
Wüthrich, K. Microscale NMR screening of new detergents for membrane protein
structural biology. J. Am. Chem. Soc. 130:7357, 2008.
24
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Insights Into Protein Chemistry
and Biology From Protein
Structure
E.D. Getzoff, A.S. Arvai, E.D. Garcin, C. Hitomi, K. Hitomi,
M.D. Kroeger, M.E. Pique, D.S. Shin, J.L. Tubbs
e investigate the chemistry and biology of
proteins, starting from
the determination and analysis
of protein structures. In projects
funded by the Skaggs Institute,
we have focused on understanding catalysis and regulation of
the redox-active superoxide dis- Elizabeth D. Getzoff, Ph.D.
Professor
mutase and nitric oxide synMolecular Biology
thase (NOS) metalloenzymes
that control reactive oxygen species and on proteincofactor interactions that regulate the response of photoactive proteins to light. We integrate high-resolution
crystallographic results with those from spectroscopy,
hydrogen-deuterium exchange mass spectrometry, and
x-ray scattering to probe chemical, conformational, and
dynamic changes in proteins and their cofactors. On the
basis of our integrated results, we propose comprehensive mechanistic models that explain how proteins function as efficient catalysts and molecular machines. We
test these hypotheses with biochemical and mutational
analyses, to improve understanding of how proteins
achieve and regulate their activities and to aid applications of this knowledge for the design of proteins
and inhibitors.
This year, we achieved major advances in isozymeselective inhibition of NOS, including the development
of the anchored plasticity approach for the design of
selective inhibitors. Our research on the light-activated
DNA-repair of (6-4) photoproducts by the enzyme (6-4)
photolyase also shed light on how human cryptochromes
function in circadian clocks to control biological rhythms.
W
ISOZYME-SELECTIVE INHIBITION OF NITRIC
OXIDE SYNTHASE
The 3 human NOS isozymes offer key therapeutic
targets for neurotransmission (neuronal NOS), regulation
of blood pressure (endothelial NOS), and the immune
response (inducible NOS). These highly similar, but differently regulated, isozymes all synthesize the diatomic
molecule nitric oxide, which is both a molecular signal
(at low concentrations) and a cytotoxin (at high concentrations). The aims of our ongoing cross-disciplinary
mutational, biochemical, and structural investigations of
NOS are to (1) determine the bases for functional domain
interactions, cofactor recognition, and tuning for electron
transfer and catalysis; (2) characterize the diverse regulatory mechanisms that differentially control the NOS
isozymes; and (3) elucidate distinguishing features for
isozyme-specific inhibitors. Isozyme-specific NOS inhibitors are sought for medicinal purposes and for advancing
understanding of basic human physiology but present a
huge challenge because of active-site conservation.
Our comprehensive structural and mutagenesis
analyses of NOS in complexes with isozyme-selective
inhibitors revealed determinants for isozyme selectivity. In inducible but not endothelial NOS, bulky inhibitors promote a cascade of conformational changes up
to 20 Å away from the substrate and inhibitor-binding
site (Fig. 1). Correlated side-chain rotations accommo-
F i g . 1 . Isozyme-selective inhibition of NOS. In both isozymes, the
aminopyridine core of the inhibitor (orange) stacks above the heme
and forms hydrogen bonds (black dots) mimicking those of the arginine
substrate. In human endothelial NOS (left), the long tail of the inhibitor protrudes between the heme carboxylate groups (foreground). In
inducible NOS (right), the inhibitor tail projects upward, inducing a
cascade of side-chain conformational changes leading to the opening
of a new selectivity pocket that enhances binding affinity. In endothelial NOS, conformational changes in the conserved glutamine (Q246)
and arginine (R249) side chains are prevented by bulky isozymespecific amino acids distant from the substrate-binding pocket.
date the rigid bulky tails of the selective inhibitors and
expose a new specificity pocket for enhanced inhibitor
binding in inducible NOS. Although first-shell (touching the inhibitor) and second-shell (touching the first
shell) residues that begin this conformational cascade
are invariant, in endothelial NOS their correlated rotations leading to the opening of the specificity pocket
are precluded by bulky isozyme-specific residues at the
far end. Thus, isozyme differences in the plasticity of
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
25
second- and third-shell residues modulate conformational changes of invariant first-shell residues to determine inhibitor selectivity.
Our combined results allowed us to propose, and
successfully test, the anchored plasticity approach for
the design of selective inhibitors. With this approach,
we exploit conserved binding sites coupled to distant
isozyme-specific residues via cascades of conformational
changes; the inhibitor core is designed to mimic the
binding of a substrate or cofactor in a conserved binding site. This anchor is extended by rigid bulky substituents oriented along pathways leading to sequence
or structural variations needed for selectivity. This
anchored plasticity approach exemplifies general principles for the development of novel selective inhibitors
that overcome active-site conservation.
PROTEIN PAR TNERSHIP WITH FAD TO REPAIR DNA
OR CONTROL BIOLOGICAL CLOCKS
We are investigating how living things use cofactorprotein partnerships to transduce environmental changes
into appropriate biological responses. Proteins of the
cryptochrome/photolyase family share not only the same
protein fold but also the redox-active FAD cofactor
bound in an unusual U-shaped conformation beneath
a positively charged groove designed for DNA binding.
Through structural and functional studies of diverse
members of the cryptochrome/photolyase families, we
are deciphering how the similarities and differences in
these molecules direct the same cofactor and protein
fold to produce different biological responses to light:
cryptochromes control biological rhythms, whereas photolyases repair DNA damage.
To develop and test hypotheses for structure-function
relationships in the cryptochrome/photolyase family, we
determined the x-ray crystallographic structure of (6-4)
photolyase, which confers UV protection to plants and
has high sequence similarities (~50% identity) to human
cryptochromes (Fig. 2). In humans and other vertebrates,
cryptochromes are essential components of the circadian
clock, which regulates sleep-wake cycles and other daily
biological rhythms. The eukaryotic (6-4) photolyase
structure revealed a substrate recognition site specific
for the UV-induced DNA lesion, the (6-4) photoproduct,
and cofactor binding sites different from those of the bacterial photolyase, consistent with distinct mechanisms
for activities and regulation. The entrance to the activesite cavity above FAD is constricted by adjacent phosphate-binding and protrusion motifs that correlate with a
phosphorylation site and nuclear localization sequence
F i g . 2 . Structure of (6-4) photolyase. Ribbon diagram shows the
N-terminal α/β domain (top), the helical domain with bound FAD
cofactor (yellow), and the C-terminal extension (lower right) for a
eukaryotic photolyase that also serves as an improved model for
human cryptochromes.
for cryptochrome. We coupled our structural studies
with site-directed mutagenesis and functional assays.
We tested (6-4) photolyase mutants for DNA-repair activity and tested mouse cryptochrome mutants for clock
functions by transient transfection assays done in collaboration with L. DeHaro and S. Panda, the Salk Institute
for Biological Studies.
PUBLICATIONS
Biskup, T., Schleicher, E., Okafuji, A., Link, G., Hitomi, K., Getzoff, E.D., Weber, S.
Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor. Angew. Chem. Int. Ed. 48:404, 2009.
Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson,
G., Andrews, G., Hamley, P.J., Mallinder, P.R., Nicholls, D.J., St-Gallay, S.A., Tinker, A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuehr, D.J.,
Åberg, A., Wallace, A.V., Tainer, J.A., Getzoff, E.D. Anchored plasticity opens
doors for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol.
4:700, 2008.
Shin, D.S., Didonato, M., Barondeau, D.P., Hura, G.L., Hitomi, C., Berglund, J.A.,
Getzoff, E.D., Cary, S.C., Tainer, J.A. Superoxide dismutase from the eukaryotic
thermophile Alvinella pompejana: structures, stability, mechanism, and insights
into amyotrophic lateral sclerosis. J. Mol. Biol., in press.
26
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Structure-Based Design of
Bioactive Agents
M.R. Ghadiri, J.M. Beierle, A. Chavochi, L. Leman,
A. Montero, C.A. Olsen
e are interested in advancing rational structure-based strategies
for the design of bioactive agents.
W
G E N E R AT I O N O F T U R N
MIMETICS
Many protein-protein interactions are mediated through
the recognition of β-turn sec- M. Reza Ghadiri, Ph.D.
ondary structures. Consequently, Professor
Chemistry
small-molecule β-turn mimetics are valuable probes for assessing bioactive ligand
conformations, establishing pharmacophoric requirements, and pursuing rational drug designs. Although
effective drug scaffolds have been developed to precisely position up to 4 functional groups primarily in
2 dimensions, an analogous rigid scaffold capable of
predictably juxtaposing 4 amino acid side chains in
3 dimensions has not been readily available. In order
to meet this deficiency, diverse approaches have been
taken to constrain peptides or peptidelike structures
into turn conformations.
One strategy for generating turn mimics is the use
of cyclic tetrapeptides. Because of their appropriate
size, shape, and useful synthetic modularity, cyclic
tetrapeptides in principle offer an attractive platform
to mimic β-turn regions. However, these tetrapeptides
remain largely unexplored because of poor synthetic
efficiency in constructing the strained 12-membered
ring, an inability to control cis-trans backbone geometry, and the apparent requirement to sacrifice 1 of 4
amino acid residues to incorporate a proline or other
turn-forming residue.
To confront the limitations associated with cyclic
tetrapeptides as β-turn mimics, we have designed and
structurally analyzed 2 alternative classes of 13- or
14-membered ring pseudotetrapeptides containing
either 1 or 2 triazole moieties, respectively (Fig. 1).
Moreover, we have completed the design, syntheses,
structural analyses, and determination of the somatostatin receptor binding activities of a library of all 16
possible strereoisomeric pseudotetramers incorporating
F i g . 1 . Chemical and molecular structures of representative mem-
bers of 2 classes of cyclic pseudotetrapeptide scaffolds. All 4 compounds had 1H NMR spectra consistent with a single conformational
species in solution. Structures were determined by using multidimensional NMR (1–3) or x-ray crystallography (4). Dmb = dimethoxybenzyl.
the somatostatin pharmacophore. In these studies, we
exploited the 1,4-disubstituted 1,2,3-triazole as a trans
peptide-bond surrogate. Structural analysis of the diastereomeric library with nuclear magnetic resonance (NMR)
spectroscopy indicated that each peptide scaffold adopts
a distinct, rigid, conformationally homogeneous turnlike
structure in solution. The 3-dimensional pharmacophoric
display of the pseudotetrapeptides is systematically
altered by varying the stereochemistry around the otherwise constitutionally identical scaffolds, yielding both
compounds with broad-spectrum activity against the 5
human somatostatin receptor subtypes and compounds
with receptor selectivity. Our studies provide a basic
set of scaffolds with subtle but predictable differences
in the spatial display of amino acid side chains that are
useful for rational, structure-based drug design.
INHIBITORS OF HISTONE DEACETYLASES
A fundamental strategy in rationally designing synthetic compounds to bind a protein of interest is to use a
known ligand as a structural model to specify the precise conformational and pharmacophoric requirements
for binding. Despite the remarkable success of this
approach, a major difficulty is that compared to the
receptor-bound structure, free ligands (in the absence of
their cognate receptors) often adopt multiple conformations in solution or in the solid state. These occurrences can make design models based on the free ligand
structure difficult to obtain or even misleading.
Using the rigid scaffold strategy described earlier,
we have gathered evidence that the more potent conformation of apicidin, an archetypal member of a fam-
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
ily of naturally occurring cyclic tetrapeptide inhibitors
of histone deacetylases (HDACs), is not the previously
believed all-trans (t-t-t-t) structure that predominates
in solution, but rather a cis-trans-trans-trans (c-t-t-t)
conformation (Fig. 2). Our approach relies on the design,
synthesis, structural characterization, and functional
analysis of a series of cyclic pseudotetrapeptides bearing 1,4- or 1,5-disubstituted 1,2,3-triazole amino
acids that serve as trans- or cis-amide bond surro-
27
gates, respectively. We have shown that by replacing
an amide bond with a triazole, we can fix the bond
in question in either a trans- or a cis-like configuration,
allowing us to individually probe the binding affinity of
distinct peptide conformations. The heterocyclic compounds adopt conformations that overlay closely with
the targeted conformations of apicidin and have potent
HDAC inhibitory activities, in some instances equivalent to or better than those of the natural product. This
study highlights the usefulness of triazole-modified
cyclic peptides in constructing useful bioactive probe
molecules, supports the c-t-t-t conformation as the
bioactive conformation of the cyclic tetrapeptide HDAC
inhibitors, and provides a useful 3-dimensional pharmacophoric model for use in advancing design principles for more selective HDAC inhibitors.
Bacterial Quorum Sensing
K.D. Janda, J. Ashley, K. Capková, S. De Lamo Marin,
J. Denery, T.J. Dickerson, A. Di Mola, B. Ellis, L. Eubanks,
K. Fukuchi, C. Hernandez, G. Kaufmann, C. Lowery,
S. Mahajan, A. Mayorov, G. McElhaney, J. Mee, A. Moreno,
Y. Nakai, A. Nguyen, A. Nunes, J. Park, A. Rohrbach,
C. Saccavini, N. Salzameda, S. Steiniger, J.B. Treweek,
A. Willis, Y. Xu, Y. Yoneda, B. Zhou, H. Zhou
riginally described as a
method of cell-to-cell
signaling through which
bacterial populations engaged
in coordinated behavior, quorum sensing has since been
shown to mediate several microbial processes, from formation
Kim D. Janda, Ph.D.
of biofilms and bioluminesProfessor
cence to expression of viruChemistry
lence factors, interspecies
competition, and infectivity. Thus, the study of quorum
sensing has both general relevance to the field of microbiology and medical implications in relation to combating bacterial infectivity. With respect to bacterial
infectivity, interference of quorum sensing has been
approached experimentally by designing antagonists
against autoinducers, the signaling molecules secreted
by bacteria, and by using immunopharmacotherapeutic agents to bind secreted quorum-sensing compounds,
thereby preventing signal transmission between cells.
O
F i g . 2 . NMR structures for the triazole-modified apicidin analogs
(side chains are omitted for clarity). A, Peptide 2 adopted a t-t-t-t
conformation (top) that overlays well (bottom) on the lowest energy
calculated conformation of apicidin (yellow; which reportedly closely
matches the predominant conformation of apicidin in dimethylsulfoxide). B, Peptide 11 adopted a c-t-t-t conformation; 2 families of
structures were observed that differ in the rotation of the Trp/Aoda
amide relative to the backbone (top). The structures overlay well on
the crystal structure of apicidin (bottom). C, Peptide 12 adopted a
c-t-c-t conformation (top), which overlays well on the crystal structure of the natural product dihydrotentoxin (yellow; bottom). D, Overlay of Ca atoms for compounds 2 (magenta), 11 (2 structural families,
green and cyan), and 12 (yellow). Because of the c-t-c-t conformation of 12, the Aoda, Trp, and Leu side chains of this peptide project
in the same plane as the backbone ring rather than projecting upward
and out of the plane as for the other peptides. E, Overlay of Ca and
Cb atoms for compounds 2 (magenta) and 11 (2 structural families,
green and cyan). The Ca atoms of the Ala and Ile/Leu residues are
farther apart in 2, and the Ca-Cb vector of 2 directs the Ile side chain
outward away from the ring rather than directly above the ring in 11.
28
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
M O D U L AT I O N O F Q U O R U M S E N S I N G T H R O U G H
S Y N T H E T I C 4 , 5 - D I H Y D R O X Y - 2 , 3 - P E N TA N E D I O N E
A
ANALOGS
The capacity for autoinducers to mediate a variety
of interspecies and interkingdom interactions is achieved
through the specificity of different bacterial species in
secreting and recognizing quorum-sensing molecules
within distinct structural classes. Traditionally, autoinducers have been classified within 2 major groups: acyl
homoserine lactones (AHLs), used by gram-negative bacteria, and oligopeptides, used by gram-positive bacteria.
It has since been realized that the signaling molecules
used by bacteria span a much larger chemical space;
more notable autoinducers include the Pseudomonas
quinolone signal, bradyoxetin, and AI-2, a class of
autoinducers derived from the precursor 4,5-dihydroxy2,3-pentanedione (DPD). The AI-2 quorum-sensing
system is used by both gram-negative and gram-positive bacteria, and the DPD synthase is expressed by
more than 70 bacterial species.
The prevalence of AI-2 was suggestive of its role
in interspecies signaling, and indeed the hypothesis
that a common biosynthetic pathway exists among bacteria has been confirmed chemically. We chose Vibrio
harveyi and Salmonella typhimurium as bacterial models for our investigations of AI-2–based quorum sensing because these are the only species in which the
discrete structures of DPD-based autoinducers and their
respective receptor proteins have been identified. We
hypothesized that upon DPD secretion by one species,
many DPD-based signaling molecules would be generated because of the reactivity and variable stereochemistry of the parent DPD compound.
To probe the specificity of AI-2–based quorum sensing, we synthesized a panel of C1-substituted DPD
analogs and evaluated them in 2 biological assays
(Fig. 1). A goal of this experimental plan was to identify DPD-based agonists or antagonists that could be
used in investigations of unknown AI-2 receptor proteins
and the modulation of AI-2–based signaling. In the first
assay, the effect of all DPD analogs on β-galactosidase
production in S typhimurium was measured in the
absence (agonist screen) and presence (antagonist screen)
of DPD. Excitingly, all test compounds antagonized
AI-2–based quorum sensing. In particular, the propyland butyl-substituted analogs caused potent inhibition
of quorum sensing, with IC50 values 10-fold lower than
the concentration of the natural DPD signal (Fig. 1B).
B
F i g . 1 . DPD analogs. A, Synthetic route for construction of DPD
with alkyl groups introduced at the C-1 position. B, Effect of synthesized DPD-related compounds 5a–5g on quorum sensing as
assessed in 2 biological assays: induction of β-galactosidase activity
in S typhimurium and bioluminescence of V harveyi.
Like S typhimurium, V harveyi is responsive to the
AI-2 class of autoinducers. The second screen capitalized on the inability of V har veyi cells to luminesce
through either the AHL pathway or the AI-2 pathway
in the absence of exogenous DPD. Application of the
DPD analogs to V harveyi cultures resulted in only mild
agonist activity. However, addition of 1 µM DPD to
these cultures had a dramatic synergistic effect: activation of bioluminescence with the DPD analogs was
several-fold greater than that caused by 1 µM DPD
alone (Fig. 1B).
IMPEDING BACTERIAL INFECTION THROUGH
ANTIBODIES AGAINST QUORUM-SENSING
COMPOUNDS
We have pioneered an antibody-based strategy to
combat bacterial infectivity by disrupting transmission
of quorum-sensing signals. Recently, we applied our
antibody-based technology to disruption of the quorumsensing circuits of Pseudomonas aeruginosa. This gramnegative bacterium uses various quorum-sensing systems
for more nefarious purposes with respect to interspecies
communication. Namely, the 2-alkyl-4-quinolones and
related AHLs (Fig. 2) secreted by P aeruginosa have
antibacterial activity against gram-positive bacteria,
allowing P aeruginosa to outcompete other bacterial
species within a shared environment. The clinical relevance of quorum sensing by P aeruginosa includes the
displacement of Staphylococcus aureus in the lungs of
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
29
Kaufmann, G.F., Park, J., Mee, J.M., Ulevitch, R.J., Janda, K.D. The quorum
quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of
the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone. Mol. Immunol. 45:2710, 2008.
Kravchenko, V.V., Kaufmann, G.F., Mathison, J.C., Scott, D.A., Katz, A.Z., Grauer
D.C., Lehmann, M., Meijler, M.M., Janda, K.D., Ulevitch, R.J. Modulation of gene
expression via disruption of NF-κB signaling by a bacterial small molecule. Science
321:259, 2008.
F i g . 2 . General chemical structure of ALHs (left), a major class of
autoinducers, and a representative member of this class, 3-oxo-C12HSL (right).
patients with cystic fibrosis and detrimental AHL-mediated immunomodulation in host cells, including an
altered inflammatory response, a weakened host defense
system, and induction of apoptosis. Therefore, removal
of AHLs, which are thought to mediate the cytotoxicity
in mammalian macrophages and neutrophils, may be
advantageous to controlling this aspect of quorum sensing–related pathogenicity.
To this end, we engineered monoclonal antibodies
against AHL targets, and most notably, we showed that
the monoclonal antibody RS2-1G9 had inhibitory activity in vitro against quorum sensing of P aeruginosa
based on 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL;
Fig. 2). RS2-1G9 not only protected murine macrophages exposed to 3-oxo-C 12-HSL in a concentrationdependent manner but also prevented the downstream
activation of cellular stress kinase pathways, indicating
complete sequestration of 3-oxo-C 12-HSL. Thus, using
this immunopharmacotherapy to quench expression of
bacterial virulence factors and quorum sensing holds
promise both in preventing infection and AHL-associated
cytotoxicity and in developing therapies that will not
promote the evolution of methicillin-resistant S aureus
and future “superbugs.”
PUBLICATIONS
Brogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueous
reduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952,
2007.
Capková, K., Hixon, M.S., McAllister, L.A., Janda, K.D. Toward the discovery of
potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based
assay operational from low to subnanomolar enzyme concentrations. Chem. Commun. (Camb.) Issue 30:3525, 2008.
Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structureactivity relationships of second-generation hydroxamate botulinum neurotoxin A
protease inhibitors. Bioorg. Med. Chem. Lett. 17:6463, 2007.
Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic,
G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B.,
Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.
Kaufmann, G.F., Park, J., Janda, K.D. Bacterial quorum sensing: a new target for
anti-infective immunotherapy. Expert Opin. Biol. Ther. 8:719, 2008.
Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37:1337, 2008.
Lowery, C.A., Park, J., Kaufmann, G.F., Janda, K.D. An unexpected switch in the
modulation of AI-2-based quorum sensing discovered through synthetic 4,5-dihydroxy-2,3-pentanedione analogues. J. Am. Chem. Soc. 130:9200, 2008.
Park, J., Dickerson, T.J., Janda, K.D. Major sperm protein as a diagnostic antigen
for onchocerciasis. Bioorg. Med. Chem. 16:7206, 2008.
