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 3
O n t h e f r o n t c o v e r : Acetylcholinesterase, a crucial enzyme in the cholinergic
signal transduction pathways in muscles and the brain and a well-established
therapeutic target. In a novel approach termed in situ click chemistry, the enzyme
preferentially assembles 1 pair of the reactants, each of which bears a group that
binds to adjacent positions on the protein structure (the choline-binding site of the
active center and the peripheral site), into a triazole adduct that is the most potent
noncovalent inhibitor of the enzyme yet developed. This approach is more than a
novel way to design drugs with exquisite selectivity for the native biological target and
hence fewer unwanted side effects; it also provides unique insights into protein
flexibility and how enzymes, as dynamic catalysts, truly function as fluctuating,
breathing structures and not as rigid templates. This work was carried out in the
laboratories of K. Barry Sharpless, Ph.D. Image by Flavio Grynszpan, Ph.D., and
Steven Lustig, BioDesign Communications.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY
In 1996, The Scripps Research Institute established The Skaggs Institute for Chemical
Biology, made possible by a gift of more than $100 million to The Skaggs Institute for Research
from Aline W. and L.S. Skaggs.
Scientific members of the Skaggs Institute hold dual appointments in various departments
at TSRI. These scientists have broad expertise in areas including the structure of biological
macromolecules, chemical and antibody catalysis, synthetic and combinatorial chemistry,
molecular recognition, and molecular modeling methods. With the achievements of its staff, the
Skaggs Institute has assumed its research identity in the United States and throughout the
world at the interface of biology and chemistry.
2
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
MEMBERS OF THE SKAGGS INSTITUTE
Back row, left to right
Seated, Middle row, left to right
Front row, left to right
Not present
Martha J. Fedor, Ph.D.
Ernest Beutler, M.D.
Elizabeth D. Getzoff, Ph.D.
Geoffrey Chang, Ph.D.
Ian A. Wilson, D.Phil.
Peter E. Wright, Ph.D.
Dale L. Boger, Ph.D.
Ehud Keinan, Ph.D.
Jeffery W. Kelly, Ph.D.
Kim D. Janda, Ph.D.
Erik Sorensen, Ph.D.*
Richard A. Lerner, M.D.
Carlos F. Barbas III, Ph.D.
Gerald M. Edelman, M.D., Ph.D.
Benjamin F. Cravatt, Ph.D.
Peter Schultz, Ph.D.
Stephen P. Mayfield, Ph.D.
Gerald F. Joyce, M.D., Ph.D.
K. Barry Sharpless, Ph.D.
Paul R. Schimmel, Ph.D.
Julius Rebek, Jr., Ph.D.
Chi-Huey Wong, Ph.D.
Subhash C. Sinha, Ph.D.
K.C. Nicolaou, Ph.D.
M. Reza Ghadiri, Ph.D.
Kurt Wüthrich, Ph.D.
James R. Williamson, Ph.D.
Albert Eschenmoser, Ph.D.
John A. Tainer, Ph.D.
Philip Dawson, Ph.D.
M.G. Finn, Ph.D.
* Appointment completed
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
With a contribution from the Skaggs family, TSRI 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 TSRI scientists. A broader goal is
to expand the body of knowledge related to human disease and to develop effective therapeutic interventions.
THE SKAGGS SCHOLARS
Standing, Back row, left to right
Seated, Front row, left to right
Not present
Huan B. Giap, M.D.
Alan Saven, M.D.
Faith H. Barnett, M.D., Ph.D.
George E. Dailey, M.D.
James R. Mason, M.D.
John F. Bastian, M.D.
Daniel A. Nachtsheim, M.D.
Prabhakar Tripuraneni, M.D.
Sheila F. Friedlander, M.D.
Donald D. Stevenson, M.D.
Michael P. Kosty, M.D.
Merlin L. Hamer, M.D.
Robert J. Russo, M.D., Ph.D.
Katharine M. Woessner, M.D.
Mary A. Kalafut, M.D.
Darlene J. Elias, M.D.
Williamson B. Strum, M.D.
David A. Mathison, M.D.
Jack C. Sipe, M.D.
Richard A. Smith, M.D.
Jorge J. Nieva, M.D.
Ken D. Pischel, M.D.
Clifford W. Colwell, Jr., M.D.
Paul S. Phillips, M.D.
Steven J. Poceta, M.D.
Paul J. Pockros, M.D.
Ronald A. Simon, M.D.
3
VOLUME 8
PRESIDENT’S INTRODUCTION
7
DIRECTOR’S OVERVIEW
9
I N T R A C E L L U L A R R N A A S S E M B LY A N D
C ATA LY S I S
24
M.J. Fedor, K.F. Baban, C.P. Da Costa, Y.I. Kuzmin, E.M. Mahen,
S.B. Voytek, R.S. Yadava
S TA F F
11
VIRUSES AS MOLECULAR BUILDING BLOCKS
I N V E S T I G AT O R S ’ R E P O R T S
25
M.G. Finn, D.D. Díaz, W.G. Lewis, J.-C. Meng, S. Meunier,
C A N C E R , A I D S , C ATA LY S I S , A N D T H E R E G U L AT I O N
D. Prasuhn, S. Punna, K.S. Raja, Q. Wang
OF GENES: INVENTING MOLECULES WITH DEFINED
FUNCTIONS
13
C.F. Barbas III, R. Lerner, P. Blancafort, N.S. Chowdari, R. Fuller,
T. Gräslund, N. Jendreyko, C. Lund, L. Magnenat, N. Mase,
M. Popkov, C. Rader, D.B. Ramachary, S. Sinha, H. Shim, F. Tanaka,
R. Thayumanavan, J. Turner
T R A I N I N G I N M O L E C U L A R A N D E X P E R I M E N TA L
MEDICINE
INSIGHTS INTO PROTEIN CHEMISTRY AND
BIOLOGY FROM PROTEIN STRUCTURE
27
E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau, R.M. Brudler,
J.M. Castagnetto, D. Cerutti, T. Cross, M. DiDonato, E.D. Garcin,
U.K. Genick, S.W. Hennessy, C. Hitomi, K. Hitomi, C.J. Kassmann,
I. Li, S.J. Lloyd, M.E. Pique, R.J. Rosenfeld, M.E. Stroupe,
M.M. Thayer, M.J. Thompson, J.L. Tubbs, T.I. Wood
16
SUPRAMOLECULAR APPROACHES TO DRUG DESIGN,
E. Beutler
CELL DIAGNOSTICS, AND COMPLEX SYSTEM
ENGINEERING
SYNTHETIC, MEDICINAL, AND BIOORGANIC
CHEMISTRY
18
D.L. Boger, Y. Ambroise, B. Blagg, S. Buck, K. Capps, H. Cheng,
Y. Choi, B. Crowley, P. Desai, J. Desharnais, R. Dominique, W. Du,
R. Ducray, G. Elliott, R. Fecik, Y. Ham, N. Haq, C. Hardouin,
I. Hwang, S. Ichikawa, D. Kastrinsky, G. Kim, P. Krenitsky, M. Lall,
C. McComas, Y. Mori, J. Parrish, S. Pfeiffer, A. Pichota, Y. Rew,
D. Shin, D. Soenen, J. Trzupek, W. Tse, J. Wanner, G. Wilkie,
S. Wolkenberg, Z. Yuan, B. Yeung, J. Zimpleman
STRUCTURAL BIOLOGY OF MEMBRANE-BOUND
TRANSPORTERS AND RECEPTORS
20
A. Ma, T. Nguyen, P. Szewczyk, Y. Yin, C. Reyes, S. Wada, O. Pornillos,
M. Grant, G. Chang
29
M.R. Ghadiri, M. Al-Sayah, G. Ashkenasy, A. Chavochi, J. Fletcher,
V. Haridas, W.S. Horne, R. Jagasia, L. Leman, S. Rahimipour,
M. Yadav
N E W V I S TA S I N I M M U N O P H A R M A C O T H E R A P Y
30
K.D. Janda, J.-M. Ahn, J. Ashley, G. Boldt, R. Carrera, Y. Chen,
B. Clapham, A. Coyle, T. Dickerson, Y. Ding, L. Eubanks, T. Fujimori,
R. Galve, C. Gambs, C. Gao, T. Hoffman, L. Hom, G. Kaufmann,
Y.-S. Kim, E. Laxman, B.-S. Lee, S.-H. Lee, S.-J. Lee, M. Lillo,
S. Mahajan, H. Matsushita, M. Matsushita, J. McDunn, G. McElhaney,
J. Mee, M. Meijler, J. Moss, J.-I. Park, N. Reed, A. Shafton, C. Sun,
R. Troseth, A.D. Wentworth, P. Wentworth, Jr., P. Wirsching, Y. Xu,
N. Yamamoto, K. Yoshida, B. Zhou
C ATA LY T I C N U C L E I C A C I D S F O R T R E AT I N G T H E
CHEMICAL PHYSIOLOGY
20
MOLECULAR BASIS OF DISEASE
B.F. Cravatt, G.C. Adam, K.T. Barglow, M.H. Bracey, K. Chiang,
M. Evans, G. Hawkin, M. Humphrey, N. Jessani, A. Joseph,
D. Leung, K. Masuda, M. McKinney, A. Saghatelian, A. Speers
33
G.F. Joyce, J.T. Hillman, R.M. Kumar, N. Paul, W.M. Shih
B I O M O L E C U L A R C O M P U T I N G , C ATA LY T I C
SYNTHETIC PROTEIN CHEMISTRY
21
P. Dawson, R. Balambika, J. Blankenship, M. Churchill, J. Offer,
C. Neidre, F. Topert
SYNTHETIC ENZYMES
22
M E TA B O L I T E - I N I T I AT E D P R O T E I N M I S F O L D I N G
G.M. Edelman, S. Aschrafi, A. Atkins, S. Chappell, G.W. Rogers,
F. Smart, T. Stevens, M. Tsatmali, W. Zhou
23
A. Eschenmoser, R. Krishnamurthy, Z. Wang, G.R. Vavilala, C. Mang,
V. Rajwanshi, J. Nandy, T. George
Published by TSRI Press ®.
All rights reserved.
©
Copyright 2003, The Scripps Research Institute.