Park, J., Jagasia, R., Kaufmann, G.F., Mathison, J.C., Ruiz, D.I., Moss, J.A., Meijler, M.M., Ulevitch, R.J., Janda, K.D. Infection control by antibody disruption of
bacterial quorum sensing signaling. Chem. Biol. 14:1119, 2007.
Park, J., Kaufmann, G.F., Bowen, J.P., Arbiser, J.L., Janda K.D. Solenopsin A, a
venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum sensing signaling in Pseudomonas aeruginosa. J. Infect. Dis. 198:198, 2008.
Richardson, H.N., Zhao, Y., Fekete, E.M., Funk, C.K., Wirsching, P., Janda, K.D.,
Zorrilla, E.P., Koob, G.F. MPZP: a novel small molecule corticotropin-releasing factor
type 1 receptor (CRF1) antagonist. Pharmacol. Biochem. Behav. 88:497, 2008.
Willis, B., Eubanks, L.M., Dickerson, T.J., Janda, K.D. The strange case of the
botulinum neurotoxin: using chemistry and biology to modulate the most deadly
poison. Angew. Chem. Int. Ed.47:8360, 2008.
Willis, B., Eubanks, L.M., Wood, M.R., Janda, K.D., Dickerson, T.J., Lerner, R.A.
Biologically templated organic polymers with nanoscale order. Proc. Natl. Acad.
Sci. U. S. A. 105:1416, 2008.
Xu, Y., Hixon, M.S., Dawson, P.E., Janda, K.D. Development of a FRET assay for
monitoring of HIV gp41 core disruption. J. Org. Chem. 72:6700, 2007.
Yoneda, Y., Steiniger, S.C., Capková, K., Mee, J.M., Liu, Y., Kaufmann, G.F.,
Janda, K.D. A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg. Med. Chem. Lett. 18:1632, 2008.
Zarebski, L.M., Vaughan, K., Sidney, J., Peters, B., Grey, H., Janda, K.D.,
Casadevall, A., Sette, A. Analysis of epitope information related to Bacillus
anthracis and Clostridium botulinum. Expert Rev. Vaccines 7:55, 2008.
Zhou, B., Carney, C., Janda, K.D. Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin. Bioorg. Med. Chem. 16:1903, 2008.
Zhou, B., Pellett, S., Tepp, W.H., Zhou, H., Johnson, E.A., Janda, K.D. Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal
effects. FEBS Lett. 582:1526, 2008.
Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D. Selection and characterization of human monoclonal antibodies against Abrin by phage display. Bioorg. Med.
Chem. Lett. 17:5690, 2007.
30
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Continuous In Vitro Evolution of
RNA Enzymes
G.F. Joyce, B.J. Lam, T.A. Lincoln, B.M. Paegel, K.L. Petrie
e have devised methods for evolving nucleic acid enzymes in
the test tube. These methods
have enabled us to develop
a variety of RNA and DNA
enzymes, some of which have
had applications in biomedicine. We continue both to
Gerald F. Joyce, M.D., Ph.D.
Professor
advance the technology of
Molecular Biology
directed molecular evolution
and to seek novel applications for our evolved enzymes.
W
A D VA N C E D D I R E C T E D E V O L U T I O N T E C H N O L O G Y
Most in vitro evolution studies involve a powerful
but laborious process in which a population of molecules is first challenged to perform a biochemical task,
segregated on the basis of whether or not the molecules performed the task, and then amplified to produce “progeny” molecules that resemble but are not
identical to their parents. This entire set of procedures
is repeated until the population adapts to the task at
hand, a process typically requiring weeks to months to
complete. We have developed methods that allow us
to carry out evolution in a continuous manner, within a
single reaction mixture. The only required manipulation is to refresh periodically the supply of reagents,
a task that is accomplished by either a serial transfer
or serial dilution procedure. The continuous evolution
method allows adaptation to occur within a period of
only a few days.
Recently, we developed a marked enhancement of
continuous in vitro evolution in which the process is
carried out within a computer-controlled microfluidic
chip. The circuits on the chip contain a population of
billions of RNA enzymes with RNA-joining activity, and
these molecules can be challenged to adapt to various
imposed selection constraints. The growth of the population is monitored continuously by using a laser confocal microscope. Whenever the population size reaches
a predetermined threshold, chip-based operations are
executed to isolate a fraction of the population and mix
it with fresh reagents. In a recently published article,
we described the first example of “evolution on a chip,”
in which a population of RNA enzymes underwent
500 iterations of 10-fold exponential growth followed
by 10-fold dilution, carried out during a period of 70
hours. During that time, the molecules evolved to use
progressively lower concentrations of a required substrate; each step of that adaptation was observed in
real time.
We recently devised 2 further enhancements of
the continuous evolution method. The first involves a
controlled mutagenesis technique that can be applied
throughout selective amplification. This technique allows
us to maintain a diverse population of individuals, even
in the face of stringent selection pressure, thus enabling
a more comprehensive exploration of potentially advantageous variants. The second enhancement involves a
method for isolating and then propagating individual
RNA enzymes within water-in-oil compartments within
a microfluidic chip. A novel multiport injector design
allows us to produce millions of individual fluidic compartments of precisely controlled size (Fig. 1), ranging
F i g . 1 . High-throughput production of water droplets in oil, carried
out within a microfluidic chip. The liquid (containing a blue dye)
enters the chip and fans out radially to meet the oil, where it forms
microdroplets (here 100 µm in diameter), which are then collected.
Each droplet contains, on average, 1 starting RNA enzyme and the
materials necessary for that enzyme to undergo continuous evolution.
from 20 to 100 µm in diameter (containing 4–500 pL).
These microcompartments allow each enzyme to express
a “cellular ” phenotype based on the enzyme’s catalytic function.
L I G A N D - D E P E N D E N T E X P O N E N T I A L A M P L I F I C AT I O N
OF RNA
We previously developed an RNA enzyme that catalyzes its own replication by joining 2 RNA substrates
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
to form additional copies of itself. This enzyme was
converted to a cross-catalytic format whereby 2 RNA
enzymes catalyze each other’s synthesis from a total of
4 RNA substrates. We then used in vitro evolution to
improve substantially the activity of the cross-replicating
RNA enzymes. The enzymes now can undergo efficient
exponential amplification, generating about a billion
copies in 30 hours at a constant temperature of 42°C.
Recently, we inserted a ligand-binding domain
adjacent to the catalytic domain of the cross-replicating enzymes so that the enzymes undergo exponential
amplification in the presence, but not the absence, of
the corresponding ligand. The catalytic domain assumes
its active conformation only when the ligand is present,
resulting in a large signal that can be used to detect
and quantify compounds of biomedical interest, such
as proteins, drugs, and metabolites. For example, the
cross-replicating enzymes were made dependent on
theophylline, a drug commonly used to treat respiratory diseases, for which the dose must be carefully
adjusted on the basis of its level in the serum. Strong
exponential amplification occurred in the presence of
theophylline, but amplification in the presence of caffeine was undetectable (Fig. 2), even though the 2
F i g . 2 . Exponential amplification of cross-replicating RNA enzymes
dependent on the compound theophylline. Amplification occurs in
the presence of 1 mM theophylline (filled circles) but not in the
presence of 1 mM caffeine (open circles).
compounds differ by only a single methyl group. Furthermore, the exponential growth rate of the enzymes
depended on the concentration of theophylline, a characteristic that allowed us to construct standardized
curves that could be used to determine the concentration of theophylline in an unknown sample. The method
31
is analogous to quantitative polymerase chain reaction
for the detection of nucleic acids but can be generalized
to a wide variety of targets relevant to medical diagnostics and environmental monitoring.
PUBLICATIONS
Paegel, B.M., Joyce, G.F. Darwinian evolution on a chip. PLoS Biol. 6:e85, 2008.
Synthetic Enzymes, Catalytic
Antibodies, Biomolecular
Computing, and Synthetic
Capsids
E. Keinan, O. Reany, N. Metanis, E. Kossoy, M. Soreni,
R. Piran, M. Sinha, I. Ben-Shir, T. Shekhter, T. Ratner,
T. Mejuch, E. Solel, S. Shoshani, R. Gershoni, A. Karmakar,
D. Pappo, G. Parvari
SYNTHETIC ENZYMES
fforts to generate new
enzymatic activities from
existing protein scaffolds
may not only provide biotechnologically useful catalysts but
also lead to a better understanding of the natural process of evolution. Enzymes are
Ehud Keinan, Ph.D.
usually characterized as catAdjunct Professor
alyzing a specific reaction by a
Molecular Biology
unique chemical mechanism.
However, small changes in the amino acid sequence
of some enzymes can markedly alter the catalytic properties of the enzymes, affecting the substrate selectivity and subtle aspects of the catalytic mechanism. The
catalytic promiscuity displayed in these enzymes may
be an important factor in the natural evolution of new
catalytic activities and in the development of new catalysts through protein engineering.
We are particularly interested in selenoenzymes,
which have a central role in maintaining cellular redox
potential. These enzymes have selenylsulfide bonds in
their active sites that catalyze the reduction of peroxides,
sulfoxides, and disulfides. The selenol-disulfide exchange
reaction is common to all of the enzymes, and the activesite redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation
of enzymes containing selenocysteine is experimentally
E
32
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
challenging. As a result, little is known about the kinetic
role of selenols in enzyme active sites, and the redox
potential of a selenylsulfide or diselenide bond in a protein has not been experimentally determined.
To fully evaluate the effects of selenocysteine on
oxidoreductase redox potential and kinetics, we chemically synthesized glutaredoxin 3 (Grx3) and all 3 selenocysteine variants of its conserved 11CXX14C active
site and determined their redox potentials. In particular,
the position of redox equilibrium between Grx3(C11UC14U) (–308 mV) and thioredoxin (–270 mV) suggests
a possible role for diselenide bonds in biological systems.
Kinetic analysis showed that the lower redox potentials
of the selenocysteine variants are due primarily to the
greater nucleophilicity of the active-site selenium. The
10 2 - to 10 4 -fold increase in the rate of thioredoxin
reduction by the seleno-Grx3 analogs indicates that
oxidoreductases containing either selenylsulfide or
diselenide bonds can have physiologically compatible
redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts. This research
on synthetic enzymes is a collaboration with P.E. Dawson,
Scripps Research.
C ATA LY T I C A N T I B O D I E S
A relatively unexplored opportunity in the science
of catalytic antibodies is modifying the phenotype of
an organism in vivo by incorporating the gene for a catalytic antibody into the genome of that organism. An
attractive application of this concept would be the expression of such a catalyst in transgenic plants to provide
a beneficial trait. For example, introduction of a herbicide-resistance trait in commercial plants is highly desirable because plants with the trait could be grown in the
presence of a nonselective herbicide that affects only
weeds and other undesired plant species.
We have shown that herbicide-resistant plants can be
engineered by designing both a herbicide and a catalytic
antibody that destroys the herbicide within the plants.
Such a transgenic plant was achieved via a 3-step
maneuver: (1) development of a new carbamate herbicide, one that can be catalytically destroyed by the
aldolase antibody 38C2; (2) separate expression of the
light chain and half of the heavy chain (Fab) of the
catalytic antibody in the endoplasmic reticulum of 2
plant lines of Arabidopsis thaliana; and (3) cross-pollination of these 2 transgenic plants to produce a herbicide-resistant F1 hybrid (Fig. 1). In vivo expression
of catalytic antibodies could become a useful, general
strategy to achieve desired phenotype modifications
not only in plants but also in other organisms.
F i g . 1 . Influence of a new herbicide (1) on the rooting and development of A thaliana plant lines. The control plants are shown in
A, C, and E; the hybrid plants (F1) expressing both light and heavy
chains, in B, D, and F. Plantlets grown on medium without the herbicide are shown in A and B; those grown with the herbicide, in
C and F.
BIOMOLECULAR COMPUTING DEVICES
In fully autonomous molecular computing devices,
all components, including input, output, software, and
hardware, are specific molecules that interact with each
other through a cascade of programmable chemical
events, progressing from the input molecule to the
molecular output signal. DNA molecules and DNA
enzymes have been used as convenient, readily available components of such computing devices because
the DNA materials have highly predictable recognition
patterns, reactivity, and information-encoding features.
Furthermore, DNA-based computers can become part
of a biological system, generating outputs in the form
of biomolecular structures and functions.
Our previously reported 2-symbol–2-state finite
automata computed autonomously, and all of their components were soluble biomolecules mixed in solution.
The hardware consisted of 2 enzymes, an endonuclease and a ligase, and the software and the input were
double-stranded DNA oligomers. More recently, we
designed and created 3-symbol–3-state automata that
can carry out more complex computations. In addition,
we found that immobilization of the input molecules on
chips allowed parallel computation, a system that can
be to encrypt information.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
The main advantage of autonomous biomolecular
computing devices compared with electronic computers
is the ability of the devices to interact directly with biological systems. No interface is required because all
components of molecular computers, including hardware, software, input, and output, are molecules that
interact in solution along a cascade of programmable
chemical events. We showed for the first time that the
output of a molecular finite automaton can be a visible
bacterial phenotype. Our 2-symbol–2-state finite automaton uses linear double-stranded DNA inputs prepared
by inserting a string of 6-bp symbols into the lacZ gene
on plasmid pUC18. The computation resulted in a circular plasmid that differed from the original pUC18 by
either a 9-bp (accepting state) or an 11-bp (unaccepting state) insert within the lacZ gene. Upon transformation
and expression of the resultant plasmids in Escherichia
coli, either blue colonies or white colonies, respectively,
were formed (Fig. 2).
33
constructs and molecular computing. By examining physical models of spherical virus assembly, we developed
a general synthetic strategy for producing chemical capsids at sizes between fullerenes and spherical viruses.
Such capsids can be formed by self-assembly from a
class of novel symmetric molecules developed from a
pentagonal core. By designing chemical complementarity into the 5 interface edges of the molecule, we
can produce self-assembling stable structures of icosahedral symmetry.
We considered 3 different binding mechanisms:
hydrogen bonding, metal binding, and formation of
disulfide bonds. These structures can be designed to
assemble and disassemble under controlled environmental conditions. We have conducted molecular dynamics simulation on a class of corannulene-based molecules
to demonstrate the characteristics of self-assembly and
to aid in the design of the molecular subunits. This
research was done in collaboration with A.J. Olson,
Scripps Research.
PUBLICATIONS
Ben Shir, I., Sasmal, S., Mejuch, T., Sinha, M.K., Kapon, M., Keinan, E. Repulsive interaction can be a key design element of molecular rotary motors. J. Org.
Chem. 73:8772, 2008.
Pappo, D., Mejuch, T., Reany, O., Solel, E., Gurram, M., Keinan, E. Diverse functionalization of corannulene: easy access to pentagonal superstructures. Org. Lett., in press.
Understanding and Ameliorating
Protein-Misfolding Diseases
J.W. Kelly, S. Choi, E. Culyba, M.T.A. Dendle, D. Du,
C. Fearns, A.A. Fuller, T.-W. Mu, A. Murray, D. Ong,
J. Paulsson, E.T. Powers, P. Rao, M. Saure, R. Simkovsky,
S. Siegel, J. Solomon, K. Usui, Y. Wang, I. Yonemoto, Z. Yu
F i g . 2 . Computation with aaba input in the presence (A) and
absence (B) of transition molecules results in white bacteria when
the transition molecules are present. Computation with abba input
in the presence (C) and absence (D) of transition molecules results
in blue bacteria when the transition molecules are present.
SYNTHETIC CAPSIDS
Stable structures of icosahedral symmetry can have
numerous functional roles, including chemical microencapsulation and delivery of drugs and biomolecules, a
way to observe encapsulated reactive intermediates, presentation of epitopes for efficient immunization, synthesis of nanoparticles of uniform size, and formation
of structural elements for molecular supramolecular
ur main goal is to gain
insight into the mechanisms of proteome maintenance that can be used to
develop new therapeutic strategies to ameliorate protein-misfolding diseases when deficiencies
in protein maintenance occur.
Maintenance of the proteome
Jeffery W. Kelly, Ph.D.
Professor, Chemistry
(proteostasis) both inside and
Chairman, Molecular and
outside human cells is essential Experimental Medicine
for development, reproduction,
and successful aging. Deficiencies in proteostasis lead
O
34
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
to many metabolic, oncologic, neurodegenerative, and
cardiovascular disorders. We focus mostly on neurodegenerative diseases, and we benefit greatly from collaborations with J. Buxbaum, J.R. Yates, and W.E. Balch
at Scripps Research and with A. Dillin at the Salk
Institute for Biological Studies, La Jolla, California.
A MODEL FOR PROTEIN EXPORT FROM THE
ENDOPLASMIC RETICULUM
About one-third of all eukaryotic proteins, including
all membrane proteins and secreted proteins, are folded
in and then exported from the endoplasmic reticulum.
Proteins are initially unstructured and must fold into welldefined structures to become functional. Unfortunately,
proteins can also misfold, leaving them trapped in nonfunctional, sometimes aggregated, structures. Because of
this inherent inefficiency in protein folding, the endoplasmic reticulum has a set of pathways that regulate protein
folding and export. These pathways include an export
pathway, which recognizes and exports properly folded
proteins; an endoplasmic reticulum–associated degradation pathway, which recognizes and degrades unfolded or
misfolded proteins; and a chaperoning pathway, which
recognizes and recovers misfolded proteins. The efficiency with which a given protein is exported, defined
as the rate of synthesis divided by the rate of export,
depends on the interplay between the activities of these
3 pathways and the thermodynamics and kinetics of
the folding and misfolding processes of the protein.
We recently described the FoldEx model of folding for
export from the endoplasmic reticulum; this model was
designed to semiquantitatively capture this interplay. In
FoldEx, the activities of the export, degradation, and
chaperone-mediated folding pathways are (most easily)
controlled through the concentrations of the machineries
that make up the pathways. The thermodynamics of folding are quantified by the equilibrium constant for folding,
and the kinetics of folding or misfolding are quantified
by the time required for folding or misfolding to reach its
half-way point. The FoldEx model establishes that no
single feature of protein folding energetics or endoplasmic reticulum biology dictates folding and transport efficiency. Instead, a network view provides insight into the
basis for cellular diversity, disease origins, and protein
homeostasis and predicts strategies for restoring protein
homeostasis in protein-misfolding diseases.
P R O T E O S TA S I S R E G U L AT O R S A N D P H A R M A C O L O G I C
C H A P E R O N E S I N LY S O S O M A L S T O R A G E D I S E A S E S
Lysosomal storage diseases are loss-of-function
diseases often caused by a mutation in one of the
lysosomal enzymes. The mutation results in excessive
misfolding and degradation of the enzyme within the
endoplasmic reticulum instead of proper folding and
trafficking of the enzyme to the lysosome. The resulting deficiency in lysosomal enzyme activity leads to
accumulation of the substrate of the mutant lysosomal
enzyme. At least 40 distinct lysosomal storage diseases
have been identified; the most prevalent is Gaucher
disease, which is caused by a deficiency in the activity of lysosomal glucocerebrosidase. Previously, we
showed that in fibroblasts derived from the tissue of
a patient with Gaucher disease, novel pharmacologic
chaperones enhanced glucocerebrosidase activity up
to 7.2-fold by binding directly to the enzyme and thereby
stabilizing it.
More recently, we found that the innate proteostasis capacity of a cell can be enhanced with small molecules we call proteostasis regulators to fold mutated
enzymes that would otherwise misfold and be degraded,
resulting in increased trafficking of the mutated enzyme
to the lysosome and increased function. We discovered
that inhibiting L-type calcium channels with either diltiazem or verapamil partially restored enzyme homeostasis in 3 distinct lysosomal storage diseases: Gaucher
disease, α-mannosidosis, and type IIIA mucopolysaccharidosis. The increased capacity of the endoplasmic
reticulum to fold misfolding-prone proteins probably is
due to a modest, calcium ion–mediated upregulation
of a subset of molecular chaperones, including calnexin and calreticulin.
C O R R E L AT I N G T H E F O L D I N G A N D A S S E M B LY
ENERGETICS OF TRANSTHYRETIN WITH DISEASE
PHENOTYPES
Transthyretin, a tetrameric protein, is the primary
transporter of retinol-binding protein and the secondary
transporter of the thyroid hormone thyroxine. Transthyretin can dissociate, misfold, and aggregate, forming deposits that interfere with the normal functioning
of several tissues or organs. Destabilized mutants of
transthyretin are particularly prone to aggregation,
but the precise energetic effects of the mutations are
obscured by the linked folding and assembly equilibria
of transthyretin. We used urea denaturation studies of
transthyretin and several of its mutants to quantify the
thermodynamically linked quaternary and tertiary structural stability to better understand the relationship
between mutant folding energetics and amyloid disease phenotype.
Using a method of analysis that simultaneously
accounts for the 2-step denaturation (tetramer disso-
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
ciation followed by unfolding), we analyzed the stability of quaternary and tertiary structures of wild-type
transthyretin and the V122I variant, which is linked to
late-onset familial amyloid cardiomyopathy, the most
common familial transthyretin amyloid disease. The
results indicated that V122I transthyretin has a destabilized quaternary structure and a stable tertiary structure
relative to wild-type transthyretin. We also examined 3
other variants of transthyretin: L55P, V30M, and A25T.
We found that both the L55P mutant, associated with
the most aggressive familial transthyretin amyloid disease, and the V30M mutant, the most common mutation
associated with neuropathic forms of transthyretin amyloidosis, have complex denaturation pathways that cannot be fit to the 2-step denaturation model. Nevertheless,
L55P transthyretin is clearly less stable than is wild-type
transthyretin, primarily because the tertiary structure of
L55P is unstable, although its quaternary structure is
destabilized as well. Published data suggest that V30M
transthyretin has stable quaternary structure but unstable
tertiary structure. The A25T mutant, associated with
CNS amyloidosis, is highly prone to aggregation and has
drastically reduced quaternary and tertiary structural stability. The observed differences in stability among the
disease-associated transthyretin variants highlight the
complexity and the heterogeneity of transthyretin amyloid
disease, an observation with important implications
for the treatment of these diseases.
A G G R E G AT I O N O F A M Y L I N A N D I T S P R O C E S S I N G
35
the amyloidogenicity of amylin and its processing
intermediates is negatively correlated with net charge
and charge at the C terminus. Although our conditions
may not precisely reflect those of amyloidogenesis in
vivo, the lower amyloidogenicity of the processing intermediates relative to amylin suggests that the presence
of the intermediates in intracellular amyloid deposits
in the increasingly stressed beta cells of patients with
diabetes may be a consequence of general defects in
protein homeostasis known to occur in diabetes rather
than the result of the amylin processing intermediates
acting as initiators of amyloid.