36
J.W. Kelly, S. Deechongkit, M.A. Dendle, T. Foss, D. Fowler,
K. Frankenfeld, N. Green, A. Hurshman, M.B. Huff, S. Johnson,
H.-J. Lim, E. Powers, A. Sawkar, Y. Sekijima, S. Werner, L. Wiseman,
S.-L. You, Q. Zhang
CHEMICAL ETIOLOGY OF THE STRUCTURE OF
NUCLEIC ACIDS
34
E. Keinan, H.C. Lo, H. Han, S. Sasmal, M. Arifuddin, A. Brik,
S. Saphier, G. Sklute, N. Metanis, M. Soreni, D. Vebenov, L. Kosoy,
M.K. Sinha, A. Alt, I. Ben-Shir, R. Piran, P.E. Dawson
F U N D A M E N TA L P R O C E S S E S I N N E U R A L
DEVELOPMENT
ANTIBODIES, ORGANIC SYNTHESIS, AND
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
EXPRESSION OF THERAPEUTIC PROTEINS IN
EUKARYOTIC ALGAE
MACROMOLECULAR MACHINES AS MASTER KEYS
38
S.P. Mayfield, E. Brown, M. Beligni, A. Manuell, K. Yamaguchi,
S.E. Franklin
I N G E N O M E I N T E G R I T Y, T H E C E L L C Y C L E ,
PCONTROL OF REACTIVE OXYGEN SPECIES,
A N D PAT H O G E N E S I S
48
J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras, R. Brudler,
CHEMICAL SYNTHESIS AND CHEMICAL
BIOLOGY
R.M. Cardoso, J.M. Castagnetto, B.R. Chapados, C. Chahwan, L. Craig,
38
K.C. Nicolaou, R. Baati, T. Bando, M. Bella, F. Bernal, W. Brenzovich,
S. Bulat, D. Chen, J. Chen, E. Couladouros, P. Dagneau, P. Diamandis,
A. Estrada, M. Follmann, T. Francis, M. Frederick, L. Gomez Paloma,
M. Govindasamy, D. Gray, P. Guntupalli, S. Harrison, J. Hao, X. Huang,
R. Hughes, M. Jennings, F. Kaiser, D. Kim, T. Koftis, S. Lee, Y. Li,
T. Ling, D. Longbottom, F. Marr, C. Mathison, T. Montagnon,
M. Nevalainen, A. Nalbandian, R. Noronha, . Paraselli, B. Pratt,
W. Qian, G. Rassias, M. Reddy, M. Rodriguez, A. Roecker, B. Safina,
P. Sasmal, D. Seigel, S. Snyder, X. Sun, G. Vasilikogiannakis, S. Vyskocil,
H. Xu, Y. Yamada, M. Zak
D.S. Daniels, M. DiDonato, G. Divita, L. Fan, S. Han, C. Hitomi,
K. Hitomi, J.L. Huffman, K.-P. Hopfner, D. Hosfield, C.D. Mol,
G. Moncalian, S.S. Parikh, C.D. Putnam, D.S. Shin, M.E. Stroupe,
O. Sundheim, T.I. Wood, A. Yamagata
TA R G E T I N G H I V R N A
50
J.R. Williamson, A. Bunner, J. Chao, S. Edgcomb, P.M. Funke,
M. Hennig, E. Johnson, K.A. Lehmann, P.K. Radha, M. Recht,
S.P. Ryder, L.G. Scott, E. Sperling, M.W. Trevathan
C R Y S TA L L O G R A P H I C S T U D I E S O F I M M U N E
M O L E C U L A R R E C O G N I T I O N A N D E N C A P S U L AT I O N
40
J. Rebek, Jr., T. Amaya, P. Ballester,* S. Biros, W.-D. Cho, T. Dale,
J. Friese, A. Gissot, S. Gu, F. Hof, D.W. Johnson, L. Kröck, Y. Kim,
H. Onagi, L. Palmer, B. Purse, D. Rachavi-Robinson, A. Scarso,
A. Shivanyuk, L. Trembleau, E. Ullrich, M. Yamanaka
42
K. Ewalt, J. Liu, M. Lovato, D. Metzgar, C. Myers, L. Nangle,
B. Nordin, F. Otero, J. Reader, L. Ribas de Pouplana, B. Slike,
M. Swairjo, K. Tamura, W. Waas, X. Yang
CLICK CHEMISTRY AND THE SEARCH FOR
45
K.B. Sharpless, Y. Baba, S. Boral, J. Cappiello, T. Chan, A. Converso,
M. David, A. Feldman, V. Fokin, R. Hilgraf, P. Holzer, C. Kappe,
T. Kitayama, A. Krasinski, I. Lewis, V. Litosh, R. Manetsch,
A. McPherson, J. Muldoon, S. Narayan, V. Rostovtsev, S. Silverman,
N. Stuhr-Hansen, E. Van der Eycken, P. Wu, Y. Xie
A N T I B O D Y C ATA LY S I S , O R G A N I C S Y N T H E S I S ,
A N D P R O D R U G A N D TA R G E T I N G T H E R A P I E S
S.C. Sinha, R.A. Lerner, L.-S. Li, S. Das, S. Dutta
55
44
P.G. Schultz, J.C. Anderson, M. Bose, C. Cho, J. Gildersleeve, J. Hong,
Q. Huang, J. Liu, K.H. Min, M. Mukherji, S. Santoro, F. Tian, L. Wang,
P. Yang, J. Yin, C. Yu, Z. Zhang, L.X. Zheng
BIOLOGICAL ACTIVITY
DRUG DISCOVERY
C.-H. Wong, F. Agnelli, C. Behrens, M. Best, A. Brik, M.C. Bryan,
A. Buchnyskyy, G. Cain, A. Chang, W.C. Cheng, A. Datta, S. Duron,
F. Fazio, D. Franke, J.I. Furukawa, M.C. Galan, S. Hanson, Z.-Y. Hong,
J. Hsu, C.-Y. Huang, H.K. Lee, L. Lee, F.-S. Liang, D. Lin, H. Liu,
J. Liu, M. Numa, P. Nyffeler, T. Polat, D. Thayer, S.K. Wang, G. Watt,
D. Wu, Y.Y. Yang, N. Yu, G.-W. Xing, R. Xu
P. Schimmel, J. Bacher, A. Bates, K. Beebe, V. de Crécy-Lagard,
EXPANDING THE GENETIC CODE
51
I.A. Wilson, D.A. Calarese, R.M.F. Cardoso, C.-G. Cheong, A.L. Corper,
M.D.M. Crispin, M.-A. Elsliger, S. Ferguson, A. Gakhal, P.A. Horton,
S. Ito, N.A. Larsen, J.G. Luz, E. Ollmann Saphire, J.B. Reiser,
R. Sanders, D.A. Shore, R.L. Stanfield, R.S. Stefanko, J. Stevens,
P. Verdino, D.W. Wolan, D. Wu, L. Xu, D.M. Zajonc, Y. Zhang, X. Zhu
BIOORGANIC CHEMISTRY AND
POTENTIAL MEDICAL CONSEQUENCES OF
AMBIGUITY IN THE GENETIC CODE
R E C O G N I T I O N A N D T H E R A P E U T I C TA R G E T S
46
STUDIES OF MACROMOLECULAR RECOGNITION
B Y M U LT I D I M E N S I O N A L N U C L E A R M A G N E T I C
RESONANCE
55
P.E. Wright, H.J. Dyson, M. Martinez-Yamout, J. Covalt, R. De Guzman,
T. Dunzendorfer-Matt, N. Goto, B. Hudson, J. Lansing, T. Nishikawa,
G. Perez-Alvarado, J. Wojciak, T. Zor, J. Hosea, M. Landes
S TA F F AWA R D S A N D A C T I V I T E S
58
SUBJECT INDEX
60
5
6
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
THE SKAGGS INSTITUTE
THE SCRIPPS
THE SCRIPPS
FOR RESEARCH
RESEARCH INSTITUTE
RESEARCH INSTITUTE
BOARD OF TRUSTEES
BOARD OF TRUSTEES
OFFICERS
Susie Balukoff
The Hon. Alice D. Sullivan
(Ret.)
Chair
Paul L. Herrling, Ph.D.
Vincent E. Benstead
Thomas H. Insley
Mrs. William McCormick
Blair, Jr.
Janet R. Kellogg
Ernest Beutler, M.D.
The Most Reverend Robert
H. Brom
Hubert T. Greenway, Jr.,
M.D.
Lawrence C. Horowitz, M.D.
Arnold LaGuardia
Herbert Boyer, Ph.D.
Richard A. Lerner, M.D.
Richard A. Lerner, M.D.
Thomas E.K. Cerruti, Esq.
Claudia S. Luttrell
Gary N. Coburn
Don L. Skaggs
Gerald Cohn
Frank Lowy AC
Claudia S. Luttrell
Mark S. Skaggs
Thomas E.K. Cerruti
Richard A. Lerner, M.D.
President
James R. Mellor
George H. Conrades
John J. Moores
Rod Dammeyer
John G. Davies, Esq.
The Hon. Paul G. Rogers,
Esq.
Thomas E. Dewey, Jr.
John Safer
John D. Diekman, Ph.D.
The Hon. Lynn Schenk
Charles C. Edwards, M.D.
John R. Seffrin, Ph.D.
Richard J. Elkus, Jr.
Ralph J. Shapiro
Jane E. Henney, M.D.
David Tappan
Charles C. Edwards, M.D.
Arnold LaGuardia
Chris D. Van Gorder
Arnold LaGuardia
Executive Vice President and
Treasurer
Douglas Bingham
Executive Vice President and
General Counsel and
Secretary
Donna Weston
Senior Vice President and
Chief Financial Officer
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Richard A. Lerner, M.D.
President’s Introduction
s is the norm, members of The Skaggs Institute
for Chemical Biology at The Scripps Research
Institute this year contributed a prodigious volume of work to the body of scientific knowledge in a
broad range of disciplines, work that changes the way
we think about biological mechanisms and the course
of human disease. The following merely skims the surface of the knowledge they created and the importance
of their discoveries.
A
RESEARCH ACTIVITIES
Work in the laboratory of Peter Schultz effectively
removed a billion-year constraint on the ability to manipulate the structure and function of proteins. Dr. Schultz
and his research group completed the synthesis of a
form of the bacterium Escherichia coli with a genetic
code that uses 21 basic amino acid building blocks to
synthesize proteins, instead of the 20 that occur naturally. This creation was the first one of an autonomous
organism that uses 21 amino acids and has the metabolic machinery to build those amino acids. Further, the
group introduced revolutionary changes into the genetic
code of organisms such as yeast that allow the mass
production of proteins with unnatural amino acids. By
so doing, Dr. Schultz and his team set the stage for an
entirely new approach to applying the same technology to other eukaryotic cells, and even to multicellular
organisms. Simply stated, these scientists have opened
up the whole pathway to higher organisms.
Researchers in the laboratory of Stephen Mayfield
and in my laboratory used algae to express an antibody
that targets herpesvirus. The usefulness of the antibody
lies not only in the potential production of an antiherpes topical cream or treatment but also in the development of technology that could facilitate the production of
multiple human antibodies and other proteins on a massive scale. This technology enables the generation of
antibodies, soluble receptors, and other proteins so much
more cheaply than previous technology that an entire
new class of therapeutic agents may become available.
Jeffery Kelly and his colleagues discovered a new
approach for treating amyloid diseases, particularly
transthyretin amyloid diseases, which are similar to
Parkinson’s and Alzheimer’s diseases. Amyloid diseases
are caused by misfolding of proteins into a structure
that leads the proteins to cluster, forming microscopic
fibrillar plaques that are deposited in internal organs
and interfere with normal function. Dr. Kelly and his
team showed the efficacy of using small molecules to
stabilize the normal folding of transthyretin, preventing
this protein from misfolding. By so doing, they were
able to inhibit the formation of fibrils by a mechanism
that can ameliorate disease.
In what was a first for biology, researchers in my
laboratory reported that the human body makes ozone.
Ozone appears to be produced in a process involving
human immune cells known as neutrophils and human
antibodies. The presence of ozone in the body may be
linked to inflammation, and the research may have
important ramifications for treating inflammatory diseases. In addition to killing bacteria, the neutrophils
feed singlet oxygen to the antibodies, which convert it
into ozone.
Carlos F. Barbas and his group designed a hybrid
anticancer compound that combines the efficacy of a
cancer cell–targeting agent with the long-lasting dose
of an antibody. This potent combination has a profound
effect on the size of tumors in animal models; in preclinical studies, the compound shrank both Kaposi sarcomas and colon cancers. The approach is general
enough to be used to design hybrids against numerous
different cancers; a single antibody can be mixed with
multiple small molecules, resulting in a multiplicity of
therapeutic agents.
7
8
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Scientists led by Kim D. Janda designed a new way
to make a vaccine against nicotine that could become
a valuable tool for treating nicotine addiction by helping the body clear the drug from the bloodstream. The
vaccine, which eventually would be given to patients
in smoking cessation programs, greatly suppresses the
reinforcing aspect of nicotine. The researchers used an
“immunopharmacotherapy” approach, designing a drug
that stimulates the immune system to clear the nicotine from the body.
In a related study, Dr. Janda and his group discovered that a chemical called nornicotine, a major metabolite of nicotine, modifies proteins that misfold and form
the fibril plaques found in abundance in the brains of
patients who have Alzheimer’s disease. Simply stated,
this process physically inhibits the formation of the
fibrils. The research is promising—not because nornicotine most likely would be an effective therapeutic agent,
but because it shows how a single molecule can cause
a chemical interaction that may alter a mechanism
important in Alzheimer’s disease. This research could
lead to the development of small molecules similar to
nornicotine that are not toxic but that could interact in
a similar fashion, preventing the aggregation of amyloid-β protein and perhaps Alzheimer’s disease.
A group of scientists including John Tainer of the
Skaggs Institute and Lisa Craig, Mark Yeager, and Mike
Pique at The Scripps Research Institute solved 2 key
structures of a bacterial protein called pilin, which is
required for infection by pathogens that cause diseases
such as meningitis, gonorrhea, pneumonia, and cholera.
The members of the group think that the research provides essential knowledge to help scientists develop
novel antibiotics and vaccines against these sometimes
deadly bacterial diseases. Because the structures are
too large and flexible to be solved by using the traditional techniques of structural biology, the team used
both x-ray crystallography and electron microscopy to
build a model of the pili that would have otherwise
been impossible at that level of molecular detail.
In another structural achievement, a multi-institutional group of researchers led by Ian Wilson of the
Skaggs Institute and Dennis Burton of The Scripps
Research Institute solved the structure of an antibody
that effectively neutralizes HIV, an important step toward
the goal of designing an effective vaccine against HIV
and a new means by which scientists may design antibodies in general. The structure of the antibody had
never been seen before, prompting the scientists to
speculate on whether they can use this knowledge
to engineer antibodies with higher affinity against
other antigens.
FA C U LT Y H O N O R S A N D AWA R D S
Many of our scientists, at various stages of their
careers, were honored by their peers this past year with
awards for achievement in numerous areas of scientific
endeavor. Members of the Skaggs Institute who received
recognition include Ernest Beutler, who was awarded
the E. Donnall Thomas Lecture and Prize of the American Society of Hematology, and Ben Cravatt, who won
the Eli Lilly Award in Biological Chemistry from the
American Chemical Society.
A L O O K AT T H E F U T U R E
The Skaggs Institute for Chemical Biology is an
organization that exceeds expectations on multiple levels; its faculty and staff remain at the leading edge of
science in an era in which the pace of discovery accelerates on a continual basis. Its new developments and
discoveries, all of which were made possible by the
vision and remarkable generosity of the Skaggs family
and The Skaggs Institute for Research, will provide
greater impetus for the Skaggs Institute for Chemical
Biology to play an even larger role on the international
stage of scientific discovery.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Julius Rebek, Jr., Ph.D.
Director’s Overview
he Skaggs Institute for Chemical Biology was
established in 1996, and as it enters its 8th
year, we congratulate our benefactor, L. Sam
Skaggs, on his 80th birthday. His generosity, and that
of the entire Skaggs family, supports the work of the
Institute through its 31 principal investigators, 95 postdoctoral fellows, and some 50 graduate students. The
aim of the Institute is to relieve human suffering by
moving basic research to applications that lead to cures
for diseases. Our researchers authored nearly 300 publications during the past year, loosely clustered in the
areas of organic synthesis, antibody science, nucleic
acid chemistry, and protein structure. The details of
the scientific discoveries appear in the individual reports
of the principal investigators, but I present a few of the
highlights here.
The faculty members involved with nucleic acids,
Albert Eschenmoser, Martha Fedor, Gerald Joyce, Ehud
Keinan, Peter Schultz, and Jamie Williamson, showed
that DNA isn’t just for genes anymore. DNA can fold
into a spectacular octahedral shape, visible by electron
microscopy; it can do biomolecular computing; and
its sequences can be selected and amplified to cat-
T
alyze the cleavage of RNA. The use of RNA as a target
for small-molecule medicinal agents is also growing.
Synthetic alterations in either the base pairs or the
sugar backbones indicate that nucleic acid structure is
enormously versatile and that the information encoded
can even be expanded. In the past 50 years, the seed
represented by the discovery of the double helix has
grown into an orchard.
The synthetic team, K.C. Nicolaou, Phil Baran,
Dale Boger, Barry Sharpless, and Chi-Huey Wong, created purely synthetic molecules to regulate protein-protein interactions. These interactions emerged as targets
for intervention in diseases but have not, could not, be
addressed by conventional medicinal chemistry. Specific agents for blocking angiogenesis and stimulating
production of blood cells were synthesized, and small
molecules that arrest microtubular structures are now
in clinical trials. Both plant- and marine-derived natural products were used to produce antitumor agents,
and efforts to discover carbohydrate-based drugs led
to the development of new HIV protease inhibitors. As
a result of methods developed here last year, environmentally benign reagents are now available as building blocks for combinatorial medicinal chemistry.