PUBLICATIONS
Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for disease intervention. Science 319:916, 2008.
Bieschke, J., Siegel, S.J., Fu, J., Kelly, J.W. Alzheimer’s Aβ peptides containing
an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity: evidence for a common low-abundance toxic structure(s)? Biochemistry 47:50, 2008.
Gao, J., Kelly, J.W. Toward quantification of protein backbone-backbone hydrogen
bonding energies: an energetic analysis of an amide-to-ester mutation in an α-helix
within a protein. Protein Sci. 17:1096, 2008.
Hurshman Babbes, A.R., Powers, E.T., Kelly, J.W. Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its
disease-associated variants: the relationship between stability and amyloidosis.
Biochemistry 47:6969, 2008.
Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural
evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.
Liu, F., Du, D., Fuller, A.A., Davoren, J.E., Wipf, P., Kelly, J.W., Gruebele, M. An
experimental survey of the transition between two-state and downhill protein folding scenarios. Proc. Natl. Acad. Sci. U. S. A. 105:2369, 2008.
I N T E R M E D I AT E S
Human amylin, or islet amyloid polypeptide, is a
peptide cosecreted with insulin by the beta cells of the
pancreatic islets of Langerhans. The 37-residue, C-terminally amidated human amylin peptide is derived from
a proprotein that undergoes formation of disulfide bonds
in the endoplasmic reticulum and then 4 enzymatic
processing events in the immature secretory granule.
Human amylin forms both intracellular and extracellular amyloid deposits in the pancreas of most patients
with type 2 diabetes, likely reflecting compromised function of secretory cells. In addition, amylin-processing
intermediates have been reported as components of
intracellular amyloid in beta cells.
We investigated the amyloidogenicity of amylin
and its processing intermediates in vitro. Under conditions mimicking those in immature secretory granules
(37°C, pH 6), amylin forms amyloid aggregates more
rapidly than its processing intermediates and its reduced
counterparts form aggregates. Our results indicate that
Mu, T.-W., Fowler, D.M., Kelly, J.W. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium
homeostasis. PloS Biol. 6:e26, 2008.
Mu, T.-W., Ong, D.S.T., Wang, Y.-J., Balch, W.E., Yates, J.R. III, Segatori, L.,
Kelly, J.W. Chemical and biological approaches synergize to ameliorate proteinfolding diseases. Cell 134:769, 2008.
Reixach, N., Foss, T.R., Santelli, E., Pascual, J., Kelly, J.W. Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic: a lesson in
the generation of murine models of diseases involving oligomeric proteins. J. Biol.
Chem. 283:2098, 2008.
Wiseman, R.L., Powers, E.T., Buxbaum, J.N., Kelly, J.W., Balch, W.E. An adaptable
standard for protein export from the endoplasmic reticulum. Cell 131:809, 2007.
Yonemoto, I.T., Kroon, G.J.A., Dyson, H.J., Balch, W.E., Kelly, J.W. Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state. Biochemistry 47:9900, 2008.
36
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Engineering Eukaryotic Algal
Chloroplasts for Production of
Human Therapeutic Proteins
and of Biofuels
S.P. Mayfield, M. Tran, A. Manuell, J. Marin-Navarro,
M. Muto, P. Pettersson, P. Lee, B. Rasala, M. Jager
lgae offer a tremendous
opportunity as a biotechnology platform both
for the production of protein
therapeutics and as a source
of renewable energy. Because
production of therapeutic proteins in algae can be achieved
at a fraction of the cost of traStephen P. Mayfield, Ph.D.
ditional mammalian cell culProfessor
ture, algal production has the
Cell Biology
potential to dramatically reduce
the cost of protein-based drugs. Algae can also produce biomass at 10 times the rate of terrestrial plants
and can be grown in minimal media on land not suitable for food crop production, making algae a potential source of renewable biofuels.
To realize the potential of algae for production of
biofuels and therapeutic proteins, we must understand
and control algal gene expression. In algae, both proteins and biofuel molecules are produced in chloroplasts, and understanding chloroplast gene expression
will be essential to developing algae as a biotechnology platform. Using molecular, biochemical, genetic,
and structural biology, we have identified key factors
that control gene expression within the chloroplast
and have used this knowledge to produce algae that
have strong, regulated protein expression. With this
basic understanding of the genetics and biology of
algae, we can develop eukaryotic algae as a cost-effective system for biotechnology applications, including
the production of human therapeutic proteins, industrial enzymes, and biofuels.
A
HUMAN THERAPEUTIC PROTEINS
During the past several years, we have developed
a system for the expression of recombinant proteins in
the green alga Chlamydomonas reinhardtii. We now
routinely obtain strong expression of complex mammalian proteins that are suitable as human therapeutic
agents. We have expressed a number of proteins in
algae, including monoclonal antibodies, growth factors, and a variety of other potential therapeutics.
In addition to therapeutic proteins, we have also
expressed eukaryotic protein toxins, an achievement
that is possible because chloroplasts are naturally
resistant to such toxins. We have now developed antibody-toxin fusion proteins, a class of recombinant protein molecules that can target and kill eukaryotic cells,
including human cancer cells. The production of antibody-toxin fusion proteins is unique to our expression
system; bacterial expression systems cannot efficiently
produce these complex molecules, and mammalian
cell cultures would be killed by the toxin during production. Thus, algal chloroplasts are the best system
for the production of this type of superior cancer therapeutic agent.
To examine the potential of algae to produce antibody-toxin fusion proteins as cancer therapeutics, we
engineered an antibody to CD19 to fuse with an exotoxin-A protein domain to produce the antibody-toxin
fusion protein anti-CD19–ETA, which targets human
B-cell lymphomas. This recombinant fusion protein
was expressed in algae, where it accumulated as a soluble protein. Using cell-based assays, we showed that
isolated anti-CD19–ETA efficiently binds to CD19 +
human B-cell lymphomas but does not bind to CD19–
normal human cells. Once bound to the tumor cells,
anti-CD19–ETA efficiently kills the cells. These cellbased assays are the first step in demonstrating the
potential of these fusion proteins as human anticancer
therapeutic agents.
BIOFUELS
With fossil fuel reserves dwindling, mandates
requiring the reduction of carbon dioxide emissions,
and a need for national energy independence, we face
the formidable challenge of developing sustainable
forms of carbon-neutral energy in an economically practical manner. Algae offer the potential to produce carbon-neutral liquid biofuels at a scale and cost that
can be competitive with existing fossil fuel production. In addition, production of biofuels from algae
will not compete with production of food crops for
use of arable land. Economic production of biofuels
from algae will require production of other molecules
in addition to the biofuel molecules, because all of
the biomass produced from algae will need to have a
commercial value. The technology we developed for
the production of therapeutic proteins in algae can be
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
used to produce algae with valuable protein coproducts
as well as improved biofuel characteristics, and we are
now developing algae as a renewable energy source.
PUBLICATIONS
Beligni, M.V., Mayfield, S.P. Arabidopsis thaliana mutants reveal a role for CSP41a
and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal
RNA metabolism. Plant Mol. Biol. 67:389, 2008.
Manuell, A.L., Beligni, M.V., Elder, J.H., Siefker, D.T., Tran, M., Weber, A., McDonald, T.L., Mayfield, S.P. Robust expression of a bioactive mammalian protein in
Chlamydomonas chloroplast. Plant Biotechnol. J. 5:402, 2007.
Auditory Perception and Hearing
Impairment: From Mouse
Models to Human Genetic
Disease
U. Müller, C. Barros, F. Conti, H. Elledge, S. Franco,
N. Grillet, S. Harkins-Perry, P. Kazmierczak, I. Martinez-Garay,
R. Radakovits, C. Ramos, A. Reynolds, M. Schwander,
S. Webb, W. Xiong
echanosensation, the
transduction of mechanical force into an electrochemical signal, allows living
organisms to detect touch, hear,
register movement and gravity, and sense changes in cell
volume and shape. The hair
cells of the mammalian inner
ear are the principal mechUllrich Müller, Ph.D.
Professor
anosensors for the detection
Cell Biology
of sound and head movement.
We identify and study genes that control the development and function of hair cells. We are particularly
interested in the molecules that form the mechanotransduction machinery of these cells. We also analyze the
mechanisms that establish neuronal connections between
the auditory sense organs and the cerebral cortex and
the formation of cell layers and neuronal circuits within
the cerebral cortex.
M
MOLECULAR COMPOSITION OF THE
MECHANOTRANSDUCTION MACHINERY IN HAIR CELLS
The mechanically sensitive organelle of a hair cell
is the hair bundle, which consists of dozens of stereocilia that project from the apical cell surface. Mechanotransduction channels are localized close to the tips of
37
stereocilia. Tip-links, extracellular filaments that connect
the tips of neighboring stereocilia and are visible by electron microscopy, are thought to transmit sound-induced
tension force onto the transduction channels. The molecular identity of most components of the mechanotransduction complex is still largely unknown.
To identify genes that control hair cell function, such
as the transduction channel and the tip-links, we use
genetic approaches. Approximately 1 child in 1000
children is born deaf, and a large part of the human
population experiences age-related hearing loss. Many
forms of hearing loss are of genetic origin, and mutations in more than 400 genes cause deafness. Recently,
we discovered that some of the genes linked to Usher
syndrome, the leading cause of deaf-blindness in humans,
may encode components of the mechanotransduction
machinery in hair cells. Using mouse model systems,
we have shown that the Usher syndrome proteins cadherin 23 and protocadherin 15 interact to form tiplinks in hair cells.
Intriguingly, some of the mutations in the genes for
cadherin 23 and protocadherin 15 that cause deafness in
humans disrupt interactions between the 2 proteins. Others leave interactions intact and probably change the
mechanical properties of tip-links and affect the mechanotransduction process. Currently, we are defining the biophysical properties of cadherin 23 and protocadherin
15, investigating whether other genes linked to deafness
encode components of the mechanotransduction machinery, and generating mouse models for diseases that affect
hair cell function in mechanotransduction.
MOUSE MODELS OF DEAFNESS
Deafness has a profound effect on the quality of life
of the affected individuals, yet few promising therapeutic
approaches exist to help these individuals. To identify
genes that control auditory perception and to provide
animal models for the human disease, we carried out
a genetic screen in mice. Using N-ethyl-N-nitrosourea,
we introduced point mutations in the germ line of mice.
Using phenotypic screens, we identified more than 20
mouse lines in which the mice inherit hearing defects
as recessive traits. We have mapped many of the mutations to chromosomal intervals and have used DNA
sequencing to identify mutations in single genes that
cause some of the hearing defects.
All of the genes that we have identified so far are
expressed in hair cells. One of the genes encodes the
unconventional motor protein myosin VIIa. Interestingly, mutations in myosin VIIa have been linked to
38
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Usher syndrome in humans. Our phenotypic analysis
of the mice with the myosin VIIa mutation indicated
that the mutation affects expression of the protein differentially in the ear and retina and provides a molecular explanation of why some mutations in the human
gene for myosin VIIa affect the function of the protein
in the ear and retina, whereas other mutations only
affect the function of myosin VIIa in the ear. A mutation in a second mouse line maps to a gene that encodes
an enzyme important for the control of neurotransmitter
levels. We have identified a similar mutation in human
families with deafness.
Our studies show that genetic screens in mice are
powerful tools for identifying mutations that control the
function of hair cells and that mutations in mice can
serve as models for deafness in humans. In future studies, we will define the structure and function of the
normal and pathogenic variants of the involved proteins to gain insights into disease mechanisms at the
cellular, molecular, and anatomic level.
PUBLICATIONS
Du, X., Schwander, M., Moresco, E.M., Viviani, P., Haller, C., Hildebrand, M.S.,
Pak, K., Tarantino, L., Roberts, A. Richardson, H., Koob, G., Najmabadi, H.,
Ryan, A.F., Smith, R.J., Müller, U., Beutler, B. A catechol-O-methyltransferase
that is essential for auditory function in mice and humans. Proc. Natl. Acad. Sci.
U. S. A. 105:14609, 2008.
Müller, U. Cadherins and mechanotransduction by hair cells. Curr. Opin. Cell Biol.
20:557, 2008.
Müller, U., Gillespie, P. Silencing the cochlear amplifier by immobilizing prestin.
Neuron 58:299, 2008.
Chemical Synthesis and
Chemical Biology
K.C. Nicolaou, A. Agua, R. Aversa, W. Brenzovich, J. Chen,
S. Dalby, D. Edmonds, S. Ellery, A. Estrada, B. Fraga,
M. Frederick, M. Freestone, C. Gelin, J. Goodwin-Tindall,
V. Gondi, M. Hesse, P. Huang, Z. Huang, V. Jeso, J. Jin,
M. Kar, A. Krasovskiy, A. Lemire, R. Levin, A. Li, H. Li, Y. Lim,
T. Lister, U. Majumder, C. Mathison, A. Morgan, A. Nold,
A. Ortiz, N. Patil, B. Pratt, R. Reingruber, F. Rivas,
A. Sanchez Ruiz, D. Sarlah, D. Shaw, A. Stepan, A. Talbot,
Y. Tang, V. Trépanier, G. Tria, T. Umezawa, J. Wang, T. Wu,
W. Zhan, H. Zhang
uring the past year, we
made considerable progress in a number of
areas, including the total synthesis and biological investigation of neurotoxins, antitumor
agents, and antibiotics (Fig. 1).
In an effort to resolve a conK.C. Nicolaou, Ph.D.
troversy over the structure of
Professor and Chairman
maitotoxin, the largest and most Chemistry
potent marine neurotoxin, we
synthesized the GHIJK and GHIJKMNO domains of the
molecule and provided spectroscopic support for the
originally assigned structure of this biomolecule. We
also provided synthetic azaspiracids, another group of
marine neurotoxins, to biologists who investigated the
neurotoxic properties and mechanism of action of the
molecules. During the same period, we completed the
total synthesis of several members of the artochamin
family of natural products, which have cytotoxic properties. We also developed an asymmetric synthesis of
the uncialamycins and determined their DNA-cleaving
properties and extremely high cytotoxic effects against
tumor cells and drug-resistant bacteria. Our research
in infectious diseases yielded a series of biyouyanagin
analogs, some of which had anti-HIV/AIDS properties.
In addition, we developed asymmetric total syntheses
of platensimycin and platencin, 2 naturally occurring
antibiotics that exert their activities against drug-resistant
bacteria through a novel mechanism involving inhibition
of fatty acid biosynthesis. Carbaplatensimycin, a carbon analog of platensimycin, was also synthesized and
tested. In yet another area, we developed chemistry
suitable for the eventual total synthesis of sporolide B,
D
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
39
Nicolaou, K.C., Dethe, D.H., Leung, G.Y.C., Zou, B., Chen, D.Y.-K. Total synthesis
of thiopeptide antibiotics GE2270A, GE2270T, and GE2270C. Chem. Asian J.
3:413, 2008.
Nicolaou, K.C., Frederick, M.O., Aversa, R.J. The continuing saga of the marine
polyether biotoxins. Angew. Chem. Int. Ed. 47:7182, 2008.
Nicolaou, K.C., Frederick, M.O., Burtoloso, A.C.B., Denton, R.M., Rivas, F., Cole,
K.P., Aversa, R.J., Gibe, R., Umezawa, T., Suzuki, T. Chemical synthesis of the
GHIJKLMNO ring system of maitotoxin. J. Am. Chem. Soc. 130:7466, 2008.
Nicolaou, K.C., Krasovskiy, A., Trépanier, V.É., Chen, D.Y.-K. An expedient strategy for the synthesis of tryptamines and other heterocycles. Angew. Chem. Int. Ed.
47:4217, 2008.
Nicolaou, K.C., Leung, G.Y.C., Dethe, D.H., Guduru, R., Sun, Y.-P. Lim, C.S.,
Chen, D.Y.-K. Chemical synthesis and biological evaluation of palmerolide A analogues. J. Am. Chem. Soc. 130:10019, 2008.
Nicolaou, K.C., Li, A. Total syntheses and structural revisions of α- and β-diversonolic esters and total syntheses of diversonol and blennolide C. Angew. Chem. Int.
Ed. 47:6579, 2008.
Nicolaou, K.C., Lister, T., Denton, R.M., Gelin, C.F. Total synthesis of artochamins F,
H, I, and J through cascade reactions. Tetrahedron 64:4736, 2008.
Nicolaou, K.C., Ortiz, A., Denton, R.M. Metathesis reactions in the synthesis of
complex molecules. Chem. Today 25:70, 2007.
Nicolaou, K.C., Pappo, D., Tsang, K.Y., Gibe, R., Chen, D.Y.-K. A chiral pool
based synthesis of platensimycin. Angew. Chem. Int. Ed. 47:944, 2008.
Nicolaou, K.C., Stepan, A.F., Lister, T., Montero, A., Tria, G.S., Turner, C.I., Tang,
Y., Wang, J., Denton, R.M., Edmonds, D.J. Design, synthesis, and biological evaluation of platensimycin analogues with varying degrees of molecular complexity. J.
Am. Chem. Soc. 130:13110, 2008.
Nicolaou, K.C., Sun, Y.-P., Guduru, R., Banerji, B., Chen, D.Y.-K. Total synthesis
of the originally proposed and revised structures of palmerolide A and isomers
thereof. J. Am. Chem. Soc. 130:3633, 2008.
F i g . 1 . Selected target molecules.
a metabolite of a powerful enediyne antitumor antibiotic.
Strides have also been made toward the total synthesis of nocathiacin III and lomaiviticin B, 2 potent antitumor antibiotics.
Overall, our research programs continue to sharpen
the tools of chemical synthesis and provide biologically
active molecules, some natural and some designed, for
chemical biology studies. By advancing the art of chemical synthesis, and through the preparation of biological
tools and potential drug candidates, we strengthen the
foundation of the drug discovery and development process.
PUBLICATIONS
Nicolaou, K.C., Chen, J.S., Edmonds, D.J., Estrada, A.A. Recent advances in the
total synthesis and biology of naturally occurring antibiotics. Angew. Chem. Int.
Ed., in press.
Nicolaou, K.C., Chen, J.S., Zhang, H., Montero, A. Asymmetric synthesis and biological properties of uncialamycin and 26-epi-unicialamycin. Angew. Chem. Int.
Ed. 47:185, 2008.
Nicolaou, K.C., Cole, K.P., Frederick, M.O., Aversa, R.J., Denton, R.M. Chemical
synthesis of the GHIJK ring system and further experimental support for the originally assigned structure of maitotoxin. Angew. Chem. Int. Ed. 46:8875, 2007.
Nicolaou, K.C., Dethe, D.H., Chen, D.Y.-K. Total syntheses of amythiamicins A, B,
and C. Chem. Commun. (Camb.) Issue 23:2632, 2008.
Nicolaou, K.C., Sun, Y.-P., Peng, X.-S., Polet, D., Chen, D.Y.-K. Total synthesis of
(+)-cortistatin. Angew. Chem. Int. Ed. 47:7310, 2008.
Nicolaou, K.C., Toh, Q.-Y., Chen, D.Y.-K. An expedient asymmetric synthesis of
platencin. J. Am. Chem. Soc. 130:11292, 2008.
Nicolaou, K.C., Tria, G.S., Edmonds, D.J. Total synthesis of platencin. Angew.
Chem. Int. Ed. 47:1780, 2008.
Nicolaou, K.C., Wang, J., Tang, Y. Synthesis of the sporolide ring framework through
a cascade sequence involving an intramolecular [4+2] cycloaddition reaction of an
o-quinone. Angew. Chem. Int. Ed. 47:1432, 2008.
Nicolaou, K.C., Wu, T.R., Sarlan, D., Shaw, D.M., Rowcliffe, E., Burton, D.R.
Total synthesis, revised structure, and biological evaluation of biyouyanagin A and
analogues thereof. J. Am. Chem. Soc. 130:11114, 2008.
Vale, C., Gómez-Limia, B., Nicolaou, K.C., Frederick, M.O., Vieytes, M.R.,
Botana, L.M. The c-Jun-N-terminal kinase is involved in the neurotoxic effect of
azaspiracid-1. Cell. Physiol. Biochem. 20:957, 2007.
Vale, C., Wandscheer, C., Nicolaou, K.C., Frederick, M.O., Alfonso, C., Vieytes,
M.R., Botana, L.M. Cytotoxic effect of azaspiracid-2 and azaspiracid-2-methyl
ester in cultured neurons: involvement of the c-Jun N-terminal kinase. J. Neurosci.
Res. 86:2952, 2008.
Vilariño, N., Nicolaou, K.C., Frederick, M.O., Cagide, E., Alfonso, C., Alonso, E.,
Vieytes, M.R., Botana, L.M. Azaspiracid substituent at C1 is relevant to in vitro
toxicity. Chem. Res. Toxicol. 21:1823, 2008.
40
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Filling Space at the
Molecular Level
J. Rebek, Jr., D. Ajami, M. Ams, E. Barrett, S. Beer,
T.J. Dale, R.J. Hooley, J.-L. Hou, H. Van Anda, S. Xiao,
spaces. They have revealed phenomena never before
observed, such as coiled alkanes, stabilization of reactive
intermediates, places where new forms of stereochemistry can emerge, and reaction chambers with welldefined shapes. We found that some capsules, such
as shown in Figure 2, can incorporate spacer elements
A. Lledo, H. Dube, P. Restorp, S. Kamioka
MOLECULAR MIMICRY
rotein-protein interactions
are involved in many cell
signaling events, and a
large fraction of protein surfaces
involve α-helices. This secondary structure presents side
chains of the component amino
acids along one face of the
Julius Rebek, Jr., Ph.D.
helix that are recognized by the Professor and Director
The Skaggs Institute
partner protein. To interfere
for Chemical Biology
with these protein-protein
interactions, several research groups have made α-helix
mimetics. Our efforts have gone into those that have
amphiphilic behavior, that is, ones that are hydrophobic on one side and hydrophilic on the other. We have
found rapid and efficient ways of assembling these mimics by using a pyridizine-based scaffold. The mimics
have good solubility, and their synthesis can be easily
scaled up. The pyridizines and the scheme for their
assembly are shown in Figure 1.