Research led by Richard Lerner indicated that ozone
is one of the key reactive species that antibodies can
generate to destroy antigens. Steve Mayfield developed
methods for large-scale, inexpensive production of antibodies in algae—good news at a time when antibodies
are a sizable fraction of the new drugs approved by the
Food and Drug Administration. Using x-ray crystallography, Ian Wilson and collaborators determined the
structures of antibodies responsible for resistance to
HIV; these antibodies, specifically, and structural biology, generally, will take greater roles in the ongoing
efforts to develop an AIDS vaccine.
Peter Wright used nuclear magnetic resonance to
solve protein structures as they exist in solution. Several
of these proteins are transcription factors, and their interactions with other proteins are responsible for specific
gene expression of tumor viruses. Elsewhere, structures
of enzymes that produce nitric oxide, a ubiquitous cellsignaling molecule, were determined, and the behavior
of superoxide dismutase, an enzyme involved in amyotrophic lateral sclerosis, was analyzed. The structural
details offer a starting point for the development phase
of small-molecule medicinal agents.
We are proud of these discoveries, for which a number of the Skaggs investigators received national and
9
10
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
international awards, including the Paul Ehrlich Prize
and, in the past 2 years, a share of the Nobel Prize in
Chemistry. The Skaggs family has enabled the recruitment of more than 15 principal investigators during the
Institute’s short lifetime. These new faculty members
have propelled our graduate programs to the top in
national surveys. At the international level, recognition
came through an unprecedented joint Ph.D. program
with Oxford University in England. Graduate students
in the program will receive Skaggs Scholarships. All of
us at Scripps are grateful for the continued support of
the Skaggs Institute for Research.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
THE SKAGGS INSTITUTE
FOR CHEMICAL BIOLOGY
S TA F F
Julius Rebek, Jr., Ph.D.*
Director and Professor
Carlos F. Barbas III, Ph.D.**
Professor
Janet and W. Keith Kellogg II
Chair in Molecular Biology
Ernest Beutler, M.D.***
Professor
Chairman, Department of
Molecular and Experimental
Medicine, TSRI
Dale L. Boger, Ph.D.*
Richard and Alice Cramer
Professor of Chemistry
Geoffrey Chang, Ph.D.**
Assistant Professor
Benjamin F. Cravatt,
Ph.D.****
Associate Professor
Philip Dawson, Ph.D.*****
Assistant Professor
Gerald M. Edelman, M.D.,
Ph.D. †
Professor
Chairman, Department of
Neurobiology, TSRI
Albert Eschenmoser, Ph.D.*
Professor
Martha J. Fedor, Ph.D.**
Associate Professor
M.G. Finn, Ph.D.*
Associate Professor
Elizabeth D. Getzoff, Ph.D.††
Professor
M. Reza Ghadiri, Ph.D.*
Professor
Kim D. Janda, Ph.D.*
Professor
Ely R. Callaway, Jr., Chair
in Chemistry
Gerald F. Joyce, M.D.,
Ph.D. †††
Professor
Ehud Keinan, Ph.D.**
Adjunct Professor
Jeffery W. Kelly, Ph.D.*
Vice President, Academic
Affairs, TSRI
Dean, Graduate Program, TSRI
Lita Annenberg Hazen
Professor of Chemistry
Hartmuth Kolb*
Associate Professor
Richard A. Lerner, M.D.†††
President, TSRI
Lita Annenberg Hazen
Professor of
Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
Stephen P. Mayfield,
Ph.D.*****
Associate Professor
Associate Dean, Graduate
Program, TSRI
K.C. Nicolaou, Ph.D.*
Aline W. and L.S. Skaggs
Professor of Chemical Biology
Darlene Shiley Chair in
Chemistry
Chairman, Department of
Chemistry, TSRI
John A. Tainer, Ph.D.**
Professor
James R. Williamson, Ph.D.**
Professor
Associate Dean, Graduate
Program, TSRI
Ian A. Wilson, D.Phil.**
Professor
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, TSRI
Kurt Wüthrich, Ph.D. †††
Cecil H. and Ida M. Green
Visiting Professor of
Structural Biology
Eidgenössische Technische
Hochschule Zürich
Zürich, Switzerland
RESEARCH
A S S O C I A T E S †††††
Toru Amaya, Ph.D.
Wook Dong Cho, Ph.D.
Paul R. Schimmel, Ph.D. †††
Ernest and Jean Hahn
Professor and Chair of
Molecular Biology
andChemistry
Jan Friese, Ph.D.
Peter Schultz, Ph.D.*
Professor
Scripps Family Chair
Yoonkyung Kim, Ph.D.
Arnaud Gissot, Ph.D.
Shen Gu, Ph.D.
Darren W. Johnson, Ph.D.
Hideki Onagi, Ph.D.
Alessandro Scarso, Ph.D.
K. Barry Sharpless, Ph.D.*
W.M. Keck Professor of
Chemistry
Subhash C. Sinha, Ph.D.**
Associate Professor
Erik J. Sorensen, Ph.D. ††††
Assistant Professor
Alexander Shivanyuk, Ph.D.
Laurent Trembleau, Ph.D.
Elke Ullrich, Ph.D.
Masamichi Yamanaka, Ph.D.
11
* Joint appointment in the
Department of Chemistry
** Joint appointment in the
Department of Molecular
Biology
*** Joint appointment in the
Department of Molecular and
Experimental Medicine
**** Joint appointments in the
Departments of Cell Biology
and Chemistry
***** Joint appointment in the
Department of Cell Biology
†
††
†††
††††
†††††
Joint appointment in the
Department of Neurobiology
Joint appointments in the
Departments of Molecular
Biology and Immunology
Joint appointments in the
Departments of Chemistry and
Molecular Biology
Appointment completed
Research associates in the laboratories of staff other than Dr.
Rebek are included in the lists
of the respective departments
in which the associates hold
joint appointments.
12
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Shannon Biros, Graduate Student in Chemistry,
The Skaggs Institute for Chemical Biology
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
INVESTIGATORS ’ R EPORTS
Cancer, AIDS, Catalysis, and the
Regulation of Genes: Inventing
Molecules With Defined Functions
C.F. Barbas III, R. Lerner, P. Blancafort, N.S. Chowdari,
R. Fuller, T. Gräslund, N. Jendreyko, C. Lund, L. Magnenat,
N. Mase, M. Popkov, C. Rader, D.B. Ramachary, S. Sinha,
H. Shim, F. Tanaka, R. Thayumanavan, J. Turner
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, such as
regulating a gene, 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 performed naturally 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 work is the development of
strategies to produce antibodies that efficiently form and
break carbon-carbon bonds. Much of this work centers
on the chemistry of imines and enamines and the development of antibodies that use covalent catalysis. We
are examining the aldol, Michael, and Diels-Alder reactions and related reactions such as the Mannich reaction. Many of these catalysts may someday be important
in the synthesis of enantiomerically pure drugs. Using
novel catalytic antibodies, we showed the efficient
asymmetric synthesis and resolution of a variety of molecules, including tertiary and fluorinated aldols, carbo-
13
hydrates, and insect pheromones. In our collaboration
with S.C. Sinha, the Skaggs Institute, large-scale antibody-catalyzed reactions have been key to the synthesis of epothilone anticancer drugs.
In a new approach to catalytic antibodies, we combined transition-state theory with reactive immunization
and produced antibodies with catalytic proficiencies of
almost 1014. More recently, we focused on learning how
we can use a laboratory-based evolutionary strategy to
improve these catalysts even further. A practical goal
of this study is the creation of human catalytic antibodies that can be applied therapeutically in the treatment
of metabolic diseases, cancer and HIV disease. We
showed that we can selectively release insulin from an
inactive or captured state in animal models. This result
suggests that our catalytic antibody approach can be
used in vivo to prepare and selectively deliver proteins
and peptides.
O R G A N O C ATA LY S I S : E N Z Y M E L I K E C H E M I S T R Y
WITH SMALL ORGANIC MOLECULES
In studying how proteins catalyze reactions, we
often examine how the constituent components react.
These studies 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 chemical
actions 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 measure the importance of pocket sequestration
in catalysis.
In addition, we achieved direct asymmetric catalytic
aldol reactions by using aldehydes and unmodified
ketones together with commercially available chiral
cyclic secondary amines as catalysts. This approach can
also be applied to Diels-Alder reactions wherein dienes
are catalytically generated in situ. We extended the
scope of catalytic asymmetric reactions that form carboncarbon bonds to embrace the use of aldehyde donors.
We produced catalytic asymmetric aldol, Michael, and
Mannich reactions, the first examples of their kind, that
use aldehyde donors to create highly desirable aldehydebearing synthons. 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 mol-
14
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
ecules in a manner analogous to that of natural
enzymes (Fig. 1).
range of drugs to dramatically increase their efficacy.
In cancer studies, we showed that we can use this
approach to turn weakly active anticancer drugs into
potent anticancer therapeutic agents (Fig. 2).
F i g . 2 . Chemically programmed antibodies. A, Many small mol-
F i g . 1 . Enzymelike asymmetric assembly of simple molecules
can be used to create a wide variety of chiral synthons.
TA R G E T I N G C A N C E R
In targeting cancer, we take a multidisciplinary
approach that involves gene regulation, catalytic antibodies, drug design, and combinatorial antibody libraries.
Using a combinatorial antibody strategy, we prepared
a high-affinity human antibody to an integrin. Our goal
is to use the antibody in dual antitumor-antivascular
therapy in cancer. Our most recent studies involved
Kaposi sarcoma, the most common AIDS-associated
malignant neoplasm. We showed that a laboratoryevolved antibody can substantially inhibit growth of
Kaposi tumors in mice.
Using catalytic antibodies, we are developing a
strategy to activate drugs in a highly specific fashion
at the site of cancer. Typically, the use of highly potent
chemotherapeutic agents is limited by nonspecific toxic
effects associated with the inability to direct these agents
to the appropriate targets. We showed that a wide variety of clinically relevant anticancer drugs such as doxorubicin, camptothecin, and etoposide can be modified
to less toxic prodrugs that can be specifically activated
by catalytic antibody 38C2 to kill cancer cells in vivo.
This past year, we invented a new approach to therapeutic antibodies in which small molecules are used
to chemically program the specificity of a generic catalytic antibody. This approach can be applied to a wide
ecules can be endowed with the unique pharmacokinetic properties
of antibodies as well as the ability to fix complement and direct
cellular cytotoxic reactions. Attachment of a β-diketone functional
group allows the targeting molecule to be attached to a aldolase
catalytic antibody. B, An integrin-targeting molecule. When this
molecule was attached to the catalytic antibody, the combination
had potent antiangiogenic and antitumor properties.
CONTROLLING GENES AND EXPLORING GENOMES
In all organisms, from the simplest to the most
complex, proteins that bind nucleic acids control the
expression of genes. The nucleic acids 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. Proteins that bind nucleic acids enable this coordinated
control of the genetic code; lack of coordination 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
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
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.
We showed that these domains are functionally
modular and can be recombined with one another to
create polydactyl proteins capable of binding 18-bp
sequences with subnanomolar affinity. 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. Recently, we created gene switches that can be
controlled by small molecules and showed the effectiveness of our zinc finger technology in tobacco and
Arabidopsis plants.
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. With this
powerful new strategy, we can quickly regulate a target gene or discover other genes key in disease (Fig. 3).
We are targeting a variety of other genes involved not
only in cancer and HIV disease but also in genetic diseases such as sickle cell anemia. We hope to take this
genetic strategy and our other molecular approaches
all the way to clinic trials.
15
Blancafort, P., Magnenat, L., Barbas, C.F. Scanning the human genome with combinatorial transcription factor libraries. Nat. Biotechnol. 21:269, 2003.
Chowdari, N.S., Ramachary, D.B., Barbas, C.F. III. Organocatalysis in ionic liquids:
highly efficient l-proline-catalyzed direct asymmetric Mannich reactions involving
ketone and aldehyde nucleophiles. Synlett, in press.
Chowdari, N.S., Ramachary, D.B., Barbas, C.F. III. Organocatalytic asymmetric
assembly reactions: one-pot synthesis of functionalized β-amino alcohols from
aldehydes, ketones, and azodicarboxylates. Org. Lett. 5:1685, 2003.
Chung, J., Rader, C., Popkov, M., Hur, Y.-M., Kim, H.-K., Lee, Y.-J., Barbas, C.F.
III. Integrin αIIbβ3-specific synthetic human monoclonal antibodies and HCDR3
peptides that potently inhibit platelet aggregation. FASEB J., in press.
Córdova, A., Barbas, C.F. III. Direct organocatalytic asymmetric Mannich-type
reactions in aqueous media: one-pot Mannich-allylation reactions. Tetrahedron Lett.
44:1923, 2002.
Córdova, A., Notz, W., Barbas, C.F. III. Direct organocatalytic aldol reactions in
buffered aqueous media. Chem. Commun. (Camb.) 3024, 2002, Issue 24.
Gottesfeld, J.M., Barbas, C.F. III. RNA as a transcriptional activator. Chem. Biol.
10:584, 2003.
Holbro, T., Beerli, R.R., Maurer, F., Koziczak, M., Barbas, C.F. III, Hynes, N.E. The
ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to
drive breast tumor cell proliferation. Proc. Natl. Acad. Sci. U. S. A. 100:8933, 2003.
Hyland, S., Beerli, R.R., Barbas, C.F., Hynes, N.E., Wels, W. Generation and
functional characterization of intracellular antibodies interacting with the kinase
domain of human EGF receptor. Oncogene 22:1557, 2003.
Jendreyko, N., Popkov, M., Beerli, R.R., Chung, J., McGavern, D.B., Rader, C., Barbas, C.F. III. Intradiabodies: bispecific, tetravalent antibodies for the simultaneous
functional knockout of two cell surface receptors. J. Biol. Chem. 278:47812, 2003.
Lin, Q., Barbas, C.F. III, Schultz, P.G. Small-molecule switches for zinc finger transcription factors. J. Am. Chem. Soc. 125:612, 2003.
Mase, N., Tanaka, F., Barbas, C.F. III. Rapid fluorescent screening for bifunctional
amine-acid catalysts: efficient syntheses of quaternary carbon-containing aldols
under organocatalysis. Org. Lett. 5:4369, 2003.
Notz, W., Tanaka, F., Watanabe, S.-I., Chowdari, N.S., Turner, J.M., Thayumanavan, R., Barbas, C.F. III. The direct organocatalytic asymmetric Mannich reaction:
unmodified aldehydes as nucleophiles. J. Org Chem. 68:9624, 2003.
Rader, C., Sinha, S.C., Popkov, M., Lerner, R.A., Barbas, C.F. III. Chemically programmed monoclonal antibodies for cancer therapy: adaptor immunotherapy based
on a covalent antibody catalyst. Proc. Natl. Acad. Sci. U. S. A. 100:5396, 2003.
Rader, C., Turner, J.M., Heine, A., Shabat, D., Sinha, S.C., Wilson, I.A., Lerner,
R.A., Barbas, C.F. III. A humanized aldolase antibody for selective chemotherapy
and adaptor immunotherapy. J. Mol. Biol. 332:889, 2003.
Ramachary, D.B., Chowdari, N.S., Barbas, C.F. III. The first organocatalytic heterodomino Knoevenagel-Diels-Alder-epimerization reactions: diastereoselective synthesis of highly substituted spiro[cyclohexane-1,2′-indan]-1,3′,4-triones. Synlett, in press.
F i g . 3 . Genome-wide transcriptional regulation strategies. Libraries
of transcription factors can be rapidly constructed from predefined
recognition domains. With this approach, a wide range of regulators
can be studied to quickly determine the regulator most optimal for
gene regulation. This approach can also be applied to gene discovery.
PUBLICATIONS
Berry, J.D., Licea, A., Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L.,
Kubitz, D., Barbas, C.F. III. Rapid monoclonal antibody generation via dendritic
cell targeting in vivo. Hybrid. Hybridomics 22:23, 2003.
Berry, J.D., Rutherford, J., Silverman, G.J., Kaul, R., Elia, M., Gobuty, S., Fuller, R.,
Plummer, F.A., Barbas, C.F. III. Development of functional human monoclonal single-chain variable fragment antibody against HIV-1 from human cervical B cells.
Hybrid. Hybridomics 22:97, 2003.
Ramachary, D.B., Chowdari, N.S., Barbas, C.F. III. Organocatalytic asymmetric
domino Knoevenagel/Diels-Alder reactions: a bioorganic approach to the diastereospecific and enantioselective construction of highly substituted spiro[5,5]undecane1,5,9-triones. Angew. Chem. Int. Ed. 42:4233, 2003.
Segal, D.J., Beerli, R.R., Blancafort, P., Dreier, B., Effertz, K., Huber, A., Koksch, B.,
Lund, C.V., Magnenat, L., Valente, D., Barbas, C.F. III. Evaluation of a modular
strategy for the construction of novel polydactyl zinc finger DNA-binding proteins.
Biochemistry 42:2137, 2003.
Segal, D.J., Stege, J., Barbas, C.F. III. Zinc fingers and a green thumb: manipulating gene expression in plants. Curr. Opin. Plant Biol. 6:163, 2003.
Tanaka, F., Fuller, R., Shim, H., Lerner, R.A., Barbas, C.F. III. Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection.
J. Mol. Biol. 335:1007, 2004.
16
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Tanaka, F., Thayumanavan, R., Barbas, C.F. III. Fluorescent detection of carboncarbon bond formation. J. Am. Chem. Soc. 125:8523, 2003.
Tanaka, F., Thayumanavan, R., Mase, N., Barbas, C.F. III. Rapid analysis of solvent effects on enamine formation by fluorescence: how might enzymes facilitate
enamine chemistry with primary amines? Tetrahedron Lett., in press.
Wantanabe, S., Córdova, A., Tanaka, F., Barbas, C.F. III. One-pot asymmetric synthesis of β-cyanohydroxymethyl α-amino acid derivatives: formation of three contiguous stereogenic centers. Org. Lett. 4:4519, 2002.
Training in Molecular and
Experimental Medicine
E. Beutler
he Skaggs Institute for Chemical Biology is currently supporting the training of 5 young scientists
in the Department of Molecular and Experimental Medicine at The Scripps Research Institute. Each of
the 5 is studying a problem that is important for human
health under the direct supervision of a mentor who is
an established expert in the field.
ISG15 is an ubiquitin-like protein that is strongly
induced and forms protein conjugates in mammalian
cells after viral infection. Kenneth Ritchie and his mentor, Dong-Er Zhang, have identified UBP43 as an ISG15
protease and have produced UBP43-deficient mice.
They have been investigating the response of UBP43–/–
mice to infection with lymphocytic choriomeningitis
virus (LCMV). In previous studies, UBP43–/– mice were
found to have increased levels of ISG15 in ependymal
cells and the choroid plexus of the brain. Recently,
Dr. Ritchie extended these observations to include the
meninges. After intracerebral inoculation with LCMV,
viral replication primarily occurs in the ependyma,
choroid plexus, and meninges. He found that such infection induces ISGylation with these cell types and the
brain microvasculature.
In order to investigate if UBP43–/– mice have altered
susceptibility to intracerebral viral infection, LCMV was
injected intracerebrally and the mice were monitored
daily. Confirming successful infection, 100% of wildtype mice died before day 7 postinfection. Contrastingly,
no UBP43 –/– mice died up to day 11 postinfection.
Investigations into the presence of LCMV in UBP43 –/–
brain tissue and serum revealed no detectable virus by
plaque assay. Supporting in vitro studies with mouse
embryo fibroblasts cells revealed that UBP43 deficiency
also confers resistance to infection by Sindbus virus.
Dr. Ritchie has therefore provided the first direct evi-
T
dence that ISGylation has an important role in innate
immune defense against viral infection. In doing so,
he also extended the repertoire of functions performed
by ubiquitin-like molecules to viral defense.
Xianghong Li, under the direction of Xiaohua Wu,
has participated in the study of DNA replication initiation control and cell-cycle checkpoint control. She has
mainly focused on studying a replication licensing factor, Cdt1, in the control of DNA replication initiation.
She has shown that Cdt1 degrades when cells enter
S phase. On the basis of the observation that overexpression of Cdt1 leads to re-replication, cell cycle–dependent Cdt1 degradation is likely to play a crucial role in
restricting DNA replication to once per cell cycle. Dr. Li
investigated the mechanisms of human Cdt1 degradation and found that human Cdt1 degrades through the
SCF Skp2 -mediated ubiquitination pathway. She also
made the observation that Cdt1 is phosphorylated by
cyclin-dependent kinase (Cdk) at multiple sites on Cdt1.
This Cdk-mediated phosphorylation stimulates Skp2
binding and triggers human Cdt1 for degradation. The
biological significance of the Cdk-mediated phosphorylation events is currently under investigation. In addition, Dr. Li investigated Cdt1 function in DNA damage
responses and performed experiments to test the association of Cdt1 with other proteins involved in DNA
replication initiation. Her findings have contributed
significantly to the understanding of cellular mechanisms that control DNA replication and checkpoints
and will help to reveal how genome stability, which is
essential for preventing cancer, is maintained.
The production of reactive oxygen species represents
the major microbicidal mechanism of professional
phagocytes. The aim of the research of Natalia Sigal,
mentored by Bernard Babior, is to study the recruitment
of cytosolic components of NADPH oxidase (proteins
Rac2, p67 phox, p47 phox, and p40 phox) to the phagosomal membrane that forms around ingested particles.
To do this, she has used deconvolution microscopy
instead of confocal microscopy, thus making it possible to take images of 3-dimensional objects, allowing
her to see proteins associated with the phagosome.
Neutrophils were settled on serum-coated coverslips;
opsonized particles were then incubated with adhered
neutrophils for various periods (0–15 minutes). After
fixation with 10% formalin and permeabilization with
acetone, cells were blocked with mouse or rabbit serum
and incubated with primary antibody and then with
secondary fluorescent antibody. Dr. Sigal was able to
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
determine the time course of association of p47 phox ,
p67 phox, p40 phox, and Rac2 assembly with the phagosomal membrane.
Cytosolic proteins p47 phox and p40 phox were found
to interact with phosphoinositides that are under control of phosphatidylinositol-3′-kinase (PI3K). PI3K has
been known to be required for oxidase function, and
PI3K products are believed to be responsible for translocation of cytosolic proteins to the membranes. Dr. Sigal
studied the involvement of PI3K in the targeting of
cytosolic proteins to the phagosomal membrane and
the ability of PI3K inhibitors to inhibit superoxide production in assays with whole cells. She found that
wortmannin and LY294002 have a strong inhibitory
effect on neutrophils activated by N-formylmethionine–
leucine–phenylalanine, but not on cells activated by
phorbol myristate acetate. To better characterize the
ability of opsonized zymosan particles to activate the
NADPH oxidase in the forming phagosome, Dr. Sigal
activated neutrophils with opsonized zymosan particles
and measured superoxide production. PI3K inhibitors
were found to inhibit the zymosan-activated superoxide
production in a dose-response manner. Previous studies have shown a major role of protein kinase C in
NADPH oxidase activation. To investigate the involvement of protein kinase C in the translocation of cytosolic protein to the phagosomal membrane and to
compare the results with the inhibition of translocation
by PI3K inhibitors, Dr. Sigal has begun to study the
ability of protein kinase C inhibitors to affect translocation of p47phox and p40 phox.
Human platelets are the smallest cellular component of blood, but they play a very important role in
the prevention of bleeding. At sites of injury, they do
this by sticking to exposed substances in the blood
vessel wall, such as collagens. Platelets have specific
binding sites or receptors on their surface that mediate the attachment to collagens and other substances
in the blood vessel wall. Kazunobu Kato, under the
supervision of Jerry Ware and Thomas Kunicki, is currently investigating the genetic differences between
platelet receptors that can modify the ability of platelets to function properly. One of these receptors for
collagen, glycoprotein VI, is unique to platelets. It is
important to identify and study natural products that
bind to this receptor so that they can be used to develop
therapeutic or diagnostic reagents. One such natural
product is a substance called convulxin, produced in
the venom of a certain South American rattlesnake.
17
Convulxin normally binds to platelets and induces
platelet clumping or clot formation. By carefully studying the structure of convulxin, Dr. Kato hopes to learn
ways to modify its effect and to turn it into an inhibitor. Such an inhibitor may prove useful for the control
of unwanted and dangerous clot formation, as seen in
stroke or heart disease. This research involves the isolation of DNA that encodes convulxin, the mutation of
this DNA, and the expression of the convulxin protein
by using recombinant DNA technology.
Sharookh Kapadia, under the guidance of Frank
Chisari, is studying hepatitis C virus (HCV), a member
of the Flaviviridae family of viruses and a major cause
of chronic hepatitis and hepatocellular carcinoma.
Viral clearance during acute HCV infection is usually
associated with a multispecific CD4+ and CD8 + T-cell
response that is weak or undetectable in subjects who
do not control the infection. Importantly, most chronically infected patients do not resolve HCV infection
after combination therapy with IFN and ribavirin.
Recent data have shown that a number of genes
involved in lipid metabolism are transcriptionally regulated preferentially in acutely HCV-infected chimpanzees
that had the highest acute titers of virus. This finding
suggests that these genes may, theoretically, be important under baseline conditions for efficient viral replication to occur. Although there is considerable evidence
suggesting that the lipid metabolism pathways may
play a role in HCV replication and infection, it is not
known whether changes in cholesterol and/or fatty acid
biosynthesis directly regulate viral replication or whether
these changes merely reflect a pathologic host response
to HCV infection. Furthermore, drugs that are known
to inhibit key steps in these biosynthetic pathways were
also shown to regulate replication of the HCV subgenomic replicon in Huh-7 cells. However, the specificity
of these drugs was not assessed.
Therefore, Dr. Kapadia wanted to specifically determine whether the cholesterol and/or fatty acid biosynthetic pathways regulated HCV replication by using the
subgenomic HCV replicon cell system. The liver X receptors (LXRs) belong to a family of nuclear hormone
receptors that play a key role in regulating cholesterol
and fatty acid metabolism. Dr. Kapadia demonstrated
that activation of the LXRs by a nonsteroidal LXR agonist, T0901317, dramatically regulates HCV replication. Further characterization of the expression of genes
encoding enzymes involved in cholesterol and fatty acid
biosynthetic pathways are in progress. She is currently
18
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
using short interfering RNAs to specifically inhibit
expression of key genes that encode enzymes involved
in these pathways.
PUBLICATIONS
Kapadia, S.B., Brideau-Andersen, A., Chisari, F.V. Interference of hepatitis C virus RNA
replication by short interfering RNAs. Proc. Natl. Acad. Sci. U. S. A. 100:2014, 2003.
Li, X., Zhao, O., Liao, R., Sun, P., Wu, X. The SCFSkp2 ubiquitin ligase complex
interacts with the human replication licensing factor Cdt1 and regulates Cdt1
degradation. J. Biol. Chem. 278:30854, 2003.
Synthetic, Medicinal, and
Bioorganic Chemistry
by using combinatorial libraries to target protein-protein
interactions. Each example defined or validated new
therapeutic targets and provided the first small-molecule leads for each target. We identified an effective
inhibitor of Myc-Max that inhibits protein dimerization,
subsequent DNA binding, and gene transcription (Fig. 1).