P
F i g . 2 . Size and shape of the space inside a capsule show the
tapered ends and the constricted center. Center, Encapsulated 1-hexadecene inside the assembly. Right, Encapsulated 1-hexadecyne. The
thin alkene and alkyne groups penetrate deep into the bottom of the
capsule. The central parts of the guests are in fully extended conformations, and compression of the alkane appears near the top.
known as glycolurils in response to the presence of
guests. The expanded capsules shown are present only
when a suitable guest is able to fill the space inside.
The shape of the space inside is shown in the figure,
and narrow functional groups such as primary alkenes
and acetylenes can fit in the tapered ends of the space.
C O E N C A P S U L AT I O N
With 2 different guests inside a capsule, the contact points between the guests can be mapped out
by using nuclear magnetic resonance techniques. For
example, with ethane and heptane coencapsulated, as
shown in Figure 3, we found that the 2 ends of heptane are alternately in contact with the ethane. This
contact is achieved by the flipping of the heptane
F i g . 1 . A, Overlay of an idealized α-helix protein structure with a
new scaffold. B, Synthetic scheme shows the component parts that
present the amino acid side chains on the scaffold and the atoms
that make up the hydrophilic surface.
EXPANDED CAPSULES
Reversible encapsulation complexes are synthetic
receptors that more or less completely surround their
target guests. They provide a window through which
molecular behavior can be seen in extremely small
F i g . 3 . Ethane (gray) and heptane (rainbow colored) are coen-
capsulated. Tumbling motions of the heptane can be detected by
nuclear magnetic resonance techniques that show contact between
the 2 ends of the longer guest with ethane.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
inside the capsule, rather than by the exchange of places
of the 2 guests inside.
C AV I TA N D S W I T H I N T R O V E R T E D F U N C T I O N A L I T Y
Cavitands are open-ended molecular vessels that
allow relatively rapid motions of guests inside and out.
Figure 4 shows a system held together by hydrogen
41
Role of Mistranslation
in Disease
P. Schimmel, X.-L. Yang, R. Belani, Y. Chong, Z. Druzina,
J. Frater, M. Guo, R.-T. Guo, M. Hanan, W. He, I.L. Jung,
M. Kapoor, S.H. Lee, J. Liu, E. Merriman, M.H. Nawaz,
R. Shapiro, Y.Z. Song, M.-N. Vo, W. Zhang, Q. Zhou
istranslation occurs
when the wrong amino
acid is inserted into
a growing polypeptide chain
during protein synthesis. Most
usually, proteins are error-free;
that is, each specific protein
has its own, specific amino acid
sequence that is defined by the
gene that encodes the protein. Paul R. Schimmel, Ph.D.
Professor
(The process of protein synthesis Molecular Biology
“translates” the sequence of a
gene into the corresponding protein sequence.) When
an error is made, so that the wrong amino acid occasionally appears at a specific location in the sequence
of a protein, this aberration can lead to a protein with
altered biological activity. Additionally or alternatively,
the error-containing protein may misfold. Recently, we
showed that mistranslation that causes altered protein
structure and function is connected to disease.
Normally, errors of translation are prevented by
the editing activities of tRNA synthetases. These synthetases attach amino acids to tRNAs, matching each
amino acid to its cognate tRNA partner. The attached
amino acid is then carried to the ribosome, where it is
inserted into a growing polypeptide chain at the position
specified by the anticodon of the tRNA. If the wrong
amino acid is accidentally attached to a particular
tRNA and is not corrected by the editing activity of the
appropriate tRNA synthetase, then that amino acid is
carried in the same way to the ribosome and inserted
into a growing polypeptide. This insertion is at the place
normally occupied by the correct amino acid.
Mutations in the editing centers of tRNA synthetases
can thus lead to mistranslation. Recently, we showed
that mistranslation arising from such mutations is deleterious to bacterial and mammalian cells. In addition,
we found that a mild editing defect in a specific tRNA
synthetase (alanyl-tRNA synthetase) led to neurodegeneration in mice.
M
F i g . 4 . A deep vaselike structure presents a cavity, and the carboxylic acid (red) is directed into the space. Isonitriles are captured
in the cavity and react with the acid to give stabilized intermediates, such as A.
bonding that features a seam of hydrogen bonds that
maintain the vaselike shape. The cavitand is attached
to an anthracene that delivers a carboxylic acid to the
inside space. We have used this system to trap reactive intermediates such as those involved in reactions
of isonitriles. The intermediates have only microsecond lifetimes in solution but are stabilized inside the
cavitand for up to 15 minutes, long enough to characterize them by using nuclear magnetic resonance and
infrared spectroscopic techniques.
PUBLICATIONS
Ajami, D., Rebek, J., Jr. Gas behavior in self-assembled capsules. Angew. Chem.
Int. Ed. 47:6059, 2008.
Ajami, D., Rebek, J., Jr. Longer guests drive the reversible assembly of hyperextended capsules. Angew. Chem. Int. Ed. 46:9283, 2007.
Ajami, D., Rebek, J., Jr. Reversible encapsulation of terminal alkenes and alkynes.
Heterocycles 76:169, 2008.
Ajami, D., Schramm, M.P., Rebek, J., Jr. Translational motion inside self-assembled encapsulation complexes. Tetrahedron, in press.
Mann, E., Moisan, L., Hou, J.-L., Rebek, J., Jr. Synthesis of pyridazines functionalized with amino acid side chains. Tetrahedron Lett. 49:903, 2008.
Moisan, L., Odermatt, S., Gombosuren, N., Carella, A., Rebek, J., Jr. Synthesis of
an oxazole-pyrrole-piperazine scaffold as an α-helix mimetic. Eur. J. Org. Chem.
10:1673, 2008.
Restorp, P., Rebek, J., Jr. Reaction of isonitriles with carboxylic acids in a cavitand:
observation of elusive isoimide intermediates. J. Am. Chem. Soc. 130:11850, 2008.
Restorp, P., Rebek, J., Jr. Synthesis of α-helix mimetics with four side-chains.
Bioorg. Med. Chem. Lett. 18:5909, 2008.
42
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Currently, we are focusing on the possibility that
editing defects are causally connected to the etiology
of some cancers. Because cancer is mostly a disease
of aging, the random occurrence of mutations in the
editing domains of tRNA synthetases in somatic cells
can lead to mistranslation in those tissues. In other
studies, we established that in aging bacteria, an editing-defective tRNA synthetase (the defect itself caused
by a mutation in its editing domain) can lead to mutations in the error-prone DNA repair apparatus. When
error-prone repair is perturbed, random errors spontaneously occur more frequently in the genome. We envision that a similar situation happens in mammalian
cells; that is, an editing-defective tRNA synthetase can
itself induce more mutational errors in the genome
as the organism ages. Some of these mutations might
occur in oncogenes that when activated lead to transformation to the oncogenic state.
With this possibility in mind, we established a collaboration with P. Vogt, Scripps Research, to see whether,
in a model system developed in Dr. Vogt’s laboratory,
oncogenesis can be induced by an editing-defective
tRNA synthetase. At the same time, we are investigating activities of enzymes associated with DNA repair
to see if in mammalian cells carrying an editing defect,
one or more of these enzymes is affected by mistranslation. A perturbation of one of the enzymes associated
with DNA repair could lead to the fixing of random
mutations into the genome. Some of these mutations
might occur in oncogenes.
PUBLICATIONS
Beebe, K., Mock, M., Merriman, E., Schimmel, P. Distinct domains of tRNA synthetase recognize the same base pair. Nature 451:90, 2008.
Cheng, G., Zhang, H., Yang, X.-L., Tzima, E., Ewalt, K.L., Schimmel, P., Faber,
J.E. Effect of mini-tyrosyl-tRNA synthetase on ischemic angiogenesis, leukocyte
recruitment, and vascular permeability. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295:R1138, 2008.
Chong, Y.-E., Yang, X.-L., Schimmel, P. Natural homology of tRNA synthetase
editing domain rescues conditional lethality caused by mistranslation. J. Biol.
Chem. 283:30073, 2008.
Greenberg, Y., King, M., Kiosses, W.B., Ewalt, K., Yang, X.-L., Schimmel, P., Reader,
J. S., Tzima, E. The novel fragment of tyrosyl-tRNA synthetase, mini-TyrRS, is secreted
to induce an angiogenic response in endothelial cells. FASEB J. 22:1597, 2008.
Guo, M., Ignatov, M., Musier-Forsyth, K., Schimmel, P., Yang, X.-L. Crystal structure of novel tetrameric form of human lysyl-tRNA synthetase: implications for multisynthetase complex formation. Proc. Natl. Acad. Sci. U. S. A. 105:2331, 2008.
Kapoor, M., Zhou, Q., Otero, F., Myers, C.A., Bates, A., Belani, R., Liu, J., Luo,
J.-K., Tzima, E., Zhang, D.-E., Yang, X.-L., Schimmel, P. Evidence for annexin IIS100A10 complex and plasmin in mobilization of cytokine activity of human
TrpRS. J. Biol. Chem. 283:2070. 2008.
Park, S.G., Schimmel, P., Kim, S. Aminoacyl tRNA synthetases and their connections to disease. Proc. Natl. Acad. Sci. U. S. A. 105:11043. 2008.
Schimmel, P. Development of tRNA synthetases and connection to genetic code
and disease. Protein Sci. 17:1643, 2008.
Schimmel, P. An editing activity that prevents mistranslation and connection to disease. J. Biol. Chem. 283:28777, 2008.
Zhou, Q., Kiosses, W.B., Liu, J., Schimmel, P. Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine.
Methods 44:190, 2008.
New Amino Acid
Building Blocks
P.G. Schultz, E.M. Brustad, J. Grünewald, J. Guo, H.S. Lee,
C.C. Liu, F.B. Peters, T.S. Young
lmost all processes of
living cells, from gene
regulation and information processing to photosynthesis, are carried out by proteins.
These are large molecules synthesized from 20 amino acid
building blocks. This set of 20 Peter Schultz, Ph.D.
amino acids is the basis for the Professor
Chemistry
genetic code, the code that specifies the relationship between the nucleotide sequence
of a gene and the amino acid sequence of a protein.
This fact leads to rather interesting questions: Why does
every form of life have the same set of building blocks?
Why not 21 or more? Moreover, if we can add new
amino acid building blocks to the genetic code, will we
be able to create proteins or even cells with enhanced
chemical, physical, or biological properties?
We continue to address this issue by using chemical and molecular biological methods to add new components to the protein biosynthetic machinery of bacteria.
Using this approach, we effectively expanded the genetic
code of both prokaryotes and eukaryotes by genetically
encoding new amino acids (including photoaffinity labels,
chemically reactive amino acids, posttranslationally modified amino acids, and amino acids with altered electronic and steric properties) in response to unique 3and 4-base codons.
Currently, we are exploring additional amino acids
with novel biological and physicochemical properties,
other organisms, and a number of biomedical applications of this technology. For example, in the past year,
we showed that tolerance against the self-antigen TNF-α
in mice can be broken by the selective introduction of
p-nitrophenylalanine. This research promises to greatly
A
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
facilitate the generation of cancer vaccines and vaccines against third-world pathogens; we are currently
extending it to lung and breast cancer vaccines and
vaccines against tuberculosis, malaria. and HIV disease. We also genetically encoded a boronate-containing amino acid for the scarless purification of proteins,
selective protein modification, and the development
of selective antibodies to both glycoproteins and serine
proteases involved in viral infections and cancer. In
addition, we genetically encoded a hydroxyquinoline
metal ion–binding amino acid for heavy-atom phasing
in protein structure determination and for the introduction of radioisotopes into antibodies for cancer therapy
and imaging.
In other studies, we used a phenylselenide-containing amino acid to generate proteins containing posttranslationally modified lysine residues to study the
role of histone modification in the epigenetics of cancer
and developmental biology. We also showed that phage
display can be used to select anti-gp120 sulfotyrosinecontaining antibodies with enhanced affinities relative
to the naturally sulfated anti-gp120 antibody. This
result shows for the first time that an expanded genetic
code can confer an evolutionary advantage and may
lead to the generation of therapeutic proteins or peptides with improved biological or pharmacologic properties due to the presence of unnatural amino acids.
We also genetically encoded the fluorophore prodan
in mammalian cells to facilitate cellular studies of protein structure, function, and localization; genetically
encoded an unnatural amino acid that can cleave the
polypeptide backbone in a light-dependent fashion as a
cell biological tool; and biosynthesized DNA-binding
proteins that can oxidatively cleave the DNA backbone
in a sequence-specific fashion as a probe of proteinDNA recognition. Furthermore, we showed that unnatural amino acids can be used to selectively introduce
fluorescence resonance energy transfer pairs into proteins for single-molecule imaging studies, and we developed a high-yield Pichia expression system to produce
therapeutic proteins containing unnatural amino acids.
PUBLICATIONS
Brustad, E., Bushey, M.L., Brock, A., Chittuluru, J., Schultz, P.G. A promiscuous
aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine
homologs into proteins. Bioorg. Med. Chem. Lett. 18:6004, 2008.
Brustad, E., Bushey, M.L., Lee, J.W., Groff, D., Liu, W., Schultz, P.G. A genetically
encoded boronate-containing amino acid. Angew. Chem. Int. Ed. 47:8220, 2008.
Grünewald, J., Tsao, M.-L., Perera, R., Dong, L., Niessen, F., Wen, B.G., Kubitz,
D.M., Smider, V.V., Ruf, W., Nasoff, M., Lerner, R.A., Schultz, P.G. Immunochemical
termination of self-tolerance. Proc. Natl. Acad. Sci. U. S. A. 105:11276, 2008.
43
Guo, J., Wang, J., Anderson, J.C., Schultz, P.G. Addition of an α-hydroxy acid to
the genetic code of bacteria. Angew. Chem. Int. Ed. 47:722, 2008.
Guo, J., Wang, J., Lee, J.-S., Schultz, P.G. Site-specific incorporation of methyland acetyl-lysine analogues into recombinant proteins. Angew. Chem. Int. Ed.
47:6399, 2008.
Lee, J., Hong, J., Nam, T.-G., Peters, E.C., Orth, A.P., Geierstanger, B.H., Goldfinger, L.E., Ginsberg, M.H., Cho, C.Y., Schultz, P.G. A small molecule inhibitor of α4
integrin-dependent cell migration. Bioorg. Med. Chem., in press.
Lemke, E.A., Summerer, D., Geierstanger, B.H., Brittain, S.M., Schultz, P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid.
Nat. Chem. Biol. 3:769, 2007.
Tippmann, E.M., Liu, W., Summerer, D., Mack, A.V., Schultz, P.G. A genetically
encoded diazirine photocrosslinker in Escherichia coli. ChemBioChem 8:2210, 2007.
Click Chemistry and
Biological Activity
K.B. Sharpless, V.V. Fokin, J. Culhane, J. Fotsing, S. Grecian,
N. Grimster, J. Hein, T. Horneff, J. Kalisiak, K. Korthals,
S.-W. Kwok, S. Pitram, J. Raushel, B. Stump, J. Tripp,
C. Valdez, T. Weide
e aim to discover new
chemical processes
that allow rapid and
efficient synthesis of molecules
with a desired function from
diverse building blocks. Since
1996, the support of the Skaggs
Institute for Chemical Biology
has been instrumental to the
K. Barry Sharpless, Ph.D.
Professor
development of the concept
Chemistry
and the applications of click
chemistry. Click chemistry embodies an attitude that
function matters most and that tools that enable researchers
to achieve function are to be prized. Thus, click chemistry relies on the use of a few near-perfect chemical
reactions for the synthesis and assembly of specially
designed building blocks. These building blocks have
a high built-in energy content that drives a spontaneous, selective, and irreversible linking reaction with
complementary sites in the reactive partners.
The power of click chemistry lies in the ability to
rapidly generate novel structures that may not necessarily resemble known biologically active compounds.
For discovering compounds that may be useful as drugs,
this strategy provides a means for the rapid exploration
of the chemical space. For optimization of compounds,
the method enables rapid structure-activity profiling
W
44
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
through generation of analog libraries. Click chemistry
does not replace existing methods for drug discovery;
rather it complements and extends them. It works well
in conjunction with structure-based design and combinatorial chemistry techniques.
Click chemistry is both enabled and constrained by
its reliance on a few nearly perfect reactions, and this
characteristic raises concerns about limitations on the
access of click chemistry to chemical diversity. However, the pool of druglike compounds may be as large
as 1063. Currently, only a few million compounds that
fulfill these criteria are known, implying that only an
infinitesimal part of the potential medicinal chemistry
universe has been explored so far.
These facts have staggering implications for drug
discovery. First and foremost, most molecules with useful properties remain to be discovered. Second, the
majority of useful new compounds likely will be found
in unconventional structure space. Thus, with click
chemistry, we have the interesting proposition that
greater diversity can be achieved with fewer reactions,
because it is not the number of reactions that is important, but the reach of the reactions, which is determined
by the tolerance to variations in the nature of their
components. Click chemistry approaches have already
proved themselves in biomedical research, ranging from
synthetic chemistry to bioconjugation strategies, polymer chemistry, and materials science.
R A P I D M O D I F I C AT I O N O F A N T I B I O T I C S T O
O V E R C O M E R E S I S TA N C E
Although macrolides, including erythromycin A have
been widely prescribed for more than 50 years, the
emergence of widespread bacterial resistance to these
molecules is a serious and expanding problem. Thirdgeneration macrolides, such as telithromycin, have been
developed in recent years as effective means to overcome
resistant bacterial strains. However, despite these efforts,
only a few macrolide candidates with activity against
methicillin-resistant Staphylococcus aureus (MRSA)
have been identified to date. Clearly, the medical need
for new antibiotics to combat strains of MRSA is urgent.
In our collaboration with scientists at the Kitasato
Institute, Tokyo, Japan, we have reexamined the activity
of various derivatives of erythromycin A against 12 types
of gram-positive bacteria, including macrolide-resistant
strains, and 1 gram-negative organism. We found that
11,12-di-O-iso-butyryl-8,9-anhydroerythromycin A 6,9hemiketal has moderate activity against 4 strains of
MRSA and 2 strains of vancomycin-resistant entero-
cocci (VRE). Further modification of an alkynylated derivative of this lead compound by using the copper-catalyzed azide-alkyne cycloaddition, the flagship click
reaction, quickly led to identification of several triazole-containing erythromycin A analogs with improved
activity against MRSA and VRE strains (Fig. 1). These
promising antibacterials are currently undergoing further evaluation.
F i g . 1 . Novel erythromycin A derivatives with activity against strains
of MRSA and VRE. MIC = minimum inhibitory concentration.
IN SITU CLICK CHEMISTRY
Although click chemistry allows rapid assembly of
diverse collections of molecules that may serve as lead
structures, further evolution of the molecules is traditionally achieved by iterative cycles of screening for
biological activity and synthetic modification. Can direct
involvement of the target, usually a specific receptor
or enzyme, in the selection and evolution of possible
drug candidates accelerate this drug discovery cycle?
The aim of using in situ click chemistry is to engage
an enzyme in the selection and covalent assembly of
its own “best-fitting” inhibitor. Although the concept has
been previously tested by several researchers, the in situ
click chemistry approach is unique because it relies on
the completely bioorthogonal 1,3-dipolar cycloaddition
of organic azides and alkynes. This highly exergonic reaction produces 5-membered nitrogen heterocycles, 1,2,3triazoles, which are exceedingly stable to acidic and
basic hydrolysis and to severe reduction-oxidation conditions. At the same time, the triazoles produced can
actively participate in hydrogen-bonding, dipole-dipole,
and π-stacking interactions.
The efficacy of in situ click chemistry has been
demonstrated by the discovery of novel, highly potent
inhibitors of acetylcholinesterase, carbonic anhydrase,
and HIV protease. During the past year, we extended
the approach to more challenging targets. Among these
are the enzyme β-secretase, which is involved in the
progression of Alzheimer’s disease; several members of
the vast kinase family; metalloproteases; and nicotinic
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
acetylcholine receptors, the family of ligand-gated ion
channels responsible for key neurotransmission events.
PUBLICATIONS
Finn, M.G., Kolb, H.C., Fokin, V.V., Sharpless, K.B. Concept and applications of
click chemistry from the standpoint of advocates. Kagaku to Kogyo 60:976, 2007.
Hawker, C.J., Fokin, V.V., Finn, M.G., Sharpless, K.B. Bringing efficiency to materials synthesis: the philosophy of click chemistry. Aust. J. Chem. 60:381, 2007.
Kalisiak, J., Sharpless, K.B., Fokin, V.V. Efficient synthesis of 2-substituted-1,2,3triazoles. Org. Lett. 10:3171, 2008.
Kwok, S.-W., Hein, J.E., Fokin, V.V., Sharpless, K.B. Regioselective synthesis of
either 1H- or 2H-1,2,3-triazoles via Michael addition to α,β-unsaturated ketones.
Heterocycles 76:1141, 2008.
Liu, Y., Díaz, D.D., Accurso, A.A., Sharpless, K.B., Fokin, V.V., Finn, M.G. Click
chemistry in materials synthesis, III: metal-adhesive polymers from Cu(I)-catalyzed
azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 45:5182, 2007.
Radić, Z., Manetsch, R., Fournier, D., Sharpless, K.B., Taylor, P. Probing gorge
dimensions of cholinesterases by freeze-frame click chemistry. Chem. Biol. Interact.
175:161, 2008.
Sugawara, A., Sunazuka, T., Hirose, T., Nagai, K., Yamaguchi, Y., Hanaki, H.,
Sharpless, K.B., Omura, S. Design and synthesis via click chemistry of 8,9-anhydroerythromycin A 6,9-hemiketal analogues with anti-MRSA and -VRE activity.
Bioorg. Med. Chem. Lett. 17:6340, 2007.
Van der Eycken, E., Sharpless, K.B. Click chemistry. QSAR Comb. Sci. 26:1115,
2007.
Vestberg, R., Malkoch, M., Kade, M., Wu, P., Fokin, V.V., Sharpless, K.B., Drockenmuller, E., Hawker, C.J. Role of architecture and molecular weight in the formation of tailor-made ultrathin multilayers using dendritic macromolecules and click
chemistry. J. Polym. Sci. A Polym. Chem. 45:2835, 2007.