In addition to being the first small-molecule inhibitor
of a transcription factor dimerization, the lead compound also inhibited Myc-induced cell transformation
in a functional cellular assay (the transformation from
a normal to a tumor phenotype), validating the target
for antineoplastic intervention via small molecules.
D.L. Boger, Y. Ambroise, B. Blagg, S. Buck, K. Capps,
H. Cheng, Y. Choi, B. Crowley, P. Desai, J. Desharnais,
R. Dominique, W. Du, R. Ducray, G. Elliott, R. Fecik, Y. Ham,
N. Haq, C. Hardouin, I. Hwang, S. Ichikawa, D. Kastrinsky,
G. Kim, P. Krenitsky, M. Lall, C. McComas, Y. Mori, J. Parrish,
S. Pfeiffer, A. Pichota, Y. Rew, D. Shin, D. Soenen, J. Trzupek,
W. Tse, J. Wanner, G. Wilkie, S. Wolkenberg, Z. Yuan,
B. Yeung, J. Zimpleman
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 chemistry, the study of DNA-agent
interactions, and the 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 of the agents.
T
TA R G E T I N G P R O T E I N - P R O T E I N I N T E R A C T I O N S
W I T H C O M B I N AT O R I A L L I B R A R I E S
Receptor activation and signal transduction by protein-protein homodimerization, heterodimerization, and
higher order homo- and hetero-oligomerization have
emerged as general mechanisms of initiating and transmitting intracellular signals. We are investigating the
fundamental principles and structural features embodied in such receptor activation and signal transduction
events with the receptor for erythropoietin. Additional
targets under examination include ErbB2; the transcription factor Myc-Max; LEF-1/β-catenin; the androgen
receptor; and angiogenesis inhibitors (αvβ3 and αvβ5),
including those that act by inhibiting binding of matrix
metalloproteinase 2 to αvβ3.
This past year, we reported 3 important instances
in which we successfully modulated cellular signaling
F i g . 1 . Inhibition of transcription factor heterodimerization and
aberrant gene transcription: antitumor target validation. ELISA indicates enzyme-linked immunosorbent assay; FRET, fluorescent resonance energy transfer.
Similarly, we discovered a potent inhibitor of paxillin binding to the intracellular tail of the cell-surface
α4 by screening our combinatorial library of compounds.
This discovery provided the first inhibitor of paxillin-α4
protein-protein binding and allowed a critical assessment of small-molecule positional scanning libraries.
The most potent lead compound inhibited cell migration, validating the protein-protein interaction as one
useful for treatment of chronic inflammatory and
autoimmune diseases.
Finally, we discovered a series of small-molecule
erythropoietin agonists that act by promoting (not
inhibiting) the dimerization of the cell-surface erythropoietin receptor, providing a general model for the
synthesis, screening, and design of such molecules
(agonists from antagonists) (Fig. 2).
STRUCTURE-BASED DRUG DESIGN
In collaboration with I.A. Wilson, the Skaggs Institute, we are using x-ray crystallographic structures of
(1) the apo forms of glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide
transformylase inosine monophosphate cyclohydrolase
and (2) complexes of the 2 enzymes with their substrates, folate cofactors, and inhibitors to design and
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
19
antitumor agent, involves a hydrogen-bonding triplexlike interaction in the minor groove of duplex DNA. We
are extending and exploiting these developments.
PUBLICATIONS
Ambroise, Y., Boger, D.L. The DNA phosphate backbone is not involved in catalysis of the duocarmycin and CC-1065 DNA alkylation reaction. Bioorg. Med. Chem.
Lett. 12:303, 2002.
Ambroise, Y., Yaspan, B., Ginsberg, M.H., Boger, D.L. Inhibitors of cell migration
that inhibit intracellular paxillin/α4 binding: a well-documented use of positional
scanning libraries. Chem. Biol. 9:1219, 2002.
F i g . 2 . An erythropoietin (EPO) agonist that functions by pro-
moting dimerization of the erythropoietin receptor (EPOR).
examine potent inhibitors of the enzymes as antineoplastic agents. One such inhibitor is undergoing extensive in vivo and preclinical evaluation.
After the discovery of oleamide, a fatty acid primary
amide with sleep-inducing properties, and anandamide,
the endogenous ligand for the cannabinoid receptor, the
study of these prototypical members of a new class of
endogenous chemical messengers led to the identification of fatty acid amide hydrolase, an enzyme responsible for their degradation and regulation. In the past year
we produced a series of exceptionally potent α-ketoheterocycle inhibitors of the hydrolase that may be useful
in the treatment of sleep disorders or as novel analgesics.
We also investigated the substrate specificity of pure
recombinant fatty acid amide hydrolase against a complete series of fatty acid primary amides. We identified
required structural features and additional potential
endogenous members of this class of chemical messengers subject to regulation by the hydrolase. With
the determination of the x-ray structure of the enzyme,
conducted by R. Stevens, The Scripps Research Institute,
and B.F. Cravatt, the Skaggs Institute, retrospective
active-site modeling of the inhibitors and substrates
has provided new inhibitor classes.
DNA-BINDING ANTITUMOR ANTIBIOTICS
We are continuing our synthesis and evaluation of
naturally occurring antitumor antibiotics, including
CC-1065/duocarmycins, yatakemycin, bleomycin A 2 ,
sandramycin/luzopeptins, and isochrysohermidin, and
are studying their sequence-selective interaction with
duplex DNA. In the past year, we identified the source
of catalysis for the CC-1065/duocarmycin DNA alkylation
reaction based on a DNA binding–induced conformational
change in the agent, a process we termed “shape-dependent catalysis.” Similarly, we established that the source
of the polynucleotide recognition of bleomycin A2, an
Berg, T., Cohen, S.B., Desharnais, J., Sonderegger, C., Maslyar, D.J., Goldberg,
J., Boger, D.L., Vogt, P.K. Small-molecule antagonists of Myc/Max dimerization
inhibit Myc-induced transformation of chicken embryo fibroblasts. Proc. Natl. Acad.
Sci. U. S. A. 99:3830, 2002.
Blagg, B.S.J., Boger, D.L. Total synthesis of (+)-camptothecin. Tetrahedron
58:6343, 2002.
Boger, D.L., Castle, S.L. Synthesis of cycloisodityrosine peptides. In: Synthesis of
Peptides and Peptidomimetics, Vol. E22c. Goodman, M., et al. (Eds.). Thieme Medical, New York, 2002, p. 194. Houben-Weyl: Methods of Organic Chemistry, 4th ed.
Chen, Y., Bilban, M., Foster, C.A., Boger, D.L. Solution-phase parallel synthesis of
a pharmacophore library of HUN-7293 analogues: a general chemical mutagenesis
approach to defining structure-function properties of naturally occurring cyclic (depsi)peptides. J. Am. Chem. Soc. 124:5431, 2002.
Goldberg, J., Jin, Q., Ambroise, Y., Satoh, S., Desharnais, J., Capps, K., Boger.
D.L. Erythropoietin mimetics derived from solution phase combinatorial libraries. J.
Am. Chem. Soc. 124:544, 2002.
Jiang, W., Wanner, J., Lee, R.J., Bounaud, P.-Y., Boger, D.L. Total synthesis of the
ramoplanin A2 and ramoplanose aglycon. J. Am. Chem. Soc. 124:5288, 2002.
Krenitsky, P.J., Boger, D.L. Preparation of the 14-membered L,L-cycloisodityrosine
subunit of RP 66453. Tetrahedron Lett. 43:407, 2002.
Lewy, D., Gauss, C.-M., Soenen, D.R., Boger, D.L. Fostriecin: chemistry and biology. Curr. Med. Chem. 9:2005, 2002.
Marsilje, T.H., Labroli, M.A., Hedrick, M.P., Jin, Q., Desharnais, J., Baker, S.J.,
Gooljarsingh, L.J., Ramcharan, J., Tavassoli, A., Zhang, Y., Wilson, I.A., Beardsley, G.P., Benkovic, S.J., Boger. D.L. 10-Formyl-5,10-dideaza-acyclic-5,6,7,8tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by
selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 10:2739, 2002.
McAtee, J.J., Castle, S.L., Jin, Q., Boger, D.L. Synthesis and evaluation of vancomycin and vancomycin aglycon analogues that bear modifications of the residue
3 asparagine. Bioorg. Med. Chem. Lett. 12:1319, 2002.
Wilkie, G.D., Elliott, G.I., Blagg, B.S.J., Wolkenberg, S.E., Soenen, D.R., Miller,
M.M., Pollack, S., Boger, D.L. Intramolecular Diels-Alder and tandem intramolecular Diels-Alder/1,3-dipolar cycloaddition reactions of 1,3,4-oxadiazoles. J. Am.
Chem. Soc. 124:11292, 2002.
Wolkenberg, S.E., Boger, D.L. Mechanisms of in situ activation of DNA targeting
antitumor agents. Chem. Rev. 102:2477, 2002.
Wolkenberg, S.E., Boger, D.L. Total synthesis of anhydrolycorinone utilizing
sequential intramolecular Diels-Alder reactions of a 1,3,4-oxadiazole. J. Org. Chem.
67:7361, 2002.
Woods, C.R., Faucher, N., Eschgfaller, B., Bair, K.W., Boger, D.L. Synthesis and
DNA-binding properties of saturated distamycin analogues. Bioorg. Med. Chem.
Lett. 12:2647, 2002.
Woods, C.R., Ishii, T., Boger, D.L. Synthesis and DNA binding properties of iminodiacetic acid-linked polyamides: characterization of cooperative extended 2:1
side-by-side parallel binding. J. Am. Chem. Soc. 124:10676, 2002.
20
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Woods, C.R., Ishii, T., Wu, B., Bair, K.W., Boger, D.L. Hairpin versus extended
DNA binding of a substituted β-alanine linked polyamide. J. Am. Chem. Soc.
124:2148, 2002.
Zhang, Y., Desharnais, J., Greasley, S.E., Beardsley, G.P., Boger, D.L., Wilson,
I.A. Crystal structures of human GAR Tfase at low and high pH and with substrate
β-GAR. Biochemistry 41:14206, 2002.
Structural Biology of
Membrane-Bound
Transporters and Receptors
A. Ma, T. Nguyen, P. Szewczyk, Y. Yin, C. Reyes, S. Wada,
important drug targets. Nearly all G protein–coupled
receptors are predicted to have 7 transmembrane helices
with domains that bind ligand and activate G proteins.
A structure of these receptors will reveal the mechanisms
of cell signaling on the molecular level.
PUBLICATIONS
Chang, G. Structure of MsbA from Vibrio cholerae: a multidrug resistance ABC
transporter homolog in a closed conformation. J. Mol. Biol. 330:419, 2003.
Chang, G., Roth, C.B. Signal transduction and integral membrane proteins. In:
Handbook of Cell Signaling. Bradshaw, R.A., Dennis, E.A. (Eds.). Academic Press,
San Diego, 2003, p. 115.
Roth, C.B., Chang, G. X-ray structure of an intact ABC-transporter, MsbA. In: ABC
Proteins: From Bacteria to Man. Holland, I.B., et al. (Eds.). Academic Press, San
Diego, 2003, p. 135.
O. Pornillos, M. Grant, G. Chang
-ray crystallography of receptors and multidrugresistance (MDR) efflux pumps embedded in the
cell membrane is an important frontier in structural biology and medicine. We are interested in the
structural basis for the transport of hydrophobic substrates across the cell membrane by MDR transporters
and the signal transduction by receptors. We use several
biophysical techniques, including molecular cloning,
detergent/lipid protein chromatography, membrane protein crystallization, and protein x-ray crystallography.
Methods for determining structure include the overexpression, large-scale purification, and 3-dimensional
crystallization of these integral membrane proteins.
Recently, we determined the structures of the open
and closed conformations of an MDR transporter from
the ATP-binding cassette family called MsbA. MsbA is
a bacterial homolog of human MDR1, which causes
MDR in the treatment of cancers. In collaboration with
Ronald Milligan, The Scripps Research Institute, we are
using electron cryomicroscopy to study other possible
conformational changes. Through the help of the Skaggs
Institute, we are expanding our structure determination
efforts to incorporate mammalian ATP-binding cassette
transporters that confer the MDR phenotype and other
ATP-binding cassette transporters that are involved in
human diseases, including cystic fibrosis and macular
dystrophy. In addition, we are elucidating the structures
of H +-drug antiporters from both the major facilitator
superfamily and the small multidrug transporter family.
We are also interested in the structures of membrane-bound receptors that are important models for
signal transduction across the lipid bilayer. One of the
most widely studied families of receptors is the family of
G protein–coupled receptors, which are pharmaceutically
X
Chemical Physiology
B.F. Cravatt, G.C. Adam, K.T. Barglow, M.H. Bracey,
K. Chiang, M. Evans, G. Hawkin, M. Humphrey, N. Jessani,
A. Joseph, D. Leung, K. Masuda, M. McKinney,
A. Saghatelian, A. Speers
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 act as a molecular mode for
intersystem communication. 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 are for the most part still a mystery.
Currently, we are focusing on a family of chemical
messengers termed the fatty acid amides, which affect
many physiologic functions, including sleep and pain. In
particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired animals.
This finding suggests that oleamide may be a molecular
indicator of the organism’s need for sleep. Another fatty
acid amide, anandamide, is a postulated endogenous
ligand for the cannabinoid receptor in the brain.
The in vivo levels of chemical messengers such as
the fatty acid amides must be tightly regulated to maintain proper control over the influence of the messengers
on brain and body physiology. We are characterizing a
mechanism by which the level of fatty acid amides can
be regulated in vivo. The enzyme fatty acid amide hydrolase (FAAH) degrades the fatty acid amides to inactive
metabolites. Thus, FAAH effectively terminates the sig-
W
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
naling messages conveyed by fatty acid amides, possibly ensuring that these molecules do not generate physiologic responses in excess of their intended purpose.
We are using transgenic and synthetic chemistry
techniques to study the role of FAAH in the regulation
of fatty acid amide levels in vivo. We created transgenic
mice that lack the hydrolase and found that these animals have highly elevated brain levels of fatty acid
amide 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, The Scripps Research
Institute, we recently solved the first 3-dimensional structure of FAAH. The structure will serve as a template
for the design of potent and selective inhibitors of the
enzyme. In collaboration with D.L. Boger, the Skaggs
Institute, we identified potent FAAH inhibitors, and using
a functional proteomic screen developed by our group,
we showed that these inhibitors are highly selective
for this enzyme. We are also interested in proteins
responsible for the biosynthesis of fatty acid amides.
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 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
read out the collective catalytic activities of entire
classes of enzymes.
Using activity-based probes that target the serine
hydrolases, we identified several enzymes with altered
activities in human cancer. Additionally, we are developing chemical probes that target many other enzyme
families. To date, we have identified activity-based proteomics probes for more than 10 mechanistically distinct enzyme classes.
PUBLICATIONS
Adam, G.C., Vanderwal, C.D., Sorensen, E.J., Cravatt, B.F. (–)-FR182877 is a
potent and selective inhibitor of carboxylesterase-1. Angew. Chem. Int. Ed.
42:5480. 2003.
Egertova, M., Cravatt, B.F., Elphick, M.R. Comparative analysis of fatty acid amide
hydrolase and CB1 cannabinoid receptor expression in the mouse brain: evidence
of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid
signaling. Neuroscience 119:481, 2003.
Kustedjo, K., Deechongkit, S., Kelly, J.W., Cravatt, B.F. Recombinant expression,
purification, and comparative characterization of torsinA and its torsion dystoniaassociated variant ∆E-torsinA. Biochemistry 42:15333, 2003.
Leung, D., Hardouin, C., Boger, D.L., Cravatt, B.F. Discovering potent and selective
reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21:687, 2003.
McKinney, M.K., Cravatt, B.F. Evidence for distinct roles in catalysis for residues of
the serine-serine-lysine catalytic triad of fatty acid amide hydrolase. J. Biol. Chem.
278:37393, 2003.
Speers, A.E., Adam, G.C., Cravatt, B.F. Activity-based protein profiling in vivo
using a copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc.
125:4686, 2003.
Synthetic Protein Chemistry
P. Dawson, R. Balambika, J. Blankenship, M. Churchill,
J. Offer, C. Neidre, F. Topert
ur focus is the development and use of methods
to incorporate unnatural chemical groups into
proteins. We developed a chemical approach
for producing the large polypeptide chains that make
up protein molecules, enabling us to change the structure of a protein in ways impossible by natural means.
This chemical ligation approach greatly facilitates the
synthesis of proteins of moderate size and has opened
the world of proteins to the synthetic tools of organic
chemistry. Chemical ligation can be extended to biologically expressed proteins, enabling the semisynthesis of
proteins of unlimited size that contain fluorophores or
cross-linking agents at defined positions. Our goal is
to introduce noncoded amino acids and other chemical
groups into proteins to better understand the molecular basis of protein function.
O
SYNTHESIS OF PROTEINS
Proteins synthesized by the chemical ligation
approach are assembled from several smaller polypeptides. The modular nature of this approach provides the
opportunity to assemble large numbers of modified proteins through combinatorial assembly of the individual
peptide components (Fig. 1). We are developing a sys-
Aebersold, R., Cravatt, B.F. Proteomics: advances, applications, and the challenges
that remain. Trends Biotechnol. 20(12 Suppl.):S1, 2002.
Clement, A.B., Hawkins, E.G., Lichtman, A.H., Cravatt, B.F. Increased seizure
susceptibility and proconvulsant activity of anandamide in mice lacking fatty acid
amide hydrolase. J. Neurosci. 23:3916, 2003.
Cravatt, B.F., Lichtman, A.H. The enzymatic inactivation of the fatty acid amide
class of signaling lipids. Chem. Phys. Lipids 121:135, 2002.
Cravatt, B.F., Lichtman, A.H. Fatty acid amide hydrolase: an emerging therapeutic
target in the endocannabinoid system. Curr Opin. Chem. Biol. 7:469. 2003.
21
F i g . 1 . A, Combinatorial assembly of polypeptide fragments. B,
Array of folded proteins on a solid support.
22
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
tem in which proteins can be assembled in parallel on
a sheet of cellulose by using our chemical ligation reactions. After these proteins are removed from the cellulose, they can be assayed in solution or covalently
attached to glass slides to facilitate rapid screening.
T O TA L S Y N T H E S I S O F A V I R A L C O AT
Many viruses encapsulate themselves in a protein
coating composed of multiple copies of a small number
of protein subunits. The process through which individual protein-protein interactions grow into a spherical
protein assembly is essential to the life cycle of these
viruses and may be an important target of antiviral
therapies. To study these interactions in more detail,
we are synthesizing the coat protein from the MS2
bacteriophage, which self-assembles with 180 copies
of itself to encapsulate the genetic material of the
bacterial virus. We plan to introduce site-specific fluorophores and cross-linking agents into this synthetic
viral coat in order to understand viral coat assembly.
PUBLICATIONS
Blankenship, J.W., Balambika, R., Dawson, P.E. Probing backbone hydrogen bonds in
the hydrophobic core of GCN4. Biochemistry 41:15676, 2002.
Blankenship, J.W., Dawson, P.E. Thermodynamics of a designed protein catenane.
J. Mol. Biol. 327:537, 2003.
Brik, A., Dawson, P.E., Keinan, E. The product of the natural reaction catalyzed by
4-oxalocrotonate tautomerase becomes an affinity label of its mutant. Bioorg. Med.
Chem. 10:3891, 2002.
Dawson, G., Dawson, S.A., Marinzi, C., Dawson, P.E. Anti-tumor promoting effects
of palmitoyl:protein thioesterase inhibitors against a human neurotumor cell line.
Cancer Lett. 187:163, 2002.
Zwick, M.B., Parren, P.W.H.I., Saphire, E.O., Church, C., Wang, M., Scott, J.K.,
Dawson, P.E., Wilson, I.A., Burton, D.R. Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency
virus type 1 gp120. J. Virol. 77:5863, 2003.
Fundamental Processes in
Neural Development
G.M. Edelman, S. Aschrafi, A. Atkins, S. Chappell,
G.W. Rogers, F. Smart, T. Stevens, M. Tsatmali, W. Zhou
e focus our efforts on primary cellular processes of development, with emphasis on
the development of the vertebrate nervous
system. Our recent work, which originated from the
study of the structure, function, and regulation of cell
adhesion molecules, led to approaches for examining
the fundamental mechanisms that control transcription
and translation. We are continuing our research on the
W
structure and function of adhesion molecules in neural
development and at synapses. The training grant from
the Skaggs Institute supports the work of several postdoctoral fellows. Their efforts in various projects with
senior investigators are summarized here.
Development and morphogenesis require multiple
rounds of differential gene expression. A variety of factors regulates this expression, including elements within
the genes themselves and protein factors that bind to
the elements. In examining such elements in genes for
cell adhesion molecules, Fred Jones and his colleagues
discovered a homeobox protein, Barx2, that affects a
wide variety of differentiation processes. Tracy Stevens
is working with Robyn Meech to explore the activity of
Barx2 and to identify its target genes. These studies
have opened new front lines related to breast cancer,
muscle development, and chondrogenesis.
In examining the messenger RNAs expressed in
response to neural cell adhesion, Vince Mauro noted
that many mRNAs had sequences that matched or were
complementary to ribosomal RNA. By analyzing a subset of these sequences, he and his colleagues, Stephen
Chappell, Wei Zhou, and George Rogers, Jr., defined
the characteristics of novel sequences within mRNAs
that can connect the translation machinery to the message in the absence of the traditional cap sequence.
These internal ribosome entry sites (IRESs) were known
for viral RNAs but had not been extensively defined in
cellular mRNAs. Dr. Mauro and his colleagues went on
to show that cellular and viral IRESs differ; the cellular
sites are made up of small modules, and the action of
cellular IRESs does not necessarily depend on specific
secondary structures in the mRNA.
Regulation of translation appears to play an important role in the nervous system, and IRESs may play a
special role in the functioning of the synapse. Peter
Vanderklish and Fiona Smart have been studying the
factors involved in synaptic events thought to provide
the basis of memory and learning in the area of the
brain called the hippocampus. Translation of mRNA in
dendrites plays a special role in this process. Dr. Vanderklish and his colleagues found that specific messages
at the synapse are differentially translated, depending
on whether the messages are regulated by cap- or IRESdependent processes. In addition, Ann Atkins and Armaz
Aschrafi are working with Bruce Cunningham to define
and characterize granules containing ribosomes, proteins,
and mRNAs that are transported from the cell body to
the synaptic area in order to allow translation to occur
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
in response to synaptic activity. They are also characterizing the mechanisms involved in neural cell adhesion molecule binding and signaling.
The development of the nervous system begins with
the differentiation of stem cells to neural progenitors,
which in turn develop at the proper time and place into
neurons and supporting glia. Moreover, neural progenitor cells remain in the brain in adults, where they presumably repopulate specific brain regions. Kathryn
Crossin and her colleagues found that the neural cell
adhesion molecule can act as a neurotrophin to differentiate the neural stem cells preferentially into neurons.
Moreover, using special multielectrode plates, Dr. Crossin
and her colleagues defined conditions under which a
stem cell population in culture can be converted into
neurons capable of exchanging action potentials in a
functioning neural network. Marina Tsatmali is following up these observations and is investigating the
influence of oxidative state on neuronal differentiation
and activity.
All of these activities are aimed at the study of the
molecular and cellular events that define and regulate
the development of the nervous system. Our efforts
remain focused on fundamental processes rather than
on specific diseases. This strategy is based on the belief
that understanding even a single primary process can
provide the necessary framework for defining the mechanisms underlying not just one but a variety of diseases.
23
Chemical Etiology of the
Structure of Nucleic Acids
A. Eschenmoser, R. Krishnamurthy, Z. Wang, G.R. Vavilala,
C. Mang, V. Rajwanshi, J. Nandy, T. George
D
uring the past year we worked on the following
projects.
NITROGENOUS ANALOGS OF (L)-α-THREOFURANOSYL(3′→2′) OLIGONUCLEOTIDES
We continued our comprehensive experimental
scrutiny of informational oligomeric systems containing
threose-derived backbones. To that end, we synthesized
the 2′,3′-diamino analogs of (L)-α-threofuranosyl-(3′→2′)
oligonucleotide (TNA) building blocks containing adenine and thymine as nucleobases. We are investigating
the chemistry of the analogs in order to make oligoamides; the information will be used in studies of the
base-pairing properties of the oligoamides (Fig. 1).
A LT E R N AT I V E N U C L E O B A S E S
We studied the chemistry of 7,9-dicarba-5,8diaza-2,6-diaminopurine, a constitutional isomer of
2,6-diaminopurine. This isomer smoothly undergoes
C-nucleosidations with cyclic iminium ions derived
from L-threose derivatives to form nucleosides at the
C-9 carbon of the nucleobase that are isosteric to
normal N-nucleosides.
PUBLICATIONS
Chappell, S.A., Mauro, V.P. The internal ribosome entry site (IRES) contained
within the RNA-binding motif protein 3 (Rbm3) mRNA is composed of functionally
distinct elements. J. Biol. Chem. 278:33793, 2003.
Jones, F.S., McKean, D.M., Meech, R., Edelman, D.B., Oakey, R.J., Jones, P.L.
Regulation of vascular smooth muscle cell growth and adhesion by paired-related
homeobox genes. Chest 121(3 Suppl.):89S, 2002.
Makarenkova, H.P., Meech, R., Edelman, D.B., Jones, F.S. The homeobox transcription factor Barx2 is expressed during limb development, modulates chondrogenesis, and is regulated by GDF5. Development, in press.
Meech, R., Makarenkova, H., Edelman, D.B., Jones, F.S. The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors. J. Biol. Chem. 278:8269, 2003.
Smart, F., Edelman, G.M., Vanderklish, P.W. BDNF induces translocation of initiation factor 4E to mRNA granules in neurons by a mechanism involving synaptic
microfilaments and integrins. Proc. Natl. Acad. Sci. U. S. A. 100:14403, 2003.
Zhou, W., Edelman, G.M., Mauro, V.P. Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae. Proc.
Natl. Acad. Sci. U. S. A. 100:4457, 2003.
F i g . 1 . The oligoamide trimer ATT derived from 2′,3′-dideoxy-
2′,3′-diamino-TNA. A indicates adenine; T, thymine.
24
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
5,8-Diaza-7,9-dicarba-2,6-diaminopurine is just
one member of a family of alternative purine nucleobases that potentially can undergo C-nucleosidation
with iminium ions and aldosugars. We are expanding
our investigation to other members of this family, including an extension of the C-nucleosidation chemistry to
pyrimidines and still another family of purinoids.
Jungmann, O., Beier, M., Luther, A., Huynh, H.K., Ebert, M.O., Jaun, B., Krishnamurthy, R., Eschenmoser, A. Pentopyranosyl oligonucleotide systems: communication No. 13. The α-L-arabinopyranosyl-(4′→2′)-oligonucleotide system: synthesis
and pairing properties. Helv. Chim. Acta 86:1259, 2003.
Schöning, K-.U., Scholz, P., Wu, X., Guntha, S., Delgado, G., Krishnamurthy, R.,
Eschenmoser, A. The α-(L)-threofuranosyl-(3′→2′)-oligonucleotide system (‘TNA’):
synthesis and pairing properties. Helv. Chim. Acta. 85:4111, 2002.
Wang, Z., Huynh, H.K., Han, B., Krishnamurthy, R., Eschenmoser, A. 2,6-Diamino5,8-diaza-7,9-dicarba-purine. Org. Lett. 5:2067, 2003.