Yoo, E.J., Ahlquist, M., Bae, I., Sharpless, K.B., Fokin, V.V., Chang, S. Mechanistic studies on the Cu-catalyzed three-component reactions of sulfonyl azides, 1alkynes and amines, alcohols, or water: dichotomy via a common pathway J. Org.
Chem. 73:5520, 2008.
Molecular Biology of Olfaction
L. Stowers, P. Chamero, K. Flanagan, D. Logan, T. Martin,
F. Papes, A. Kaur
ppropriate behavior relative to the surroundings
is necessary for survival;
however, the neural mechanisms
that detect important cues in
the environment, process the
meaning of the cues, and initiate the corresponding behavior
are largely unknown. The neural Lisa S. Stowers, Ph.D.
Assistant Professor
code of behavior is difficult
Cell Biology
to study because most future
behavior is determined by previous experience, which
often differs subtly among individuals. These differences
in experience lead to variable perceptions and unpre-
A
45
dictable behavior outcomes across individuals placed
in the same environment. The underlying neural circuits
are therefore correspondingly variable and dynamic. To
ensure experimental clarity and reproducibility, we study
the neural circuits that underlie innate behavior.
Olfactory stimuli are known to elicit innate behaviors in rodents. For example, when a male encounters
another individual in his environment, he detects and
processes the emitted chemical cues to determine the
age and sex of the intruder. If the cues signal that the
intruder is a juvenile, the male will respond appropriately and not alter his behavior. If the intruder is a
female, he will court her, and if he detects a male, he
will respond with aggression. Innate behaviors are strong
and universal, suggesting they are driven by genetically
programmed, invariant neural circuits.
We are investigating innate mouse behaviors that
are stereotyped and quantifiable. Just as a biochemical
assay is used to map and elucidate a metabolic pathway, we use innate behavior as a functional assay to
identify the corresponding ligand cues and mediating
neurons. Although this approach requires an unconventional research plan, it can be used to sort out and identify the unique biological tools, ligands, and receptors
essential for studies at the cellular and molecular levels, the neural circuit that detects the environment and
generates appropriate behavior.
We have been isolating the chemical ligands, pheromones, that specifically govern social behaviors such as
aggression and mating. These ligands are detected in
rodents by neurons and mechanisms that are active in
all terrestrial vertebrates but are not functional in humans.
To expand our investigation of innate neural circuits, we
are also elucidating the ligands and responsive sensory
mechanisms that promote maternal-infant behavior.
This behavior is the defining behavior of mammals,
including humans. We expect that identification of the
neuronal code that generates this innate behavior in
mice will enable us to directly investigate mechanisms
of innate behavior in humans.
We have isolated the source of pheromones that
promote maternal-infant behavior, a step allows us
now to specifically activate, and thereby identify, the
population of sensory neurons dedicated to promoting
this behavior. Our findings suggest that this fundamental
social behavior is governed by a unique olfactory mechanism, unlike other pheromone-mediated behaviors, that
may have orthologous counterparts that are present and
functional in humans. To further investigate these mech-
46
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
anisms, we are manipulating activation of the sensory
circuit by activating and inactivating ligand receptors,
signal transduction elements, and ion channels to identify
the neurons we find essential for behavior. We are also
fractionating the natural source of pheromones to purify
the ligands. Once purified, these molecules will allow
us to investigate the kinetics of their response and
manipulate their properties to validate their role in promoting behavior. We expect that our studies will provide the tools to expand our understanding of the logic
of neuronal coding of innate behaviors in mice and, additionally, investigate the molecular mechanisms that
underlie human social behavior.
PUBLICATIONS
Logan, D.W., Marton, T.F., Stowers, L. Species specificity in major urinary proteins
by parallel evolution. PLoS ONE 3:e3280, 2008.
Macromolecular Master Keys for
Genome integrity, Reactive
Oxygen Control, and
Pathogenesis
J.A. Tainer, A.S. Arvai, B.R. Chapados, L. Fan, C. Hitomi,
K. Hitomi, J.J. Perry, M.E. Pique, D.S. Shin, J.L. Tubbs,
R.S. Williams
o date, funds from the
Skaggs Institute for Chemical Biology has supported
the training of 15 graduate
students and 33 postdoctoral
fellows and contributions to
176 publications. In particular, Skaggs funding is used for
medically relevant structureJohn A. Tainer, Ph.D.
function investigations of macro- Professor
Molecular Biology
molecular master keys for genome
integrity, reactive oxygen control, and pathogenesis.
Skaggs funding also supports our synchrotron beam line,
at the Advanced Light Source, University of California,
Berkeley/Lawrence Berkeley National Laboratory. The
synchrotron is used to characterize macromolecular complexes, conformations, and interactions in solution via
small-angle x-ray scattering (SAXS) and at high resolution via macromolecular x-ray crystallography. We are
furthermore developing and using new data interpreta-
T
tion tools for the detailed visualizations of protein complexes and modified proteins that undergo functionally
important changes in shape and assembly. Working
with P. Kuhn, Scripps Research, for example, we used
our SAXS technologies to characterize solution structures that explain guanine nucleotide exchange mediated by the T-cell essential Vav1. We are working to
develop SAXS for drug discovery so that the technology can be used to identify small molecules that bind
and inhibit enzymes.
We are continuing research on pathogenic bacteria, including drug and vaccine design for the type IV
pilin system. We have new structures for the fiberforming type IV pilin protein, the assembly ATPase,
and associated machinery. Type IV pilin proteins are
critical bacterial virulence factors for cholera, pneumonia, gonorrhea, meningitis, and severe diarrhea. Our
combined SAXS and macromolecular x-ray crystallography methods are providing a mechanistic understanding relevant to controlling these virulence factors.
In collaboration with M.N. Boddy, Scripps Research,
we have identified the SUMO-targeted ubiquitin ligase
family of proteins. These proteins provide communication between the sumoylation and ubiquitination pathways and act as master regulators of DNA damage
responses. Thus, the proteins are of obvious value for
the development of novel cancer drugs. We also defined
new structure-function relationships for the multicomponent Smc5-Smc6 complex in genome stability.
Our work on macromolecules with key roles in pathways controlling reactive oxygen species and DNA damage responses is important for preserving the nervous
system and controlling cancer. In our structure-based
design projects, we collaborate with E.D. Getzoff, Skaggs
Institute, to characterize inhibitors of nitric oxide synthase. In this collaborative work, we have discovered
a new general method for designing inhibitors to specific isozymes of nitric oxide synthase that promise to
reduce unwanted side effects and allow targeted interventions for arthritis, stroke, and cancer. Defining master
keys to regulate reactive oxygen species such as superoxide and nitric oxide help provide an informed basis to
avoid the oxidative death of neurons in neurodegenerative diseases and to use reactive oxygen species to kill
cells involved in cancer and pathogenesis.
In other cancer related research, we characterized
O6-alkylguanine-DNA alkyltransferase activity on x-linked
DNA. This alkyltransferase is a target both for the prevention of cancer and for chemotherapy, because it
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
repairs mutagenic lesions in DNA and limits the effectiveness of alkylating chemotherapies. We also used
high-resolution crystal structures and mutational analyses to explain how endonuclease IV uses 3 metal ions
to remove a damaged DNA site and then hold the product to avoid the release of toxic repair intermediates. We
similarly characterized the role of active-site metal ions
for flap endonuclease-1, which removes DNA flaps generated during both replication and repair.
In recent investigations on the nucleotide excision
repair system, which repairs bulky lesions in DNA, we
have focused on the helicase XPD. Our results define
XPD helicase structures and activities to provide new
insights into the cancer and aging phenotypes of XPD
mutations (Fig. 1). Our findings also help explain the
severe developmental problems associated with Cockayne syndrome, which includes defects in both DNA
repair and transcription. Moreover, the effects of hijacking transcription factors and repair shielding associated
with some XPD mutants may be the basis for the success of cisplatin as an anticancer agent. These results
may therefore provide a mechanistic basis for the design
if novel cancer agents that extend the therapeutic usefulness of cisplatin to other tumor types.
To understand the Mre11-Rad50-Nbs1 (MRN) complex that initiates repair of DNA double-strand breaks
and homologous recombination, we are collaborating
with P. Russell, Scripps Research. Our results help show
how MRN mutations cause the Nijmegen breakage syndrome and ataxia telangiectasia–like disorder, diseases
in humans that predispose individuals to cancer. Our
new Mre11-DNA structures and mutants reveal key
Mre11 roles in DNA end synapsis and nuclease processing (Fig. 2). We also identified Rad50 mutants that form
meiotic DNA double-strand breaks and revealed an
essential structural role for Rad50 in axial element and
synaptonemal complex formation. In related research on
homologous recombination, we used our new SAXS
methods to define the domain structure interactions for
the homologous recombination protein BARD1. We are
now investigating the structural basis for homologous
recombination; our results may have implications for
improving cancer interventions by reprogramming cells
for death vs repair in response to double-strand breaks.
PUBLICATIONS
Acharya, S.N., Many, A.M., Schroeder, A.P., Kennedy, F.M., Savytskyy, O.P.,
Grubb, J.T., Vincent, J.A., Friedle, E.A., Celerin, M., Maillet, D.S., Palmerini,
H.J., Greischar, M.A., Moncalian, G., Williams, R.S., Tainer, J.A., Zolan, M.E.
Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in
axial element and synaptonemal complex formation, homolog pairing and meiotic
recombination. Genetics 180:1889, 2008.
47
F i g . 1 . Structural placement of disease-causing mutations in XPD
helicase. Mapping the 3 classes of mutations onto the SaXPD structure reveals patterns associated with each disease defect. A, Stereopair mapping the distribution of disease-causing mutations on a XPD
Cα trace. Disease-causing mutation sites (Cα colored sphere): red (XP),
greenish yellow (XP/CS), and purple (TTD). Residue F136 is also
shown (cyan). B, XPDcc fold and domain architecture (ribbons)
with labeled disease-causing mutation sites as spheres colored as in
A. C, XP mutations affect DNA- and ATP-binding regions. D, XP/CS
mutations affect HD1-HD2 conformational changes. E, TTD mutations affect the overall framework stability. Reprinted from Fan, L.,
Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel, M., Roberts, V.A.,
Cooper, P.K., Tainer, J.A. XPD helicase structures and activities:
insights into the cancer and aging phenotypes from XPD mutations.
Cell 133:789, 2008, copyright 2008, with permission from Elsevier.
Chrencik, J.E., Brooun, A., Zhang, H., Mathews, I.I., Hura, G.L., Foster, S.,
Perry, J.J., Streiff, M., Ramage, P., Widmer, H., Bokoch, G.M., Tainer, J.A.,
Weckbecker, G., Kuhn, P. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1. J. Mol. Biol. 380:828, 2008.
Edwards, R.A., Lee, M.S., Tsutakawa, S.E., Williams, R.S., Tainer, JA., Glover,
J.N.M. The BARD1 C-terminal domain structure and interactions with polyadenylation factor, CstF-50. Biochemistry 47:11446, 2008.
Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel, M., Roberts, V.A., Cooper,
P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer
and aging phenotypes from XPD mutations. Cell 133:789, 2008.
48
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Chemical Approaches
to Disease
P. Wentworth, Jr., R.K. Grover, B.D. Song, M.M.R. Peram,
J. Rogel, R.P. Troseth, D. Angrish, V. Dubrovskaya,
A.D. Wentworth
ur interdisciplinary
research focus involves
aspects of bioorganic,
biophysical, physical organic,
synthetic, and analytical chemistry coupled with biochemical
techniques, cell-based assays,
and animal models. Ongoing
Paul Wentworth, Jr., Ph.D.
projects include studies on
Professor
atherosclerosis, neurodegener- Chemistry
ative diseases, ischemia-reperfusion injury, macular degeneration, cancer, inflammation,
and infectious diseases. Currently, we are validating a
new antileishmania drug target and increasing the
scope of the role of inflammatory aldehydes in protein
misfolding diseases.
The genomic DNA of kinetoplastid parasites contains a unique modified base, 5-(β-D -glucopyranosyloxymethyl)-2′-deoxyuridine or base J. Recently, we
reported the first in-depth analysis of the molecular
recognition between the O-linked glycoside component
of deoxyribose J (dJ) in telomeric dJ-containing double-stranded DNA and J-binding protein 1 (JBP1) of
Crithidia fasciculata. Comparison between the molecular
dynamics snapshots and the free energy of binding of
JBP1 to a panel of duplex oligonucleotides containing
telomeric modified dJ revealed that JBP1 binding to
dJ-containing oligonucleotides occurs preferentially
when the β-D-glucopyranosyl moiety adopts a conformation within the major groove wherein the C-2 and C-3
hydroxyl groups of the glucoside make hydrogen-bond
contacts to the nonbridging pro-R phosphoryl oxygen of
the J-1 nucleotide phosphate group. If this orientation is
perturbed even slightly, JBP1 binding affinity drops to
the level of replacement of dJ by deoxythymidine.
In the past year, we have expanded this work and
shown that this same edge-on conformation occurs in
JBP1 in nonpathogenic Leishmania tarantolae. We
also designed phosphorothioate probes to replace the
J-1 residue in the duplex DNA. We hypothesized that
replacement of the J-1 phosphoryl oxygen with sulfur
O
F i g . 2 . ATLD missense mutations W210C and N117S, which
impair Nbs1 binding, cluster to a single Mre11 dimer surface
opposite the DNA binding cleft.
Fang, Q., Noronha, A.M., Murphy, S.P., Wilds, C.J., Tubbs, J.L., Tainer, J.A.,
Chowdhury, Q., Guengerich, F.P., Pegg, A.E. Repair of O6-G-alkyl-O6-G interstrand
cross-links by human O6-alkylguanine-DNA alkyltransferase. Biochemistry 47:10892,
2008.
Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson,
G., Andrews, G., Hamley, P.J., Malinder, P.R., Nicholis, D.J., St-Gallay, S.A., Tinker,
A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuhr, D.J., Alberg,
A., Wallace, A.V., Tainer, J.A., Getzoff, E.D. Anchored plasticity opens doors for
selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol. 4:700, 2008.
Garcin, E.D., Hosfield, D.J., Desai, S.A., Haas, B.J., Björas, M., Cunningham,
R.P., Tainer, J.A. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.
Pebernard, S., Perry, J.J., Tainer, J.A., Boddy, M.N. Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability. Mol. Biol. Cell
19:4099, 2008.
Perry, J.J., Tainer, J.A. Structural biology of Cockayne syndrome proteins, their
interactions and insights into DNA repair mechanisms. In: Molecular Mechanisms of
Cockayne Syndrome. Ahmad, S.I. (Ed.). Landes Biosciences, Austin, TX, in press.
Perry, J.J.P., Tainer, J.A, Boddy, M.N. A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem. Sci. 33:201, 2008.
Syson, K., Tomlinson, C., Chapados, B.R., Sayers, J.R., Tainer, J.A., Williams,
N.H., Grasby, J.A. Three metal ions participate in the reaction catalyzed by T5 flap
endonuclease. J. Biol. Chem. 283:28741, 2008.
Williams, R.S., Moncalian, G., Williams, J.S., Yamada, Y., Limbo, O., Shin, D.S.,
Groocock, L.M., Cahill, D., Hitomi, C., Guenther, G., Moiani, D., Carney, J.P.,
Russell, P., Tainer, J.A. Mre11 dimers coordinate DNA end bridging and nuclease
processing in double-strand-break repair. Cell 135:97, 2008.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
would have a clear effect on the strength of the C-2
and C-3 hydrogen bonding. This hypothesis was proved
by the finding that JBP1 does not bind to the phosphorothioate-containing duplex DNA as tightly as to
normal duplex DNA.
In conclusion, the use of new phosphorothioate J-1
DNA as a chemical tool has validated our proposed
binding hypothesis for JBP1 to J-DNA. This study further emphasizes the importance of our earlier proposed
hypothesis that glucose conformation in the major groove
of DNA is due to hydrogen bonds between the quasiequatorial C-2 and C-3 hydroxyl groups of the sugar
and the pro-R phosphoryl oxygen of the J-1 nucleotide.
This understanding has clear ramifications for structure-based drug design of therapeutics against Leishmania parasites.
Recently, we discovered a process that we are studying in the context of several disease-related sporadic
amyloidoses. We have shown that in vitro, certain inflammatory-derived lipidic aldehydes, when adducted to
proamyloidogenic proteins in the proteins’ native state,
can induce misfolding and aggregation of the native
protein sequences. This past year, we expanded our
studies to aggregation of antibody light chains. In vivo,
such aggregation leads to the systemic deposition of
immunoglobulin light-chain domains in the form of either
amyloid fibrils (AL-amyloidosis) or amorphous deposits
(light-chain deposition disease), mainly in cardiac or
renal tissue, and is a pathologic condition that is often
fatal. Molecular factors that may contribute to the propensity of antibody light chains to aggregate in vivo, such
as the protein primary structure or local environment, are
intensive areas of study.
We have now shown that aggregation of human
antibody κ (κ-MJM) and λ (λ-L155) light chains can
be accelerated in vitro when they are incubated under
physiologically relevant conditions in the presence of a
panel of biologically relevant lipid-derived aldehydes:
4-hydroxynonenal, malondialdehyde, glyoxal, atheronal-A,
and atheronal-B. Thioflavin-T and Congo red binding
assays coupled with turbidity studies revealed that this
aldehyde-induced aggregation can be associated with
alteration of protein secondary structure to an increased
β-sheet conformation. We found that the nature of the
conformational change depends primarily on the lipidic
aldehyde, not the protein sequence. Thus, the cholesterol 5,6-seco-sterols, atheronal-A and atheronal-B,
caused amorphous aggregations that did not bind
thioflavin-T or Congo red for both light chains, whereas
49
4-hydroxynonenal, malondialdehyde, and glyoxal induced
aggregates that bound both thioflavin-T and Congo red.
Transmission electron microscopy revealed that amyloid fibrils were formed during the aggregation of κ-MJM
and λ-L155 light chains mediated by 4-hydroxynonenal (Fig. 1), whereas aggregates induced by atheronal-B
were amorphous.
F i g . 1 . Transmission electron microscopy image of fibrillar aggre-
gates of antibody light chains induced by 4-hydroxynonenal in vitro.
Kinetic profiles of light-chain aggregation revealed
clear differences between the aldehydes. Atheronal-A
and atheronal-B caused a classic nucleated polymerization-type aggregation, with a lag phase (~150 hours)
followed by a growth phase that plateaued, whereas
4-hydroxynonenal, malondialdehyde, and glyoxal triggered a seeded-type aggregation that has no lag phase.
Studies of the accelerated aggregation of κ-MJM and
λ-L155 induced by 4-hydroxynonenal revealed a clear
dependence on the concentration of the aldehyde and a
process that can be inhibited by the naturally occurring
osmolyte trimethylamine N-oxide. On the basis of these
data, we think that our recently discovered model of
protein misfolding induced by inflammatory aldehydes
may now extend to aggregation of antibody light chains.
PUBLICATIONS
Nieva, J., Shafton, A., Altobell, L.J. III, Tripurenani, S., Rogel, J.K., Wentworth,
A.D., Lerner, R.A., Wentworth, P., Jr. Inflammatory aldehydes accelerate antibody
light chain amyloid and amorphous aggregation. Biochemistry 47:7695, 2008.
50
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Scanlan, C.N., Ritchie, G.E., Baruah, K., Harvey, D.J., Crispin, M.D., Singer, B.B.,
Lucka, L., Wormald, M., Wentworth, P., Jr., Zitzmann, N., Rudd, P.M., Burton, D.R.,
Dwek, R.A. Inhibition of mammalian glycan biosynthesis produces non-self antigens
for broadly neutralising, HIV-1 specific antibody. J. Mol. Biol. 372:16, 2007.
Scheinost, J.C., Boldt, G.E., Wentworth, P., Jr. The chemical biology of protein
misfolding. In: Encyclopedia of Chemical Biology. Wiley-VCH, New York, in press.
Scheinost, J.C., Wang, H., Boldt, G.E., Offer, J., Wentworth, P., Jr. Cholesterol secosterol-induced aggregation of methylated amyloid β-peptides—insights into aldehydeinitiated fibrillization of amyloid-β. Angew. Chem. Int. Ed. 47:3919, 2008.
Temperinini, C., Cecchi, A., Boyle, N.A., Scozzafava, A., Cabeza, J.E., Wentworth, P., Jr., Blackburn, G.M., Supuran, C.T. Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12
mammalian isoforms: kinetic and x-ray crystallographic studies. Bioorg. Med.
Chem. Lett. 18:999, 2008.
Wentworth, P., Jr., Witter, D. Antibody-catalyzed water-oxidation pathway. Pure
Appl. Chem. 80:1849, 2008.
Pathway Engineering for
Enzymatic Synthesis
J.R. Williamson, W. Anderson, F. Agnelli, A. Beck, C. Beuck,
A. Bunner, A. Carmel, S. Chen, S. Edgcomb, D. Kerkow,
S. Kwan, E. Menicelli, W. Ridgeway, G. Ring, H. Schultheisz,
L.G. Scott, Z. Shajani, E. Sperling, M. Sykes, B. Szymczyna,
J. Wu
nzymes are the protein
factors in cells that are
responsible for effecting
chemical transformations of
metabolites and macromolecules. Enzymes are responsible
for synthesizing the thousands
of small molecules in a cell that
are necessary for a functioning
metabolism and for synthesizJames R. Williamson, Ph.D.
ing diverse natural products
Professor
that can have important medi- Molecular Biology
cinal properties.
Enzymatic synthesis in the laboratory is a powerful alternative to organic chemical synthesis for certain
types of molecules. Because enzymes have complex 3dimensional folds, they can bind specifically to substrates and catalyze complex chemical reactions that
are sometimes difficult to achieve with organic synthesis. A series of enzymes can be used simultaneously to effect a series of chemical reactions without
isolation of the intermediate products. Thus, enzymatic
synthesis is a powerful tool that can be applied to certain synthetic problems.
E
We are interested in the structure of RNA molecules and RNA-protein complexes that are important
for translation or regulation of protein expression. One
of the structural biology tools we use is nuclear magnetic resonance (NMR) spectroscopy, which can be
used to determine the structure of macromolecules in
solution. Application of NMR requires the incorporation of the stable isotope labels 13C and 15N, which
can be difficult with RNA molecules. One aspect of
our research program is developing methods to incorporate these stable isotope labels into RNA molecules,
to enable structural studies.
We have developed a flexible and powerful enzymatic
synthesis of the purine nucleotides ATP and GTP. The
method is, to our knowledge, the most complex and intricately engineered enzymatic synthesis that has been
carried out in a laboratory to date. The process requires
28 enzymes, each of which was overproduced in Escherichia coli and purified before synthesis. The longest linear series of reactions has 19 sequential steps, but the
process can be carried out with a yield of about 60%.
The scheme requires input of 3 types of reagents (Fig. 1).
First, the starting material substrates are incorporated
into the final nucleotide product. Second, trace amounts
of catalytic cofactors, such as NAD, glutamine, aspartate,
and tetrahydrofolate, are supplied. The cofactors are
recycled by using metabolic enzymes. Third, excess
amounts of fuel reagents such as creatine phosphate and
α-ketoglutarate are added to drive the recycling reactions
and to drive the overall synthesis from the starting materials to the products (ATP or GTP). Almost every one of
the 19 key steps involves recycling of a cofactor and consumption of fuel molecules.
The overall synthesis results in the efficient synthesis
of ATP or GTP starting from glucose, carbon dioxide,
ammonia, and serine. These precursors are available in
isotopically labeled forms, and a variety of different labeling patterns can be constructed by using different combinations of the labeled precursors. Using this approach,
we were able to synthesize isotopically labeled ATP and
GTP with novel labeling patterns that have useful properties for NMR analysis of RNA structure.
Although preparing all of the enzymes for this biosynthetic approach is time-consuming, the resulting
labeled nucleotides cannot easily be synthesized in any
other way. The enzymatic synthesis approach we have
developed should have broad applicability in the synthesis of other high-value biochemicals that can be used
for structural biology or metabolic profiling experiments.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
51
Szymczyna, B.R., Gan, L., Johnson, J.E., Williamson, J.R. Solution NMR studies
of the maturation intermediates of a 13 MDa viral capsid. J. Am. Chem. Soc.
129:7867, 2007.
Tahmassebi, D.C., Williamson, J.R. Balancing teaching and research in obtaining
a faculty position at a predominantly undergraduate institution. ACS Chem. Biol.
2:521, 2007.
Williamson, J.R. Biophysical studies of bacterial ribosome assembly. Curr. Opin.
Struct. Biol. 18:299, 2008.
Williamson, J.R. Cooperativity in macromolecular assembly. Nat. Chem. Biol.
4:458, 2008.
X-ray Crystallographic Studies of
Therapeutically Important
Macromolecular Targets
I.A. Wilson, M.A. Adams, C.H. Bell, R.M.F. Cardoso,
S. Connelly, B.J. Droese, D.C. Ekiert, M.-A. Elsliger,
Z. Fulton, B.W. Han, M. Hong, M.J. Jimenez-Dalmaroni,
R.N. Kirchdoerfer, R. Pejchal, G.P. Porter, A. Schiefner,
F i g . 1 . One-pot de novo enzymatic synthesis of purine nucleotides.
Glucose, carbon dioxide (CO2), ammonia (NH3), and serine, the stoichiometrically consumed reagents incorporated into products, are
highlighted in yellow. A, Enzymes from the glycolytic pathway and
purine biosynthetic pathways convert glucose into ATP and GTP.
Intermediates are shown in the vertical sequence, and the abbreviation for each enzyme is shown in italics. Cofactors (e.g., ATP, NAD,
THF) are shown to the left or the right of the main reaction sequence.
The circular arrow symbols indicate the enzymatic regeneration of
the cofactors, which are color coded for the reactions shown in the
cofactor regeneration schemes (B). Red = ATP recycling, green = NAD
recycling, purple = glutamine recycling, orange = aspartate recycling, blue = folate recycling.
PUBLICATIONS
Edgcomb, S.P., Aschrafi, A., Kompfner, E., Williamson, J.R., Gerace, L., Hennig.
M. Protein structure and oligomerization are important for the formation of exportcompetent HIV-1 Rev-RRE complexes. Protein Sci. 17:420, 2008.
Hennig, M., Scott, L.G., Sperling, E., Bermel, W., Williamson, J.R. Synthesis of
5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J. Am.
Chem. Soc. 129:14911, 2007.
Kiessling, L.L., Doudna, J.A., Johnsson, K., Mapp, A.K., Marletta, M.A., Seeberger, P.H., Williamson, J.R., Wedde, S.G. A higher degree of difficulty. ACS
Chem. Biol. 2:197, 2007.
Naidoo, N., Harrop, S.J., Sobti, M., Haynes, P.A., Szymczyna, B.R., Williamson,
J.R., Curmi, P.M.G., Mabbut, B.C. Crystal structure of Lsm3 octamer from Saccharomyces cerevisae: implications for Lsm ring organisation and recruitment. J.
Mol. Biol. 377:1357, 2008.
Schultheisz, H.L., Szymczyna, B.R., Scott, L.G., Williamson, J.R. Pathway engineered de novo enzymatic purine nucleotide synthesis. ACS Chem. Biol. 3:499,
2008.
Sperling, E., Bunner, A., Sykes, M.T., Williamson, J.R. Quantitative analysis of
isotope distributions in proteomic mass spectrometry using least-squares Fourier
transform convolution. Anal. Chem. 80:4906, 2008.
R.L. Stanfield, R.S. Stefanko, J.A. Vanhnasy, R. Xu,
X. Xu, S.I. Yoon, X. Zhu
H I V T Y P E 1 VA C C I N E D E S I G N
IV type 1 (HIV-1) continues to constitute a
major worldwide health
threat, with approximately 2.1
million HIV-related deaths and
2.5 million new HIV-1 infections
in 2007. Currently, more than
Ian A. Wilson, D.Phil.
20 anti-HIV drugs approved by
Professor
the Food and Drug Administration Molecular Biology
are on the market. Although
these drugs can be effective at lowering the levels of
circulating virus, they cannot completely eliminate the
virus, are expensive, and must be taken daily for life.
Clearly, an effective vaccine against HIV-1 is needed
to control the rampant spread of this devastating pandemic. An effective vaccine likely must elicit a vigorous
antibody response to block or neutralize viral infection.
However, in many studies of patients infected with HIV-1,
only a handful of potent, broadly neutralizing antibodies have been discovered that recognize a large percentage of the circulating viral strains. Our goal has
been to understand how these rare antibodies are able
to combat the virus.
We are elucidating the 3-dimensional structures of
these rare antibodies in complexes with the antibodies’
H
52
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
viral epitopes from the envelope proteins gp120 and
gp41. The antibodies under study include b12, which
recognizes a highly conserved, but deeply recessed pocket
on gp120 that is the receptor-binding site for CD4;
2G12, which binds to a mannose-rich carbohydrate cluster on the normally immunologically silent face of gp120;
several antibodies that recognize the hypervariable V3
region of gp120; and 4E10 and Z13e1, which interact
with overlapping epitopes on gp41, just proximal to
its membrane-spanning domain.
Our original structural studies of antibody 2G12 in
complex with mannose sugars have led to design of
many nonnatural carbohydrates, peptides, and smallmolecule mimics for testing as potential immunogens.
Recently, in collaboration with B. Davis, University of
Oxford, Oxford, England, we determined a 2.8-Å crystal structure for 2G12 in complex with a novel, nonself mimic of the D1 arm of Man4/Man9GlcNac2, the
type of carbohydrate commonly found on the gp120
silent face. This nonnatural mannose variant contains
a C-6 methyl substitution of the mannose at the terminus of the D1 arm and inhibits binding of 2G12 to
gp120 better than does the D1 arm itself. This compound is the first nonself D1 arm derivative to demonstrate inhibition of 2G12/gp120 binding better than
that of the natural D1 arm. Preliminary diffraction
data have also been collected from crystals of 2G12
in complex with a C-6 methyl monosaccharide compound. Further optimization of crystallization conditions for both complexes to obtain higher resolution
diffraction data are under way.
We are currently refining the crystal structure for
Fab Z13e1, a neutralizing antibody that recognizes an
epitope in the gp41 membrane proximal external region
that overlaps the Fab 4E10 epitope (Fig. 1). Z13e1 has
been evolved to have higher affinity than the parent
antibody Z13; the higher affinity is due to 5 mutations
in complementarity-determining region L3 (residues
L90–L97). Although Z13e1 and 4E10 recognize similar epitopes, the antibody-bound conformations for the
epitopes differ markedly, perhaps giving some insight
to conformational changes that may occur during viral
entry into cells. The differences in the key contact residues also correlate with the breadth and neutralization
potency of 4E10 vs Z13e1.
Our studies on HIV are done in collaboration with
D.R. Burton, M. Zwick, R. Pantophlet, P.E. Dawson, and
C.-H. Wong, Scripps Research; B. Davis, University of
Oxford; L. Cavacini and J.K. Scott, Simon Fraser Univer-
F i g . 1 . Structure of Fab Z13e1 in complex with its epitope peptide
on the HIV-1 gp41 membrane proximal external region. The Fab is
shown in a solid surface representation, with the light and heavy
chains in cyan and blue, respectively. The peptide, shown in a balland-stick representation, binds primarily to the heavy chain of the
antibody and adopts a different conformation than do the corresponding residues bound to broadly neutralizing antibody 4E10. This epitope occurs in gp41 just before the protein enters the membrane,
so Z13e1 may have to contact the membrane to bind its epitope.
sity, Burnaby, British Columbia; J. Moore, Weill Medical
College of Cornell University, New York, New York;
H. Katinger, R. Kunert, and G. Stiegler, University für
Bodenkultur, Vienna, Austria; R. Wyatt and P. Kwong,
Vaccine Research Center, National Institutes of Health,
Bethesda, Maryland; W. Olson and K. Kang, Progenics
Pharmaceuticals, Inc., Tarrytown, New York; the National
Institutes of Health; and the Neutralizing Antibody Consortium of the International AIDS Vaccine Initiative.
I N F L U E N Z A V I R U S G LY C O P R O T E I N S
The 1918 influenza pandemic, which was responsible for more than 20 million deaths worldwide, and
the more recent “bird flu,” with its even higher mortality rate (about 60% of patients in whom it is diagnosed),
are constant reminders of the potential devastation that
could ensue if a new influenza pandemic were to occur.
To aid in the design of a vaccine to protect against such
highly virulent strains of influenza, we are carrying out
structural and functional studies of envelope proteins
of influenza virus in complex with neutralizing antibodies
to the virus.
All known antibodies that neutralize influenza virus
recognize the hemagglutinin viral envelope protein. In
general, antibodies to hemagglutinin generally recognize highly variable epitopes at the membrane distal
end of the hemagglutinin trimer. However, a small proportion of the host repertoire of antibodies is directed
against other sites on hemagglutinin, including several
antibodies that bind on the side of the hemagglutinin
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
trimer and recognize highly invariant epitopes. Several
of these unusual antibodies neutralize the hemagglutinin of different strains and subtypes of influenza virus,
both in vivo and in vitro. To gain insight into the mechanism of virus neutralization and the nature of the epitopes recognized, we are investigating several of these
broadly neutralizing antibodies in complex with hemagglutinins that represent different pandemic strains and
subtypes (H1, H2, H3) of human influenza virus as well
as avian H5N1 influenza viruses. Understanding how
these more broadly neutralizing antibodies interfere
with viral entry and subsequent infection, as well as
the nature of the highly conserved epitopes, will provide insights into the functional and antigenic constraints
on the hemagglutinin of influenza virus.
Currently, we are working with 2 Fabs, H5M11 and
H5M9, that neutralize the H5N1 avian influenza virus
A/Goose/Guangdong/1/96 and more recent avian strains
that have infected humans and with antibodies to the
influenza virus that were isolated from survivors of
the 1918 pandemic by J. Crowe, Vanderbilt University,
Nashville, Tennessee. The structure of Fab H5M11 has
been determined, and we are working toward crystallization of other Fabs in complex with hemagglutinins from
the H5N1 and 1918 H1N1 viruses.
Hemagglutinin facilitates cell fusion through interactions with host membranes. Although crystal structures
of hemagglutinin ectodomains have been extensively
studied, little is known about the conformation or function
of the membrane-interacting regions. We are working
toward the determination of crystal structures of the
full-length hemagglutinin in its states before and after
fusion. In collaborative research with G. Tobin, Biological Mimetics, Inc., Frederick, Maryland, crystallization
of the full-length hemagglutinin from A/Wyoming/3/03
(H3 subtype) and bacterial expression of the postfusion
form of the protein are under way. We also propose to
isolate and structurally analyze the hemagglutinin from
the pandemic A/Japan/305/57 (H2 subtype) virus.
These studies will advance our understanding of the
mechanism of hemagglutinin-induced fusion and provide novel targets for design of fusion inhibitors.
The crystal structure of the neuraminidase of the
1918 H1N1 virus has been determined to 1.45 Å. A
large cavity in the active site in the neuraminidase offers
new opportunities for structure-based drug design. Crystal
structures of the 1918 neuraminidase in complex with
the antiviral drugs oseltamivir (Tamiflu) and zanamivir
(Relenza) show that the loop bordering the cavity is
53
extremely flexible in binding substrates, a characteristic
that may indicate that the 1918 neuraminidase can
bind more chemically diverse ligands than can neuraminidases from some other subtypes of the virus. This
high-resolution structural information is being used for
rational design of inhibitors against influenza virus.
Additional collaborators in the influenza research
include our colleagues in the flu consortium funded by the
National Institute of Allergy and Infectious Diseases; scientists at Crucell, Leiden, the Netherlands; J. Crowe, Vanderbilt University; A. Lanzavecchia, Institute for Research
in Biomedicine, Bellinzona, Switzerland; and X. Che,
Southern Medical University China, Guangzhou, China.
T H E I N N AT E I M M U N E R E S P O N S E A G A I N S T
M I C R O B I A L PAT H O G E N S
Toll-like receptors (TLRs) are glycoproteins that
are essential for innate immune recognition of microbial pathogens. The TLR extracellular domains are
horseshoe-shaped molecules consisting of leucine-rich
repeat domains that begin and end with an N- and a
C-terminal cap domain. Recently, we have been studying the TLR4, which plays an essential role in recognition and signaling of bacterial lipopolysaccharide. Among
the TLR family members, TLR4 is unique in requiring
another molecule, myeloid differentiation protein-2
(MD-2), for its function. MD-2 directly binds lipopolysaccharide and induces dimerization and activation of
TLR4 for signaling.
To provide insights into the structural mechanism
used by lipopolysaccharide to activate TLR4, we have
expressed the extracellular domain of TLR4 (sTLR4) in
complex with MD-2 in a baculovirus expression system.
Biophysical studies suggest that purified 1:1 complexes
of sTLR4–MD-2 homodimerize to form 2:2 complexes
in the presence of lipopolysaccharide. X-ray crystallographic studies are being carried out to reveal the structural architecture of the sTLR4–MD-2 assembly induced
by lipopolysaccharide.
Jawless fish, such as the lamprey, do not have
immune receptors, such as antibodies, T-cell receptors,
or MHC molecules, yet the fish still have an adaptive
immune response to antigen. Recently, it was shown
that cell-surface molecules, termed variable lymphocyte receptors (VLRs) are responsible for the adaptive
immune response in jawless fish. These receptors
resemble the mammalian innate system TLRs, with an
overall horseshoe shape made up of a variable number
of different leucine-rich repeat domains. In collaboration with M. Cooper, Emory University, Atlanta, Geor-
54
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
gia, we recently determined the first crystal structure
of a VLR-antigen complex, RBC36, in complex with the
H trisaccharide derived from the H antigen of human
type O erythrocytes (Fig. 2). This structure reveals for
the first time the location and nature of the VLR antigen-binding site.
F i g . 3 . Crystal structure of CD1d with designed agonists. The
ligand-presenting α1α2 platform of mouse CD1d is shown in gray,
overlaid by the transparent molecular surface, with the bound superimposed ligands C6Ph (red), C8Ph (yellow), C8PhF (green), and
the short α-galactosylceramide agonist PBS-25 (blue). The phenyl
substitutions protrude deeply into the A′ pocket (see inset on right
for view of ligands only). The 2 main A′ and F′ pockets in CD1d for
ligand binding are indicated. A spacer lipid (likely palmitic acid) also
partially fills the A′ pocket when the glycolipid ligand itself does not
fully occupy the pocket. The Cα positions of the ligands show an
overall root mean square displacement of 0.42 Å.
F i g . 2 . Structure of the lamprey VLR red blood cell in complex
with its H trisaccharide antigen. Left, The VLR is shown as a solid
surface, with the antigen depicted in a ball-and-stick representation.
Right, The same view of the structure but highlighting the secondary
structural elements of the VLR. The antigen binds to the concave
side of the horseshoe-shaped VLR and is cradled on one side by a
large fingerlike insert in the C-terminal leucine-rich repeat.
The CD1 family of innate receptors consists of MHC
class I–like, antigen-presenting molecules that present
lipids, glycolipids, and lipopeptides to effector T cells.
The receptors are expressed on antigen-presenting cells
and are involved in host defense and in immunoregulatory functions. Glycolipids presented by CD1d are capable of stimulating natural killer T cells. Natural killer
T cells are of clinical interest because when stimulated
by CD1, they rapidly secrete a number of cytokines that
either promote or suppress different immune responses.
One of the most potent agonists for natural killer T cells
is α-galactosylceramide. On the basis of our structural
studies during the past 5 years, a series of glycolipids
have been synthesized by scientists in the laboratory of
C.-H. Wong, Skaggs Institute. These new ligands, which
have phenyl ring substitutions in the fatty acid part of
α-galactosylceramide, are more potent than the native
ceramide and have an altered efficacy in T-cell assays.
We have now determined the structures for 3 of the
most stimulating glycolipids in complex with CD1d. Our
analysis revealed that only minor structural changes
occur in the A′ pocket (Fig. 3). The phenyl-ring derivatives have better packing than the native α-galactosyl-
ceramide and thereby increase the overall stability of
the CD1d-ligand complexes.
PUBLICATIONS
Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson,
I.A., Blixt, O., Dwek, R.A., Wong, C.H., Burton, D.R. A glycoconjugate antigen
based on the recognition motif of a broadly neutralizing human immunodeficiency
virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the
self glycans of gp120. J. Virol. 82:6359, 2008.
Bell, C.H., Pantophlet, R., Schiefner, A., Cavacini, L.A., Stanfield, R.L., Burton,
D.R., Wilson, I.A. Structure of antibody F425-B4e8 in complex with a V3 peptide
reveals a new binding mode for HIV-1 neutralization. J. Mol. Biol. 375:969, 2008.
Burton, D.R., Wilson, I.A. Immunology: square-dancing antibodies. Science
317:1507, 2007.
Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic,
G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B.,
Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.
Debler, E.W., Müller, R., Hilvert, D., Wilson, I.A. Conformational isomerism can
limit antibody catalysis. J. Biol. Chem. 283:16554, 2008.
Demartino, J.K., Hwang, I., Connelly, S., Wilson, I.A., Boger, D.L. Asymmetric
synthesis of inhibitors of glycinamide ribonucleotide transformylase. J. Med. Chem.
51:5441, 2008.
Dhillon, A.K., Stanfield, R.L., Gorny, M.K., Williams, C., Zolla-Pazner, S., Wilson,
I.A. Structure determination of an anti-HIV-1 Fab 447-52D-peptide complex from an
epitaxially twinned data set. Acta Crystallogr. D Biol. Crystallogr. 64:792, 2008.
Huang, C.C., Lam, S.N., Acharya, P., Tang, M., Xiang, S.H., Hussan, S.S., Stanfield, R.L., Robinson, J., Sodroski, J., Wilson, I.A., Wyatt, R., Bewley, C.A., Kwong,
P.D. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with
HIV-1 gp120 and CD4. Science 317:1930, 2007.
Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural
evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.
Menendez, A., Calarese, D.A., Stanfield, R.L., Chow, K.C., Scanlan, C.N., Kunert,
R., Katinger, H., Burton, D.R., Wilson, I.A., Scott, J.K. A peptide inhibitor of HIV1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate
epitope on gp120. FASEB J. 22:1380, 2008.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Relloso, M., Cheng, T.Y., Im, J.S., Parisini, E., Roura-Mir, C., DeBono, C., Zajonc,
D.M., Murga, L.F., Ondrechen, M.J., Wilson, I.A., Porcelli, S.A., Moody, D.B. pHdependent interdomain tethers of CD1b regulate its antigen capture. Immunity
28:774, 2008.
Stevens, J., Blixt, O., Chen, L.M., Donis, R.O., Paulson, J.C., Wilson, I.A. Recent
avian H5N1 viruses exhibit increased propensity for acquiring human receptor
specificity. J. Mol. Biol. 381:1382, 2008.
Verdino, P., Aldag, C., Hilvert, D., Wilson, I.A. Closely related antibody receptors
exploit fundamentally different strategies for steroid recognition. Proc. Natl. Acad.
Sci. U. S. A. 105:11725, 2008.
Wei, C.J., Xu, L., Kong, W.P., Shi, W., Canis, K., Stevens, J., Yang, Z.Y., Dell, A.,
Haslam, S.M., Wilson, I.A., Nabel, G.J. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza
virus. J. Virol. 82:6200, 2008.
Zajonc, D.M., Savage, P.B., Bendelac, A., Wilson, I.A., Teyton, L. Crystal structures
of mouse CD1d-iGb3 complex and its cognate Vα14 T cell receptor suggest a model
for dual recognition of foreign and self glycolipids. J. Mol. Biol. 377:1104, 2008.
Zajonc, D.M., Wilson, I.A. Architecture of CD1 proteins. Curr. Top. Microbiol.
Immunol. 314:27, 2007.
Zhu, X., Xu, X., Wilson, I.A. Structure determination of the 1918 H1N1 neuraminidase from a crystal with lattice-translocation defects. Acta Crystallogr. D Biol.
Crystallogr. 64:843, 2008.
Bioorganic and
Synthetic Chemistry
C.-H. Wong, C. Bennett, S. Dean, S. Ficht, Y. Fu, W. Greenberg,
R. Guy, S. Hanson, Z. Hong, D.-R. Hwang, J.-C. Lee,
P.-H. Liang, R. Payne, M. Schelwies, S.-K. Wang
e develop new chemical and enzymatic
strategies for synthesis
of bioactive small molecules and
biomolecules. We use the methods to probe carbohydrate-mediated recognition events important
in cancer, bacterial infections,
and viral infections, including
HIV disease and influenza.