STRUCTURAL PROPERTIES OF TNAS
In collaboration with M. Egli, Vanderbilt University,
Nashville, Tennessee, we determined the crystal structure of the B-form DNA duplex [d(CGCGAA)T*d(TCGCG)]2
containing a single (L)-α-threofuranosyl-(3′→2′)-thymine
(T*) per strand. The structure reveals that single TNA
thymine nucleotides can be easily accommodated in a
B-form of DNA with little change overall relative to the
reference DNA structure (Fig. 2). Nuclear magnetic
resonance analysis of the TNA duplex containing the
self-complementary sequence t-(CGAATTCG) was carried
out by B. Jaun and H. Ebert at the Swiss Federal Institute of Technology Zürich in Switzerland. The analysis
shows that TNA forms a right-handed double helix
resembling that of RNA.
F i g . 2 . The x-ray structure analysis of the modified Dickerson
Drew dodecamer duplex [d(CGCGAA)T*d(TGCGCG)]2 shows the
C4′-exo conformation of the L-(α)- threofuranose nucleoside (T*7)
in the modified duplex.
PUBLICATIONS
Ebert, M.O., Luther, A., Huynh, H.K., Krishnamurthy, R., Eschenmoser, A., Jaun,
B. NMR solution structure of the duplex formed by self-pairing of α-(L)-arabinopyranosyl-(4′→2′)-(CGAATTCG). Helv. Chim. Acta. 85:4055, 2002.
Han, B., Wang, Z., Jaun, B., Krishnamurthy, R., Eschenmoser, A. C-Nucleosidations with 2,6-diamino-5,8-diaza-7,9-dicarba-purine. Org. Lett. 5:2071,2003.
Wilds, C.J., Wawrzak, Z., Krishnamurthy, R., Eschenmoser, A., Egli, M. Crystal
structure of a B-form DNA duplex containing (L)-α-threofuranosyl (3′→2′) nucleosides: a four-carbon sugar is easily accommodated into the backbone of DNA. J.
Am. Chem. Soc. 124:13716, 2002.
Intracellular RNA Assembly
and Catalysis
M.J. Fedor, K.F. Baban, C.P. Da Costa, Y.I. Kuzmin,
E.M. Mahen, S.B. Voytek, R.S. Yadava
NAs are essential components of the molecular
machinery that underlies growth and development. Virtually all important RNA-mediated
reactions, such as those involved in gene regulation,
splicing, and protein synthesis, involve assembly and
dissociation of RNA complexes. Formation of RNA complexes often occurs through recognition between complementary RNA nucleobases and annealing of base-paired
helices. Reaction pathways for many RNA enzymes also
involve assembly and dissociation of ribozyme complexes that contain intermolecular base-paired helices.
Unlike the complicated RNA-mediated reactions that
participate in gene expression, however, ribozyme
cleavage of RNA substrates gives a simple, quantitative signal that a functional ribozyme-substrate complex has formed.
Enzymological studies of ribozymes have been used
to dissect RNA assembly processes in vitro and have
defined how reaction conditions and structural features
of ribozyme and substrate RNAs influence the kinetics
and equilibria of assembly and dissociation in ribozyme
reaction pathways. However, establishing how the properties of RNA complex formation that can be measured
in simple solutions relate to the behavior of RNA molecules in the complicated environment inside living
cells has been difficult.
By exploiting the simple RNA cleavage reactions
mediated by the hairpin ribozyme, we devised a way
to learn how evidence from studies of RNA enzymes in
vitro translates to the complex environment of a living
R
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
cell. Our early studies established which features of
ribozyme structure are important for self-cleavage inside
cells. Now we are focusing on recognition and cleavage of separate RNA targets in reactions that require
assembly of functional ribozyme-substrate complexes.
To examine intracellular ribozyme activity, we express
hairpin ribozymes in yeast as chimeric U3 small nucleolar RNAs (snoRNAs) (Fig. 1). The snoRNAs are useful
carriers of ribozyme and substrate sequences because
they are stable RNAs that accumulate at high concentrations in yeast. Chimeric snoRNAs containing hairpin
ribozymes are coexpressed in yeast with chimeric substrate snoRNAs that contain the cleavage site. Chimeric
ribozyme snoRNAs are expressed under the control of
a galactose-inducible promoter so that intracellular ribozyme concentrations can be modulated by changing the
galactose concentration in yeast growth media. Formation of a functional complex between ribozyme and
substrate snoRNAs leads to substrate RNA cleavage.
25
through ribozyme-mediated cleavage. Consequently,
substrate RNAs that associate with ribozymes and
undergo cleavage disappear faster from yeast than do
substrate RNAs that are expressed in yeast without
ribozymes. The difference in decay rates between substrates expressed alone and substrates coexpressed with
ribozymes corresponds quantitatively to the intracellular
cleavage rate. By using the intrinsic snoRNA degradation
rate as a “clock” in this way, we can evaluate the effects
of RNA structure and concentration on intracellular
cleavage kinetics.
We found that intermolecular cleavage increases
with increasing RNA concentrations, as expected if
formation of the ribozyme-substrate complex is rate
determining. A second-order rate constant for the formation of RNA complexes in vivo can be calculated
from the dependence of cleavage kinetics on intracellular concentrations of ribozyme. The second-order
rate constant in yeast is significantly lower than rate
constants measured for cleavage reactions under physiologic salt conditions in vitro, but it has a similar
dependence on the length of the intermolecular basepaired helices that form during complex formation.
These studies provide the first quantitative description of the kinetics of the formation of RNA complexes
in vivo. Understanding this simple example of an interaction between ribozyme and target RNAs will provide
insights into more complicated RNA interactions that
perform essential roles in biology but are not as easily
studied in the laboratory.
PUBLICATIONS
Fedor, M.J. Determination of kinetic parameters for hammerhead and hairpin
ribozymes. Methods Mol. Biol., in press.
Fedor, M.J. RNA biochemistry from A to Z. Cell 109:20, 2002.
F i g . 1 . Chimeric ribozyme-substrate snoRNA complex. Chimeric
snoRNAs that contain either ribozyme or substrate sequences are
coexpressed in yeast to evaluate the reactions involved in the formation of RNA complexes that must precede intermolecular cleavage. Assembly of a functional ribozyme complex requires base pairing between complementary ribozyme and substrate sequences to
form H1 and H2 helices.
In the absence of chimeric ribozymes, chimeric substrates disappear from yeast over time through the endogenous snoRNA degradation pathway. Normal degradation
of snoRNA occurs at a characteristic rate. When substrates are coexpressed with ribozymes, substrates disappear through the normal degradation pathway, as do
all snoRNAs, but the substrates also can be destroyed
Fedor, M.J., Westhof, E. Ribozymes: the first 20 years. Mol. Cell 10:703, 2002.
Viruses as Molecular
Building Blocks
M.G. Finn, D.D. Díaz, W.G. Lewis, J.-C. Meng, S. Meunier,
D. Prasuhn, S. Punna, K.S. Raja, Q. Wang
tarting with plant and insect viruses that are
innocuous to mammals but well understood
structurally, our goals are to create entities useful for the detection and treatment of disease, for the
construction of functional materials, and for the catal-
S
26
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
ysis of important chemical reactions. We bring the techniques of both chemistry and biology to this endeavor,
an appropriate strategy because the size of virus particles makes them a natural bridge between these worlds.
The combination is powerful. In the past year we made
the following major advances.
F U N D A M E N TA L C H E M I S T R Y
We developed 2 new methods for making chemical
connections to viral coat proteins (and, by extension,
many other biomolecules). The more exciting of these
is the result of a collaboration with K.B. Sharpless and
V.V. Fokin, the Skaggs Institute, in which we used a copper-catalyzed connection between azides and alkynes.
C A R B O H Y D R AT E - D E C O R AT E D PA R T I C L E S
We further explored the chemistry and biology of
carbohydrate-decorated virus particles. We quantified
the enhancements obtained in interactions with cellular
receptors by displaying the binding agents in multiple
copies over the viral surface. This “polyvalency” is at
the heart of prospective uses of virus particles for drug
delivery and tissue imaging. Our measurements indicate
that the effective polyvalencies of virus particles are
as good as those of the best organic polymers made
for this purpose, and the viruses have other advantages
relative to these known systems. In collaboration with
S. Schmid and coworkers, The Scripps Research Institute, we showed endocytosis of virus particles into liver
cells mediated by carbohydrate-specific receptors.
PA R T I C L E S D E C O R AT E D W I T H
A R G I N I N E – G LY C I N E – A S PA R T I C A C I D
The tripeptide arginine–glycine–aspartic acid (RGD)
is recognized by a large family of proteins (integrins)
that are crucial to a wide variety of cellular recognition
and signaling processes. Many RGD-receptor interactions are thought to involve the simultaneous contact
of more than one RGD sequence with the target cell,
and this polyvalency has been the subject of much
investigation and debate. We expressed RGD sequences
corresponding to those used by the common cold virus
(adenovirus) on the surface of cowpea mosaic virus
(CPMV) and found that these particles bind selectively
to the cells that adenovirus itself targets. This work,
performed in collaboration with G. Nemerow, The Scripps
Research Institute, will be expanded in the coming year
to include the chemical synthesis and attachment of
active peptides.
ANTIBODY INTERACTIONS AND THE
IMMUNE RESPONSE
We measured the response of the mouse immune
system to CPMV and showed that the particle can be
made less immunogenic by covering it with polymeric
chains attached by chemical ligation. Novel polymer
synthesis and attachment strategies are currently in
development; our goal is to selective modulate or
enhance the antigenicity of tailored virus particles.
CPMV has been found in the blood, lymphatic system,
spleen, and mucosal tissues of mice after oral administration, setting the stage for the use of CPMV as an
oral delivery agent for compounds used to report on
and treat disease. This work is done in collaboration
with M. Manchester and coworkers, The Scripps
Research Institute.
N E W V I R U S P L AT F O R M S
We performed the first surveys of the chemical
stabilities and reactivities of 2 insect viruses: flock
house virus and nudaurelia capensis ω virus. Both are
potentially more useful than CPMV for biological and
catalytic purposes because of the following properties.
They are composed of 180 and 240, rather than 60,
identical protein subunits, respectively, allowing a higher
density of connection points to the structure. Mutants
of these viruses can be generated in a matter of days,
rather than weeks, as is the case with CPMV. Flock
house virus has far fewer reactive groups on its exterior than does CPMV, and nudaurelia capensis ω virus
undergoes a unique and dramatic structural change in
response to pH.
PUBLICATIONS
Lewis, W.G., Shen, Z., Finn, M.G., Siuzdak, G. Desorption-ionization on silicon
(DIOS) mass spectrometry: background and applications. Int. J. Mass Spectrom.
226:107, 2003.
Raja, K.S., Wang, Q., Finn, M.G. Icosahedral virus particles as polyvalent carbohydrate display platforms. Chembiochem 4:1348, 2003.
Raja, K.S., Wang, Q., Gonzalez, M.J., Manchester, M., Johnson, J.E., Finn, M.G.
Hybrid virus-polymer materials, 1: synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 4:472, 2003.
Ripka, A.S., Díaz, D.D., Sharpless, K.B., Finn, M.G. First practical synthesis of
formamidine ureas and derivatives. Org. Lett. 5:1531, 2003.
Smith, J.C., Lee, K.-B., Wang, Q., Finn, M.G., Johnson, J.E., Mrksich, M.,
Mirkin, C.A. Nanopatterning the chemospecific immobilization of cowpea mosaic
virus capsid. Nano Lett. 3:883, 2003.
Wang, Q., Chan, T., Hilgraf, R., Fokin, V.V., Sharpless, K.B., Finn, M.G. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem.
Soc. 125:3192, 2003.
Wang, Q., Raja, K.S., Janda, K.D., Lin, T., Finn, M.G. Blue fluorescent antibodies as
reporters of steric accessibility in virus conjugates. Bioconjug. Chem. 14:38, 2003.
Yao, S., Meng, J.-C., Siuzdak, G., Finn, M.G. New catalysts for the asymmetric
hydrosilylation of ketones discovered by mass spectrometry screening. J. Org.
Chem. 68:2540, 2003
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
27
Insights Into Protein
Chemistry and Biology
From Protein Structure
E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau,
R.M. Brudler, J.M. Castagnetto, D. Cerutti, T. Cross,
M. DiDonato, E.D. Garcin, U.K. Genick, S.W. Hennessy,
C. Hitomi, K. Hitomi, C.J. Kassmann, I. Li, S.J. Lloyd,
M.E. Pique, R.J. Rosenfeld, M.E. Stroupe, M.M. Thayer,
M.J. Thompson, J.L. Tubbs, T.I. Wood
C H E M I C A L B I O L O G Y A N D R E G U L AT I O N O F N I T R I C
OXIDE SYNTHASES
e are studying nitric oxide synthases (NOSs),
key therapeutic targets important for blood
pressure regulation (endothelial NOS), neurotransmission (neuronal NOS), and the immune response
(inducible NOS). These 3 similar, but differentially regulated, isozymes synthesize the diatomic molecule nitric
oxide, which is paradoxically both a molecular signal
(at low concentrations) and a cytotoxin (at high concentrations). Isozyme-specific NOS inhibitors are highly
desirable for medicinal purposes and to advance our
understanding of basic human physiology.
Each of the 3 isozymes has 2 major modules: (1) a
catalytic oxygenase with binding sites for heme, tetrahydrobiopterin, and substrate and (2) an electron-supplying reductase with binding sites for NADPH, FAD, and
FMN. The 2 modules are connected by a central linker
that binds the calcium-regulated protein calmodulin.
Electron transfer from the reductase to the oxygenase
and consequent production of nitric oxide are triggered
by the binding of calmodulin. Our recent crystallographic
structure of calcium-loaded calmodulin bound to a
20-residue peptide from the endothelial NOS linker
reveals how regulatory phosphorylation can counteract
this calmodulin activation (Fig. 1). The goals of our
ongoing mutational, biochemical, and structural studies of NOS are to (1) determine how the many domains
of the dimeric holoenzyme assemble, bind their cofactors, and perform complex catalytic and redox chemistry;
(2) identify and apply the features distinguishing the
3 isozymes to the design of isozyme-specific inhibitors;
and (3) understand the diverse regulatory mechanisms
that differentially control the 3 isozymes.