W
Chi-Huey Wong, Ph.D.
Professor
Chemistry
SYNTHETIC METHODS
We have developed new methods for sugar-assisted
ligation of glycopeptides for synthesis of homogenous
glycoproteins. We have used the methods in conjunction with enzymatic glycosylation techniques to assemble
complex glycopeptides by chemical synthesis, and we
are optimizing the techniques to achieve the total synthesis of therapeutic glycoproteins. Glycoproteins are
expressed in vivo as complex mixtures of glycoforms, a
situation that hinders efforts to study the role of glyco-
55
sylation in protein folding, stability, and function. By
synthesizing pure glycoforms, we can characterize in
molecular detail the effects of glycans on protein function.
Using chemical techniques such as programmable
1-pot oligosaccharide synthesis, as well as enzymatic
synthesis, we create glycoarrays on glass slides for
high-throughput quantitative analysis of protein-carbohydrate interactions. These arrays are being used to
study the binding specificity of carbohydrate-binding
receptors and antibodies. We have applied aldolases,
glycosyltransferases, glycosidases, and other enzymes
to develop practical new methods of synthesizing molecules such as iminocyclitols, which are inhibitors of
glycosidases and other enzymes; glycopeptides; and
other glycoconjugates. Using directed evolution, we are
evolving these enzymes to catalyze new reactions and
synthesize new molecules of pharmaceutical relevance.
C A R B O H Y D R AT E - M E D I AT E D R E C O G N I T I O N I N D I S E A S E
We are using our synthetic methods to discover
inhibitors and therapeutic agents in several diseases
related to carbohydrates. Current targets include bacterial transglycosidase, sulfatases, and glycoprocessing
enzymes involved in the biosynthesis of carbohydrates
that mediate cancer metastasis, inflammation, and viral
infection. Enzymatically synthesized iminocyclitols are
being investigated as treatments for osteoarthritis and
Gaucher disease. Inspired by the broadly neutralizing
anti-HIV antibody 2G12, which recognizes a dense array
of oligomannose displayed on HIV gp120, we are designing dendrimeric oligomannose structures for development
of an HIV vaccine. In collaboration with D.R. Burton,
Scripps Research, we are testing the immunogenicity
of these constructs. We have designed glycolipid ligands
for CD1 that activate natural killer T cells and are a promising new immunotherapeutic approach for treatment of
bacterial and viral infections and cancer. The ligands may
also be useful as adjuvants in vaccine development.
G LY C O P R O T E O M I C S A N D M O L E C U L A R G LY C O B I O L O G Y
Using metabolic oligosaccharide engineering, we
have developed methods for incorporating tagged sugars
into glycans expressed on mammalian cells. The engineered glycans can be labeled with a variety of molecules
by using click chemistry. One application is glycan-specific fluorescent labeling, which is used for fluorescent
imaging to compare glycosylation patterns of different
cells, such as normal vs cancer cells or cancer cells vs
cancer stem cells. We found that protein fucosylation and
sialylation are both elevated in cancer cell lines.
56
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
A second application of this chemistry is GIDmap,
a new method for glycoproteomic analysis (Fig. 1).
Hanson, S.R., Greenberg, W.A., Wong C.-H. Probing glycans with the copper(I)catalyzed [3+2] azide-alkyne cycloaddition. QSAR Comb. Sci. 26:1243, 2007.
Kinjo, Y., Pei, B., Bufali, S., Raju, R., Richardson, S.K., Imamura, M., Fujio, M.,
Wu, D., Khurana, A., Kawahara, K., Wong, C.-H., Howell, A.R., Seeberger, P.H.,
Kronenberg, M. Natural Sphingomonas glycolipids vary greatly in their ability to
activate natural killer T cells. Chem. Biol. 15:654, 2008.
Liang, P.-H., Imamura, M., Li, X., Wu, D., Fujio, M., Guy, R.T., Wu, B.-C., Tsuji,
M., Wong, C.-H. Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production. J. Am. Chem. Soc.
130:12348, 2008.
Liang, P.-H., Wu, C.-Y., Greenberg, W.A., Wong C.-H. Glycan arrays: biological
and medical applications. Curr. Opin. Chem. Biol. 12:86, 2008.
Northen, T.R., Lee, J.-C., Hoang, L., Raymond, J., Hwang, D.-R., Yannone, S.M.,
Wong, C.-H., Siuzdak, G. A nanostructure-initiator mass spectrometry-based enzyme
activity assay. Proc. Natl. Acad. Sci. U. S. A. 105:3678, 2008.
Payne, R.J., Ficht, S., Greenberg, W.A., Wong, C.-H. Cysteine-free peptide and
glycopeptide ligation by direct aminolysis. Angew. Chem. Int. Ed. 47:4411, 2008.
Wang, S.-K., Liang, P.-H., Astronomo, R.D., Hsu, T.-L., Hsieh, S.-L., Burton,
D.R., Wong, C.-H. Targeting the carbohydrates on HIV-1: interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl.
Acad. Sci. U. S. A. 105:3690, 2008.
Whalen, L.J., Greenberg, W.A., Mitchell, M.L., Wong, C.-H. Iminosugar-based glycosyltransferase inhibitors. In: Iminosugars: From Synthesis to Therapeutic Applications. Compain, P., Martin, O.R. (Eds.). Wiley-VCH, Hoboken, NJ, 2007, p. 153.
Wu, D., Fujio, M., Wong, C.-H. Glycolipids as immunostimulating agents. Bioorg.
Med. Chem. 16:1073, 2008.
F i g . 1 . GIDmap glycoproteomic analysis via metabolic oligosaccharide engineering.
Whole cells are fed with tagged sugars, and after biochemical incorporation into cellular glycoproteins, click
chemistry is used to attach a handle for purification of
tagged proteins. Mass spectrometric proteomic methods
are then used to identify proteins that are differentially
glycosylated. We are using GIDmap to identify proteins
that are aberrantly glycosylated in different stages of
cancer. These cancer-associated glycoproteins may be
useful as biomarkers for diagnostics or as targets for
therapeutic intervention.
PUBLICATIONS
Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson,
I.A., Blixt, O., Dwek, R.A., Wong C.-H., Burton D.R. A glycoconjugate antigen
based on the recognition motif of a broadly neutralizing human immunodeficiency
virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the
self glycans of gp120. J. Virology 82:6359, 2008.
Bennett, C.S., Dean, S.M., Payne, R.J., Ficht, S., Brik, A., Wong, C.-H. Sugarassisted glycopeptide ligation with complex oligosaccharides: scope and limitations.
J. Am. Chem. Soc. 130:11945, 2008.
Ficht, S., Payne, R.J., Guy, R.T., Wong, C.-H. Solid-phase synthesis of peptide
and glycopeptide thioesters through side-chain-anchoring strategies. Chem. Eur. J.
14:3620, 2008.
Giffin, M.J., Heaslet, H., Brik, A., Lin, Y.-C., Cauvi, G., Wong, C.-H., McRee,
D.E., Elder, J.H., Stout, C.D., Torbett, B.E. A copper(I)-catalyzed 1,2,3-triazole
azide-alkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1
protease variant. J. Med. Chem. 51:6263, 2008.
Studies of Macromolecular
Recognition by Multidimensional
Nuclear Magnetic Resonance
P.E. Wright, H.J. Dyson, M. Martinez-Yamout, M. Arai,
S.-H. Bae, D. Boehr, B. Buck-Koehntop, P. Deka, D. Felitsky,
J. Ferreon, P. Haberz, C.W. Lee, D. Meinhold, S.-J. Park,
M. Landes, E. Manlapaz
pecific interactions between
molecules are of fundamental importance in all
biological processes. An understanding of how biological macromolecules such as proteins and
nucleic acids recognize each
other is essential for understanding the fundamental molecular events of life. Knowledge
Peter E. Wright, Ph.D.
of the 3-dimensional structures
Professor and Chairman
of biological macromolecules
Molecular Biology
is key to understanding their
interactions and functions and also forms the basis for
rational design of new drugs. A particularly powerful
S
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
method for mapping the 3-dimensional structures and
interactions of biological macromolecules in solution is
multidimensional nuclear magnetic resonance (NMR)
spectroscopy. We are using this method to study a number of protein-protein and protein–nucleic acid interactions of fundamental importance in health and disease.
Transcriptional regulation in eukaryotes relies on
protein-protein interactions between DNA-bound factors
and coactivators that, in turn, interact with the basal
transcription machinery. A major effort in our laboratory is focused on elucidating the structural principles
that determine specificity of key protein-protein interactions involved in regulation of gene expression. The
transcriptional coactivator CREB-binding protein (CBP)
and its ortholog p300 play a central role in cell growth,
differentiation, and development in higher eukaryotes.
CBP and p300 mediate interactions between a number
of gene regulatory proteins and viral proteins, including
proteins from several tumor viruses and hepatitis B virus.
Understanding the molecular mechanisms by which
CBP recognizes its various target proteins is of fundamental biomedical importance. CBP has been implicated
in diverse human diseases such as leukemia, cancer,
and mental retardation and is a novel target for therapeutic intervention.
We have initiated a major program to determine the
structure of CBP and p300 and map their functional
interactions with other components of the transcriptional
machinery. Our research reveals that many regions of
these coactivators are intrinsically disordered, as are
many of the transcriptional regulatory proteins with which
they interact. Indeed, our results have indicated that
coupled folding and binding processes play a major role
in transcriptional regulation.
We have performed NMR relaxation experiments to
elucidate the mechanism of coupled folding and binding
processes and to identify “hot spots” in protein-protein
interfaces that could potentially be targeted by smallmolecule inhibitors. We initially used these methods to
investigate the interactions involved in the regulation of
hypoxia, namely binding of the α-subunit of the hypoxiainducible transcription factor (HIF-1α) to the TAZ1 zinc
finger motif of CBP/p300. We have now extended these
relaxation measurements to the complex formed between
the activation domain of the p160 nuclear receptor
coactivator ACTR and the nuclear coactivator binding
domain of CBP. Both proteins are intrinsically disordered and fold synergistically upon binding. Although
the free proteins are highly flexible, the complex has
the motional characteristics of a globular protein domain,
57
with no significant residual flexibility that might compensate for the loss of entropy incurred upon formation of a complex.
Some years ago, we determined the 3-dimensional
structure of the phosphorylated kinase inducible activation domain (pKID) of the transcription factor CREB
bound to its target domain (the KIX domain) in CBP. The
structure provides a starting point for design of small
molecules that can inhibit the CREB-KIX interactions,
an important goal in development of novel therapeutics
for treatment of diabetes. We have developed a new
method, using R2 relaxation dispersion experiments and
NMR titrations, to investigate the pathway by which
intrinsically disordered proteins fold into ordered structures upon binding to their biological targets. We have
used this method to study the mechanism of pKID binding to KIX.
The pKID first forms an ensemble of transient
encounter complexes at multiple sites on the surface
of KIX and then folds via a pathway involving a partially
structured intermediate. Folding of the pKID helices
occurs on the surface of KIX; the mechanism of recognition involves an induced protein folding event, rather
than selection of a small population of prefolded helical
structures from the solution conformational ensemble.
We have also used the method to study mechanisms of binding of the hydroxylated HIF-1α transactivation domain to the TAZ1 domain of CBP and have
commenced studies of the binding of the proto-oncogene cMyb to the KIX domain of CBP. This research is
leading to a new understanding of the molecular mechanisms by which intrinsically disordered proteins perform their diverse biological functions. In the course of
these studies, we have developed novel methods for
measuring the affinities with which intrinsically disordered proteins bind to their targets (Fig. 1).
F i g . 1 . Global fit of chemical-shift titration data to obtain accu-
rate dissociation constants.
58
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
CBP and p300 contain several zinc-binding domains
(ZZ domain, PHD motif, TAZ1 and TAZ2 domains) that
mediate critical interactions with numerous transcriptional regulators. We have determined the structures
of each of these domains during recent years. Our current efforts are focused on structural analysis of the
complexes formed between the TAZ1 and TAZ2 domains
and the activation domains of the numerous transcription factors with which the TAZ1 and TAZ2 domains
interact. We have determined the structures of the complexes formed between the TAZ domains and the activation domains of the signal transducer and activator
of transcription (STAT) family of transcriptional regulators. These interactions play a key role in cytokinedependent signal transduction. Structures have been
determined for the complex of TAZ1 with the STAT2
activation domain and for TAZ2 bound to STAT1 (Fig. 2).
phorylation of the p53 activation domain inhibits binding
of HDM2 and enhances binding to CBP/p300, thereby
stabilizing p53 and activating transcription of p53-regulated genes. Our findings provide novel insights into
the mechanism of p53 regulation in response to DNA
damage and genotoxic stress. In addition, we have determined the structures of the complexes formed between
the KIX domain of CBP and the p53 activation domain
and between the TAZ2 domain of CBP and the adenoviral oncoprotein E1A.
Finally, we have made major advances in understanding the mechanism by which the zinc finger
protein muscleblind recognizes both pathogenic doublestranded repeat RNA sequences and single-stranded
regulatory RNA elements. Sequestration of muscleblind by CUG- and CCUG-repeat RNA disrupts alternate RNA splicing and is the underlying molecular
cause of myotonic dystrophy, the most common form
of adult-onset muscular dystrophy. We have determined
the structure of the first 2 zinc fingers of muscleblind,
which fold into a unique globular structure (Fig. 3), and
we have mapped their interactions with single-stranded
RNA. We have identified the specific RNA sequence
required for high-affinity binding and are currently working on the structure of the RNA complex.
F i g . 2 . Structures of the TAZ1-STAT2 complex (A) and the TAZ2-
STAT1 complex (B). The protein backbones of the STAT activation
domains are shown as pink ribbons; the backbones of the TAZ1
and TAZ2 domains, as blue and green ribbons, respectively.
The STAT1 and STAT2 activation domains are intrinsically disordered and fold upon binding to the TAZ motifs,
burying a large surface area and forming a hydrophobic intermolecular core. The different structural features
of the TAZ1 and TAZ2 scaffolds dictate the conformation and sites of binding of the STAT2 and STAT1 motifs.
CBP and p300 play a critical role in the regulation
of the tumor suppressor p53. They interact directly with
p53 and are required for p53-mediated transcriptional
activation. They also function to regulate p53 stability.
We have used NMR spectroscopy and isothermal titration calorimetry to investigate the binding interactions
between the transcriptional activation domain of p53
and its target domains in CBP/p300. We found that
the p53 activation domain can bind simultaneously to
CBP/p300 and the ubiquitin ligase HDM2, which regulates p53 stability, to form a ternary complex. Phos-
F i g . 3 . Ribbon representation of the structure of muscleblind
zinc fingers.
PUBLICATIONS
Boehr, D.D., Dyson, H.J., Wright, P.E. Conformational relaxation following hydride
transfer plays a limiting role in dihydrofolate reductase catalysis. Biochemistry
47:9227, 2008.
Boehr, D.D., Wright, P.E. How do proteins interact? Science 320:1429, 2008.
Ebert, M.-O., Bae, S.-H. Dyson, H.J., Wright, P.E. NMR relaxation study of the
complex formed between CBP and the activation domain of the nuclear hormone
receptor coactivator ACTR. Biochemistry 47:1299, 2008.
Felitsky, D.J., Lietzow, M.A., Dyson, H.J., Wright, P.E. Modeling transient collapsed states of an unfolded protein to provide insights into early folding events.
Proc. Natl Acad. Sci. U. S. A. 105:6278, 2008.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Sugase, K., Landes, M.A., Wright, P.E., Martinez-Yamout, M.A. Overexpression of
post-translationally modified peptides in Escherichia coli by co-expression with
modifying enzymes. Protein Expr. Purif. 57:108, 2008.
Sugase, K., Lansing, J.C., Dyson, H.J., Wright, P.E. Tailoring relaxation dispersion experiments for fast-associating protein complexes. J. Am. Chem. Soc.
129:13406, 2007.
Automation of Nuclear Magnetic
Resonance Structure
Determination of Proteins
in Solution
59
and further add to the reliability of the results obtained. To
increase automation of NMR structure determination, a
research team directed by me at the ETH Zürich, Zürich,
Switzerland, developed new software and new NMR
experiments. In the context of our work in structural
genomics as part of the Joint Center for Structural Genomics (www.jcsg.org), we have now assembled these
software modules into a new protocol for structure determination that includes extensive automation.
In Figure 1 showing the newly introduced automated
NMR protocol for structure determination, 2 key features of the procedure are in red. First, a novel approach
K. Wüthrich, B. Pedrini, P. Serrano, B. Mohanty, R. Horst
e use nuclear magnetic resonance
(NMR) spectroscopy
in solution for studies in structural biology and structural
genomics. The following are
2 illustrations of current applications: structural characterization of the proteome of the
coronavirus that causes severe
Kurt Wüthrich, Ph.D.
acute respiratory syndrome,
Professor
which is pursued under the
Molecular Biology
auspices of the Center for Functional and Structural Proteomics of the SARS Coronavirus
(FSPS; http://visp.scripps.edu/SARS/default.aspx), and
studies of chaperone-mediated protein folding, which is a
collaboration with A. Horwich, a guest scientist at Scripps
Research from Yale University, New Haven, Connecticut.
In an effort to continually enhance the significance
of the NMR observations and the efficiency with which
NMR structures can be solved, developing methods is
an important part of our activities. During the past year,
the team members supported in part or entirely by funds
from the Skaggs Institute have made important contributions to new and improved NMR approaches. Because
of the important role of NMR in drug discovery and
drug design, these developments bear directly on many
aspects of biomedical research.
Currently, NMR determinations of protein structure
in solution are typically performed by experienced spectroscopists who use interactive informatics tools. Increased
use of fully automated steps in structure determination
promises to increase the efficiency of the procedure
W
F i g . 1 . Protocol for automated NMR structure determination.
to quality assessment of the protein solutions intended
for NMR structure determination is introduced in the
form of the “NMR profile.” The NMR profile enables
a quantitative assessment of the suitability of the sample
for the use of different NMR techniques for assignments
of the polypeptide backbone. Second, the protocol
includes fully automated structure determination that
leads reliably to an accurate determination of the polypeptide backbone fold. Two additional important aspects
of the new protocol are as follows: Once the polypeptide
backbone assignment has been obtained, the additional
information needed for assigning the amino acid side
chains and the structure calculation are obtained from
the same heteronuclear-resolved [ 1H,1H]-nuclear Overhauser enhancement spectroscopy (NOESY) data sets,
a step that ensures high internal consistency of the entire
procedure. The protocol contains 2 important interactive
steps to ensure (1) completeness of the polypeptide
backbone assignments and (2) refinement and validation
of the automatically solved structure.
We have so far applied the protocol to a number of
different target proteins. As an illustration, Figure 2 shows
60
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
the NMR structure of the hypothetical protein TM0212
from Thermotoga maritima. For this protein and several other proteins, the automated part of the structure
determination, leading to the accurate description of the
F i g . 2 . Stereoview of the protein TM0212 from T maritima determined by using the protocol of Figure 1. Color scheme: polypeptide
backbone, gray; well-structured amino acid side chains, yellow;
other amino acid side chains, blue.
polypeptide backbone fold (see Fig. 1), was achieved
within 1 week.
PUBLICATIONS
Johnson, M.A., Southworth, M.W., Herrmann, T., Brace, L., Perler, F.B., Wüthrich,
K. NMR structure of a Klba intein precursor from Methanococcus jannaschii. Protein
Sci. 16:1316, 2007.
Pedrini, B., Placzek, W.J., Koculi, E., Alimenti, C., LaTerza, A., Luporini, P.,
Wüthrich, K. Cold-adaptation in sea-water-borne signal proteins: sequence and NMR
structure of the pheromone En-6 from the antarctic ciliate Euplotes nobilii. J. Mol.
Biol. 372:277, 2007.
Placzek, W.J., Almeida, M.S., Wüthrich, K. NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase. J. Mol. Biol.
367:788, 2007.
Placzek, W.J., Etezady-Esfarjani, T., Herrmann, T., Pedrini, B., Peti, W., Alimenti,
C., Luporini, P., Wüthrich, K. Cold-adapted signal proteins: NMR structures of
pheromones from the antarctic ciliate Euplotes nobilii. IUBMB Life 59:578, 2007.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Staff Awards and Activities
61
Kelly, J.W.— Vincent du Vigneaud Award, American Pep-
tide Society.
Barbas, C.F. III— Arthur C. Cope Scholar Award, American
Lerner, R.A.— Jesse W. Beams Memorial Lecture in Bio-
Chemical Society; Tetrahedron Young Investigator Award,
Bioorganic and Medicinal Chemistry; Fellow, American
Association for the Advancement of Science; In-Cites
Highly Cited Researcher, Thomson Scientific, Philadelphia, Pennsylvania; Member, Faculty in Chemical Biology, Faculty of 1000, Biology Reports, Ltd.; Editorial
Boards, Bioorganic and Medicinal Chemistry Letters,
Bioorganic and Medicinal Chemistry.
physics, University of Virginia, Charlottesville, Virginia;
K.T. Wang Bioorganic Chemistry Lectureship, Taipei,
Taiwan; Editorial Boards, Bioorganic and Medicinal
Chemistry, Bioorganic and Medicinal Chemistry Letters,
Catalysis Technology, Drug Targeting and Delivery, Journal of Virology, Molecular Biology and Medicine, Molecular Medicine, Vaccine, Angewandte Chemie.
Bartfai, T.—Member, Royal Swedish Academy of Sciences,
Lifetime Achievement in Hematology, American Society
of Hematology; AABB Karl Landsteiner Award and Lectureship; Donald I. Feinstein Distinguished Lecturer,
University of Southern California, Los Angeles, California;
Member, National Academy of Sciences, Institute of
Medicine of the National Academies, American Academy of Arts and Sciences; Chairman, Scientific Advisory Board, Burnham Institute for Medical Research;
Member, Scientific Advisory Boards, Edwards Lifesciences,
iMetrikus, Optimer Pharmaceuticals; Contributing Editor,
Blood Cells, Molecules and Diseases; Associate Editor,
Acta Haematologica.
Meeting, American Chemical Society; August-Wilhelmvon-Hofmann-Denkmünze Award; Marvel Lecturer,
University of Illinois at Urbana, Champaign, Illinois;
Creigee Lecturer, University of Karlsruhe, Germany;
Behringer Simon Lecturer, ETH, Zürich, Switzerland;
Co-Editor-in-Chief, Chemistr y and Biology; Editorial
Boards, Tetrahedron Publications, Synthesis, Carbohydrate Letters, Chemistry—A European Journal, Perspectives in Drug Discovery and Design, Indian Journal of
Chemistry, Section B, Combinatorial Chemistry HighThroughput Screening, Current Opinion in Bioorganic
Chemistry, Current Organic Chemistry, Organic Letters,
ChemBioChem, Chemistry and Biodiversity, Bulletin
for the Chemical Society of Japan, Chemistry—An
Asian Journal, International Journal of Oncology.
Boger, D.L.—Nieuwland Lecturer, Notre Dame Univer-
Rebek, J., Jr.—Ta-shue Chou Award, Academia Sinica,
sity, Norte Dame, Indiana; Wyeth Lecturer, University of
Strathclyde, Glasgow, Scotland; Editor-in-Chief, Bioorganic
and Medicinal Chemistry Letters; Editorial Boards, Tetrahedron Publications, Organic Reactions, Current Opinion
in Drug Discovery and Development, Current Drugs.
Taiwan; Joullie Lecturer, University of Pennsylvania,
Philadelphia, Pennsylvania; Marker Lecture, University
of Maryland, College Park, Maryland; Member, Wittgenstein Prize Committee; Editorial Boards, Chemistry and
Biology, Current Opinion in Chemistry and Biology,
Journal of Supramolecular Chemistry.
Chemistry Section.
Beutler, E.— First Annual Wallace H. Coulter Award for
Fedor, M.J.— Chair, Macromolecular Structure and Func-
tion Study Section E, National Institutes of Health; Associate Editor, Journal of Biological Chemistry; Editorial
Board, RNA.
Janda, K.D.— Section Head, Faculty in Chemical Biol-
ogy, Faculty of 1000, Biology Reports, Ltd.; American
Regional Editor, Bioorganic and Medicinal Chemistry;
Editorial Boards, Chemical Reviews, Journal of Medicinal Chemistry, Combinatorial Chemistry Research
and Applications, Bioorganic and Medicinal Chemistry Letters, Combinatorial Chemistry High-Throughput Screening.
Joyce, G.F.— Member, National Academy of Sciences;
Member, Committee on International Security and Arms
Control, National Academy of Sciences; Member, External Advisory Board, Beckman Institute, California Institute of Technology, Pasadena, California; Head, Faculty
in Chemical Biology, Faculty of 1000, Biology Reports,
Ltd.; Associate Editor, Evolutionary Computation, Origins of Life and Evolution of the Biosphere.
Nicolaou, K.C.— Award of Excellence, Western Regional
Sharpless, K.B.— Honorary Doctorate, Hong Kong Uni-
versity of Science and Technology, Hong Kong, China;
Nobel Laureate Lectures, Griffith University, Brisbane,
Australia; Editorial Boards, Advanced Synthesis and
Catalysis, Arkivoc, Beilstein Journal of Organic Chemistry, Bulletin of the Chemical Society of Japan, Chemistry—An Asian Journal, Chirality, Current Opinion in
Drug Discovery and Development, Current Drug Discovery Technologies, Enantiomer, Organic Letters,
Synlett, Topics in Stereochemistry.
Williamson, J.R.—Associate Editor, Annual Reviews of
Biophysics and Biomolecular Structure; Editorial
Boards, ACS Chemical Biology, RNA, Molecular Cell,
Chemistry and Biology, Structure.
Wilson, I.A.— Honorary Doctorate of Science, University
of St. Andrews, Scotland; Fellow, Royal Society of London; Corresponding Fellow, Royal Society of Edinburgh;
Member, American Academy of Arts and Sciences; Member, Board of Directors, Keystone Symposia; Associate
62
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Editor, Journal of Molecular Biology, Immunity; Editorial Boards, Science, Journal of Experimental Medicine.
Wong, C.-H.— F.A. Cotton Medal for Excellence in Chem-
ical Research, American Chemical Society; Scientific
Advisor, Max-Planck-Institute, Dortmund, Germany;
Editor-in-Chief, Bioorganic and Medicinal Chemistry;
Chair, Executive Board of Editors, Tetrahedron Publications; Editorial Boards, Current Opinion in Chemical
Biology, Biocatalysis, Advanced Synthesis and Catalysis.
Wright, P.E.— Leach Medal, Lorne Conference on Pro-
tein Structure and Function, Lorne, Australia; Fellow,
International Society of Magnetic Resonance; Member,
National Academy of Sciences; Editor-in-Chief, Journal
of Molecular Biology; Editorial Boards, Biochemistry,
Current Opinion in Structural Biology, Journal of Biomolecular NMR.
Wüthrich, K.— Doctor of Medicine honoris causa, Univer-
sity of Pécs, Hungary; Doctor of Chemistry honoris
causa, Universidad del Norte, Asunción, Paraguay;
Doctor honoris causa, Lomonosov Moscow State University, Moscow, Russia; Laurea Specialistica in Biotechnology honoris causa, University of Verona, Italy; Docteur
honoris causa, Université René Descartes, Paris, France;
Irving L. Schwartz Lecture, Mount Sinai School of Medicine, New York, New York; Aline U. and James M. Orten
Memorial Lecture, Wayne State University, Detroit,
Michigan; Philip Handler Lecture, Duke University
Medical Center, Durham, North Carolina; Editor-in-Chief,
Journal of Biomolecular NMR; Associate Editor, Advanced
Science Letters; Editorial Boards, Biochimie, Biomolecular NMR Assignments, Biopolymers, ChemBioChem,
Chemical Physics Letters, Current Opinion in Structural
Biology, IUBMB Life, Journal of Magnetic Resonance,
Journal of Membrane Biology, Journal of Structural and
Functional Genomics, Proteins, Structure.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
AUTHOR INDEX
Boldface entries indicate
principal investigators who
are members of the Skaggs
Institute. The other authors
are colleagues and support
staff who work closely with
the members.
Adams, M.A. 51
Agnelli, F. 50
Agua, A. 38
Ajami, D. 40
Albertshofer, K. 11
Aller, S. 17
Ams, M. 40
Anderson, E. 15
Anderson, W. 50
Ando, Y. 15
Angrish, D. 48
Arai, M. 56
Arvai, A.S. 24, 46
Ashley, J. 27
Atkins, A. 18
Aversa, R. 38
Bachovchin, D. 17
Bae, S.-H. 56
Baksh, M. 22
Banerjee, D. 22
Barbas, C.F. III 11
Barrett, E. 40
Barros, C. 37
Bartfai, T. 13
Beck, A. 50
Beer, S. 40
Beierle, J.M. 26
Belani, R. 41
Bell, C.H. 51
Bennett, C. 55
Ben-Shir, I. 31
Beuck, C. 50
Beutler, E. 14
Blankman, J. 17
Boehr, D. 56
Boger, D.L. 15
Boyle, K. 15
Brenzovich, W. 38
Brustad, E.M. 42
Buck-Koehntop, B. 56
Bui, T. 11
Bunner, A. 50
Burke, C. 15
Capková, K. 27
Cardoso, R.M.F. 51
Carmel, A. 50
Chamero, P. 45
Chang, G. 17
Chapados, B.R. 46
Chavochi, A. 26
Chen, J. 38
Chen, S. 50
Chen, Y. 17
Choi, S. 33
Chong, Y. 41
Clark, R. 15
Colby, D. 15
Connelly, S. 51
Conti, B. 13
Conti, F. 37
Cottrell, J.W. 21
Crane, C. 15
Cravatt, B.F. 17
Culhane, J. 43
Culyba, E. 33
Dalby, S. 38
Dale, T.J. 40
De Lamo Marin, S. 27
Dean, S. 55
Deka, P. 56
DeMartino, J. 15
Dendle, M.T.A. 33
Denery, J. 27
Di Mola, A. 27
Dickerson, T.J. 27
Dix, M. 17
Droese, B.J. 51
Druzina, Z. 41
Du, D. 33
Dube, H. 40
Dubrovskaya, V. 48
Duncan, K. 15
Dyson, H.J. 56
Edelman, G.M. 18
Edgcomb, S. 50
Edmonds, D. 38
Ekiert, D.C. 51
Elledge, H. 37
Ellery, S. 38
Ellis, B. 27
Elsliger, M.-A. 51
Eschenmoser, A. 20
Estrada, A. 38
Eubanks, L. 27
Ezzili, C. 15
Fan, L. 46
Fearns, C. 33
Fedor, M.J. 21
Felitsky, D. 56
Ferreon, J. 56
Ficht, S. 55
Fiedler, J. 22
Finn, M.G. 22
Flanagan, K. 45
Fokin, V.V. 43
Fotsing, J. 43
Fraga, B. 38
Franco, S. 37
Frater, J. 41
Frederick, M. 38
Freestone, M. 38
Fu, Y. 55
Fukuchi, K. 27
Fuller, A.A. 33
Fuller, R.P. 11
Fulton, Z. 51
Gaj, T. 11
Garcin, E.D. 24
Garfunkle, J. 15
Gavrilyuk, J. 11
Ge, H. 15
Gelin, C. 38
Gersbach, C. 11
Gershoni, R. 31
Getzoff, E.D. 24
Ghadiri, M.R. 26
Gondi, V. 38
Gonzalez, B. 11
Goodwin-Tindall, J. 38
Gordley, R.M. 11
Grecian, S. 43
Greenberg, W. 55
Grillet, N. 37
Grimster, N. 43
Grover, R.K. 48
Grünewald, J. 42
Guo, J. 11
Guo, J. 42
Guo, M. 41
Guo, R.-T. 41
Guy, R. 55
Haberz, P. 56
Han, B.W. 51
Hanan, M. 41
Hanson, S. 55
Harkins-Perry, S. 37
He, W. 41
He, X. 17
Hein, J. 43
Hernandez, C. 27
Hesse, M. 38
Hitomi, C. 24, 46
Hitomi, K. 24, 46
Hochstatter, D. 15
Hong, M. 51
Hong, V. 22
Hong, Z. 55
Hooley, R.J. 40
Horneff, T. 43
Horst, R. 59
Hou, J.-L. 40
63
Huang, P. 38
Huang, Z. 38
Hwang, D.-R. 55
Hwang, I. 15
Jager, M. 36
Janda, K.D. 27
Jeso, V. 38
Ji, S. 17
Jimenez-Dalmaroni, M.J. 51
Jin, J. 38
Jones, R. 15
Joyce, G.F. 30
Jung, I.L. 41
Juraja, S. 11
Kakei, H. 15
Kalisiak, J. 43
Kamioka, S. 40
Kapoor, M. 41
Kar, M. 38
Karmakar, A. 31
Karyakin, A. 17
Kato, D. 15
Kaufmann, G. 27
Kaur, A. 45
Kazmierczak, P. 37
Keinan, E. 31
Kelly, J.W. 33
Kerkow, D. 50
Kim, D.H. 11
Kimball, F.S. 15
Kirchdoerfer, R.N. 51
Kislukhin, A. 22
Klein, I. 13
Koh, D.C.Y. 18
Kondreddi, R.R. 20
Kopp, F. 17
Korthals, K. 43
Kossoy, E. 31
Krasovskiy, A. 38
Krishnamurthy, R. 20
Kroeger, M.D. 24
Kwan, S. 50
Kwok, S.-W. 43
Lajiness, J. 15
Lam, B.J. 30
Landes, M. 56
Lee, C.W. 56
Lee, H.S. 42
Lee, J.-C. 55
Lee, P. 36
Lee, S. 15
Lee, S.H. 41
Leman, L. 26
Lemire, A. 38
Levin, R. 38
Li, A. 38
Li, H. 38
64
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
Li, L. 21
Li, W. 17
Liang, P.-H. 55
Lieu, S. 17
Lim, Y. 38
Lincoln, T.A. 30
Lister, T. 38
Liu, C.C. 42
Liu, J. 41
Liu, L. 21
Lledo, A. 40
Logan, D. 45
Long, J. 17
Lowery, C. 27
MacMillan, K. 15
Mahajan, S. 27
Majumder, U. 38
Manlapaz, E. 56
Manuell, A. 36
Marin-Navarro, J. 36
Martin, B. 17
Martin, T. 45
Martinez-Garay, I. 37
Martinez-Yamout, M. 56
Masuda, K. 17
Mathison, C. 38
Matsuda, D. 18
Mayfield, S.P. 36
Mayorov, A. 27
McElhaney, G. 27
McKinney, M. 17
Mee, J. 27
Meinhold, D. 56
Mejuch, T. 31
Menicelli, E. 50
Mercer, A. 11
Merriman, E. 41
Metanis, N. 31
Mittapalli, G.K. 20
Mizuta, S. 11
Mohanty, B. 59
Montero, A. 26
Moreno, A. 27
Morgan, A. 38
Mu, T.-W. 33
Müller, U. 37
Murray, A. 33
Muto, M. 36
Naidu, V.S. 20
Nakai, Y. 27
Nawaz, M.H. 41
Nguyen, A. 27
Nguyen, T. 17
Nicolaou, K.C. 38
Nold, A. 38
Nomura, D. 17
Nunes, A. 27
Olsen, C.A. 26
Ong, D. 33
Onoda, A. 11
Ortiz, A. 38
Osborn, O. 13
Osornio, Y. 20
Otrubova, K. 15
Paegel, B.M. 30
Panopoulos, P. 18
Papes, F. 45
Pappo, D. 31
Park, J. 27
Park, S.-J. 56
Parvari, G. 31
Patel, P. 15
Patil, N. 38
Paulsson, J. 33
Payne, R. 55
Pedrini, B. 59
Pejchal, R. 51
Peram, M.M.R. 48
Perry, J.J. 46
Peters, F.B. 42
Petrie, K.L. 30
Pettersson, P. 36
Pilotte, J. 18
Pique, M.E. 24, 46
Piran, R. 31
Pitram, S. 43
Porter, G.P. 51
Powers, E.T. 33
Pratt, B. 38
Radakovits, R. 37
Ramos, C. 37
Rao, P. 33
Rasala, B. 36
Ratner, T. 31
Raushel, J. 43
Reany, O. 31
Rebek, J., Jr. 40
Reingruber, R. 38
Restorp, P. 40
Revin, M. 17
Reynolds, A. 37
Ridgeway, W. 50
Ring, G. 50
Rivas, F. 38
Robertson, W. 15
Rodriguez, R. 15
Rogel, J. 48
Rohrbach, A. 27
Saccavini, C. 27
Salahuddin, S. 11
Salzameda, N. 27
Sanchez Ruiz, A. 38
Sanchez-Alavez, M. 13
Santa Marta, M. 11
Sarlah, D. 38
Sasaki, Y. 15
Saure, M. 33
Schelwies, M. 55
Schiefner, A. 51
Schimmel, P. 41
Schnermann, M. 15
Schultheisz, H. 50
Schultz, P.G. 42
Schwander, M. 37
Schwimmer, L.J. 11
Scott, L.G. 50
Serrano, P. 59
Seto, S. 15
Shajani, Z. 50
Shapiro, R. 41
Sharpless, K.B. 43
Shaw, D. 38
Shekhter, T. 31
Shimamura, H. 15
Shin, D.S. 24, 46
Shoshani, S. 31
Siegel, S. 33
Simkovsky, R. 33
Simon, G. 17
Sinha, M. 31
Slown, C. 15
Solel, E. 31
Solomon, J. 33
Song, B.D. 48
Song, Y.Z. 41
Soreni, M. 31
Sperling, E. 50
Stamm, S. 15
Stanfield, R.L. 51
Stefanko, R.S. 51
Steiniger, S. 27
Stepan, A. 38
Stover, J. 15
Stowers, L. 45
Stump, B. 43
Swingle, D. 15
Sykes, M. 50
Szewczyk, P. 17
Szymczyna, B. 50
Tabarean, I. 13
Tainer, J.A. 46
Takizawa, S. 15
Talbot, A. 38
Tam, A. 15
Tam, O. 21
Tanaka, F. 11
Tang, Y. 38
Thomas, J. 17
Tran, M. 36
Trépanier, V. 38
Treweek, J.B. 27
Tria, G. 38
Tripp, J. 43
Troseth, R.P. 48
Tubbs, J.L. 24, 46
Tully, S. 17
Udit, A. 22
Uehara, H. 11
Umezawa, T. 38
Usui, K. 33
Utsumi, N. 11
Va, P. 15
Valdez, C. 43
Van Anda, H. 40
Vanhnasy, J.A. 51
Vo, M.-N. 41
Wang, J. 38
Wang, S.-K. 55
Wang, Y. 33
Ward, A. 17
Watson, P. 21
Webb, S. 37
Weerapana, E. 17
Weide, T. 43
Wentworth, A.D. 48
Wentworth, P., Jr. 48
Whitby, L. 15
Williams, R.S. 46
Williamson, J.R. 50
Willis, A. 27
Wilson, I.A. 51
Wolfe, A. 15
Wong, C.-H. 55
Wright, P.E. 56
Wu, J. 50
Wu, T. 38
Wuellner, U. 11
Wüthrich, K. 59
Xiao, S. 40
Xie, J. 15
Xiong, W. 37
Xu, R. 51
Xu, X. 51
Xu, Y. 27
Yang, X.-L. 41
Yi, K.S. 11
Yoneda, Y. 27
Yonemoto, I. 33
Yoon, S.I. 51
Young, T.S. 42
Yu, J. 17
Yu, Z. 33
Zhan, W. 38
Zhang, H. 11
Zhang, H. 38
Zhang, W. 41
Zhou, B. 27
Zhou, H. 27
Zhou, Q. 41
Zhu, X. 51
Zimmerman, S. 21
Zuhl, A. 15
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
SUBJECT INDEX
Aging 42
Algae 36
Amylin 35
Amyloid β-peptides 14
Amyloidogenesis 35
Amyloidoses 49
Anchored plasticity 25
Anemia 14
Antibiotics 16, 38, 44
Antibodies
chemically programmed 12
Anticoagulants 23
Antitumor agents 38
Ataxia telangiectasia–like
disorder 47
Autoinducers 28
Base J 48
Biofuels 36
Biomolecular computing 32
Cancer 12
Capsids 33
Catalytic antibodies 11
in transgenic plants 32
Cavitands 41
Chloroplasts 36
Click chemistry 22, 43
Cockayne syndrome 47
Cryptochromes 25
Cytokines 13
Deafness 37
Diabetes 13, 35
Directed evolution 30
DNA damage 14
DNA repair 46
DNA replication 14
DNA-binding agents 15
DNA-binding proteins 57
Drug design 51
Drug discovery 43
Drug resistance 44
Encapsulation 40
Endocannabinoids 17
Enzymatic synthesis 50
Enzymes
synthetic 31
Fatty acid amide hydrolase 18
inhibitors of 15
Gaucher disease 34
Gene regulation 12
Genetic code 42
Glycobiology 55
Glycoproteins 55
Glycoproteomics 55
Hair cells 37
Hepcidin 14
Histone-deacetylase inhibitors 26
HIV vaccine 51
Hypoxia 57
Inflammation 13
Influenza virus 52
RNA structure 50
RNA-binding proteins 19
Self-assembly 40
Sensory neurons 45
Signaling 27
Innate behavior 45
Innate immunity 53
Iron metabolism 14
Leishmaniasis 48
Light-chain aggregation 49
Lysosomal storage diseases 34
Mechanotransduction 37
Metabolic engineering 50
Microfluidics 30
Mistranslation 41
Molecular mimicry 40
Mouse models
of deafness 37
Multidrug resistance 16, 17
Multidrug transporters 17
Natural products 38
Neural development 18
Neurodegeneration 13
Neurotoxins 38
Neutralizing antibodies
for HIV 51
for influenza virus 52
Nijmegen breakage syndrome 47
Nitric oxide synthases 24, 46
Nuclear magnetic resonance 56
Nucleic acids
chemical etiology of 20
structure of 20
Obesity 13
Olfaction 45
Oncogenesis 42
Organocatalysis 11
Pain 8, 15
Pheromones 45
Photolyases 25
Protein structure 24, 46, 56
automated NMR determination
of 59
Protein-misfolding diseases 33
Proteomics 18
Proteostasis 34
Quorum sensing 27
Ramoplanin 16
Receptors
CD1 54
Toll-like 53
variable lymphocyte 53
Ribosomes 19
Ribozymes 21
RNA 18
RNA enzymes 30
RNA folding 21
Structure-based design 26
Sugar-assisted ligation 55
Therapeutic proteins 36
Transcription 57
Transcription factors 12
Translation 19
Transthyretin 14, 34
tRNA synthetases 41
Turn mimetics 26
Type IV pilin 46
Unnatural amino acids 42
Usher syndrome 37
Vancomycin 16
Virus particles 22
X-ray crystallography 51
Yatakemycin 16
Zinc fingers 12, 58
65
66
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2008
ACKNOWLEDGMENTS
The scientists who have contributed sections to this report
wish to acknowledge the dedication and hard work of the
laboratory technicians who helped bring the research to
fruition, the administrative assistants who made it presentable for publication, and the support personnel who
provided critical specialized services and equipment.
EDITOR
Barbara L. Halliburton, Ph.D.
PROJECT MANAGER
Jann Coury
Office of Communications
P R I N T I N G A N D D U P L I C AT I O N
Precision Litho
Maryland Composition
PHOTOGRAPHY
Michael Balderas
Biomedical Graphics Department, Scripps Research
Mark Dastrup
D E PA R T M E N TA L C O O R D I N AT I O N
Janette Lundgren Beas
The Skaggs Institute for Chemical Biology
Ruby Santos
Department of Molecular Biology
Marcia McRae
Molecular and Integrative Sciences Department
Cheryl Negus
Department of Cell Biology
Department of Chemical Physiology
Vicky Nielsen
Department of Chemistry
Lynn Oleski
Department of Molecular and Experimental Medicine
Michelle Platero
Department of Neurobiology
The Skaggs Institute for Chemical Biology scientific report is
published annually by The Scripps Research Institute and is
available on request from
Office of Communications
TPC-30
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