W
ENZYME-COFACTOR INTERACTIONS
New realms of activities are made available to proteins by bound cofactors, which include metal ions,
vitamins, and other small molecules. Macrocyclic metal-
F i g . 1 . The solvent-exposed threonine phosphorylation site (T495)
on the endothelial NOS linker (dark gray ball-and-stick model) is
surrounded by glutamic acid residues (E7, E11, E127, and E498)
provided by both lobes of calmodulin (pale gray), explaining why
phosphorylation here counteracts calmodulin activation of nitric
oxide synthesis.
chelating cofactors, such as vitamin B 12 (cobalamin),
chlorophyll, heme, and siroheme, share a common
framework constructed during the initial steps of the
branched tetrapyrrole biosynthetic pathway. We determined crystallographic structures of (1) sulfite reductase, the enzyme that uses siroheme to prepare reduced
sulfur for incorporation into biomolecules, and (2) siroheme synthase, the enzyme that generates the siroheme
cofactor from uroporphyrinogen.
With our high-resolution structures of the sulfite
reductase hemoprotein, we measured the geometric distortions in the coupled siroheme and 4Fe4S cluster
cofactors that tune their redox properties for enzyme
function. Our crystallographic structures of siroheme synthase, in conjunction with biochemical results, yielded
insights for the 4 separate reactions catalyzed by this
enzyme: 2 distinct S-adenosyl-L-methionine–dependent
transmethylations, NAD +-dependent dehydrogenation,
and ferrochelation. We identified an unexpectedly phosphorylated serine residue (Fig. 2) poised to inhibit the
dehydrogenation reaction and thus modulate metabolic flux between the siroheme and cobalamin biosynthetic pathways.
Photoactive chromophores enable proteins to translate light energy into defined conformational changes
28
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
analyses suggest that this cryptochrome functions in
light-dependent transcriptional regulation.
For green and red fluorescent proteins, our high-resolution crystallographic structures provide insights into
the mechanism by which 3 component amino acid residues are spontaneously converted after translation into
fluorescent chromophores of different colors. We determined structures of trapped intermediates in fluorophore
synthesis, identified features key to fluorophore formation and spectral tuning, and proposed a novel conjugation-trapping mechanism for fluorophore synthesis.
MACROMOLECULAR ASSEMBLIES IN HUMAN
H E A LT H A N D D I S E A S E
F i g . 2 . Electron density contours (cages) reveal phosphorylated
serine (Ser128, bottom) deep within the dehydrogenase/ferrochelatase
active site of siroheme synthase. Located about 11 Å away from the
NAD+ cofactor (top), this unexpected phosphoserine appears poised
to inhibit the dehydrogenation reaction and thus modulate metabolic
flux between the siroheme and cobalamin biosynthetic pathways.
or fluorescence to send biological signals. We are studying the mechanisms of light-induced protein activities
in the blue-light receptor photoactive yellow protein;
the cryptochrome flavoproteins, which are components
of circadian clocks in animals and humans; and the
family of green and red fluorescent proteins used as
biological markers.
By combining ultrahigh-resolution crystallography
of photoactive yellow protein with different types of
computational chemistry, we defined mechanisms for
photochemical tuning, elucidated intermediates in the
light cycle, resolved controversies arising from spectroscopic studies, and identified active-site dynamics that
favor photoisomerization of the chromophore. For cryptochrome, we sequenced a new gene, identified a new
cryptochrome protein family, and determined the first
crystal structure. The structure contains the redox-active
FAD cofactor bound in an unusual U-shaped conformation with a surrounding positive electrostatic surface
consistent with a function in DNA binding. Microarray
We are studying the consequences for human health
of appropriate macromolecular recognition and assembly. In collaboration with J. Tainer, the Skaggs Institute,
we focus on enzymes (NOS and superoxide dismutase)
that control reactive oxygen species and on the pilus
virulence factors responsible for the attachment of
pathogenic bacteria to human hosts. For human superoxide dismutase, we are investigating the mechanisms
by which many different, naturally occurring, single-site
mutations cause the fatal neurodegenerative disease
amyotrophic lateral sclerosis. Through structural and
biochemical studies of recombinantly produced superoxide dismutase proteins that incorporate these human
genetic defects, we showed that the mutant proteins
are architecturally destabilized and can form amyloidlike aggregates, resembling the aggregates found in
the motor neurons of patients with amyotrophic lateral
sclerosis. For pili and their component pilin proteins
from bacteria that cause gonorrhea, meningitis, pneumonia, and cholera, we are characterizing how pili
assemble, function, and help these bacteria evade the
immune response in humans.
PUBLICATIONS
Aoyagi, M., Arvai, A.S., Tainer, J.A., Getzoff, E.D. Structural basis for endothelial
nitric oxide synthase binding to calmodulin [published correction in EMBO J.
22:1234, 2003]. EMBO J. 22:766, 2003.
Barondeau, D.P., Putnam, C.D., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by
trapped intermediate structures. Proc. Natl. Acad. Sci. U. S. A. 100:12111, 2003.
Brudler, R., Hitomi, K., Daiyasu, H., Toh, H., Kucho, K., Ishiura, M., Kanehisa,
M., Roberts, V.A., Todo, T., Tainer, J.A., Getzoff, E.D. Identification of a new cryptochrome class: structure, function, and evolution. Mol. Cell 11:59, 2003.
Craig, L., Taylor, R.K., Pique, M.E., Adair, B.D., Arvai, A.S., Singh, M., Lloyd,
S.J., Shin, D.S., Getzoff, E.D., Yeager, M., Forest, K.T., Tainer, J.A. Type IV pilin
structure and assembly: x-ray and EM analyses of Vibrio cholerae toxin-coregulated
pilus and Pseudomonas aeruginosa PAK pilin. Mol. Cell 11:1139, 2003.
DiDonato, M., Craig, L., Huff, M.E., Thayer, M.M., Cardoso, R.M., Kassmann,
C.J., Lo, T.P., Bruns, C.K., Powers, E.T., Kelly, J.W., Getzoff, E.D., Tainer, J.A.
ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization. J. Mol. Biol. 332:601, 2003.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Getzoff, E.D., Gutwin, K.N., Genick, U.K. Anticipatory active-site motions and
chromophore distortion prime photoreceptor PYP for light activation. Nat. Struct.
Biol. 10:663, 2003.
Meyer, T.E., Devanathan, S., Woo, T., Getzoff, E.D., Tollin, G., Cusanovich, M.A.
Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila.
Biochemistry 42:3319, 2003.
Stroupe, M.E., Leech, H.K., Daniels, D.S., Warren, M.J., Getzoff, E.D. CysG
structure reveals tetrapyrrole-binding features and novel regulation of siroheme
biosynthesis. Nat. Struct. Biol. 10:1064, 2003.
Sung, N.S., Gordon, J.I., Rose, G.D., Getzoff, E.D., Kron, S.J., Mumford, D.,
Onuchic, J.N., Scherer, N.F., Sumners, D.L., Kopell, N.J. Science education: educating future scientists. Science 301:1485, 2003.
Thompson, M.J., Bashford, D., Noodleman, L., Getzoff, E.D. Photoisomerization and
proton transfer in photoactive yellow protein. J. Am. Chem. Soc. 125:8186, 2003.
Wei, C.C., Wang, Z.Q., Arvai, A.S., Hemann, C., Hille, R., Getzoff, E.D., Stuehr,
D.J. Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy
intermediate in nitric oxide synthase. Biochemistry 42:1969, 2003.
Supramolecular Approaches to
Drug Design, Cell Diagnostics,
and Complex System Engineering
29
dynamics and coupling architecture is currently unanswered. This status is largely due to the lack of a powerful self-organized model system that is subject to de
novo design, predictable topological organization, experimental characterization at the level of the kinetics of
its individual pathways, and direct manipulation such
that the overall effects in the system of the changes
(e.g., input variations, network reorganization) can be
rigorously studied and understood.
We are attempting to bridge the gap between the
theoretical graph-based theories of network organization and the experimental realm. We developed a system that is simple in its basic molecular components
to allow rational design and networking predictions but
large in sequence space to provide the basis for the
construction of complex networks. The system is based
on template-directed coiled-coil peptide fragment condensation reactions that are substrate selective in
their ligase and replicase activities (Fig. 1). For our
studies, we designed 81 peptides and calculated the
free energies of all A 2B-type ensembles to determine
plausible network connectivities via autocatalysis and
cross-catalysis pathways. The figure shows clustered
M.R. Ghadiri, M. Al-Sayah, G. Ashkenasy, A. Chavochi,
J. Fletcher, V. Haridas, W.S. Horne, R. Jagasia, L. Leman,
S. Rahimipour, M. Yadav
e seek to develop novel chemical processes
for use in the design of complex functional
systems. Our multidisciplinary research program ranges from the design, prediction, and study of
complex peptide networks to synthesis and selection
of antimicrobial, anticancer, and optical cell diagnostic
agents. These ongoing efforts are expected to contribute
to a better understanding of the complex protein networks of living systems, assist in developing simple and
effective cell diagnostic tools for early detection of disease states, and provide a diverse supramolecular therapeutic approach for treating cancer and combating
multidrug-resistant microbial infections.
W
DESIGN OF COMPLEX SELF-ORGANIZED
CHEMICAL NETWORKS
In several recent theoretical studies, researchers
probed the distinctive statistical properties of complex
systems in social, technological, and biological networks.
The results revealed some of the unifying principles that
underlie the topology of complex self-organized networks. However, the central question of how interacting dynamic systems (whether animate or inanimate)
can produce collective behavior from their individual
F i g . 1 . Top, Schematic representation of the template-directed
peptide fragment ligation. The electrophilic peptide fragment E i
bearing a C-terminal thiolester moiety and the nucelophilic peptide
N bearing an N-terminal cysteine residue can preorganize on the
complementary peptide template Tj to form a coiled-coil ternary (not
shown) or quaternary (depicted) complex. The productive juxtaposition and enhanced effective concentration of the reactive functionalities
facilitate the Kent ligation process, proceeding through the transthiolester intermediate followed by intramolecular aminolysis, to give
product T i. The reaction is autocatalytic when T i = T j and crosscatalytic when T i ≠ T j. Bottom, The experimentally derived network
architecture. The arrows (edges) designate template-assisted ligation
pathways pointing from the template to the product. Numbers along
the edges are the theoretical –∆∆G free energy values (kcal mol–1).
30
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
nodes with an overall hierarchical architecture. Nine
nodes were selected for experimental evaluation of their
capacity to establish the predicted network connectivity. The resulting self-organized chemical network has
25 directed edges, including 3 autocatalytic processes,
in good agreement with predictions.
Moreover, we showed that by varying the system
inputs (substrates and templates), we can alter its network topology, even to the extremes of turning pathways
on or off. In our opinion, the system described provides
the first tool for designing and analyzing complex systems in a highly rational and informative manner. This
approach can offer a means to study and to better understand the emergent, collective behaviors of networks.
New Vistas in
Immunopharmacotherapy
SELF-ASSEMBLING PEPTIDE NANOTUBES AS A
ANTIBODY LIBRARIES
NEW CLASS OF CELL DIAGNOSTIC, ANTIMICROBIAL,
hage display antibody technology is a powerful
method for the design and discovery of antibodies that bind targets with high specificity and
affinity. Because the use of combinatorial antibody
libraries displayed on the surface of phage particles is
an entirely in vitro procedure, it obviates animal-, labor-,
and time-intensive immunization and hybridoma protocols required to isolate monoclonal antibodies. We
extended the usefulness of phage display methods, most
notably by developing fully human single-chain Fv (scFv)
and Fab antibody library formats displayed on phage.
These libraries allow the direct selection of antigenspecific, fully human antibodies that can be moved
more expeditiously into clinical trials.
The phage coat proteins pIII and pVIII have been
used extensively for the display of antibody libraries;
pIII in particular has been used to select numerous
highly specific human antibodies, which continue to
be the focus of a great deal of research in biotechnology and medicine. We expanded the sphere of phage
display antibody technology to develop antibody libraries that are combinatorial not just in composition but
also in conformational space. We accomplished this
step by using pVII and pIX, which are constitutively
separate but physically near each other, for the simultaneous display of scFv variable heavy-chain region
(V H) and variable light-chain region (VL) chains. This
past year, we extended the usefulness of in vitro antibody library design by developing a novel phage display format in which only the pIX coat protein is used.
In our model studies, this display system allowed the
selection of highly specific antibodies that recognized
a number of complex antigens (e.g., cholera toxin and
ricin) important in both medicine and defense against
AND ANTICANCER AGENTS
Supramolecular systems constitute an emerging
frontier in drug design and diagnostics. The capacity
for self-organization and cellular self-targeting offered
by self-assembling peptide nanotubes can provide a
range of functional attributes that are not generally
feasible by the traditional molecular approaches. Peptide nanotubes are thought to operate on targeted cellular membranes by membrane absorption of small cyclic
peptides followed by self-assembly into hole-punching
supramolecular tubular complexes. The class of selfassembling cyclic peptides can provide an unprecedented
level of membrane discrimination via the capability to
sense and respond to the membrane environment. We
exploited these characteristics to design self-assembling
peptide nanotubes with anticancer and potent antimicrobial activities against multidrug-resistant infections.
In our studies, we use various combinatorial cyclic
peptide library approaches to select and optimize desired
biological activities against a variety of cellular targets.
Using fluorescent-labeled cyclic peptide libraries, we
exploited the capacity of self-assembling peptide nanotubes to discriminate cell membranes to design optical systems for detecting pathogens. The new avenue
of research in the design of photonic cell recognition
agents is expected to yield a series of diagnostic tools
that complement our ongoing efforts directed at developing antimicrobial and anticancer therapeutic agents.
K.D. Janda, J.-M. Ahn, J. Ashley, G. Boldt, R. Carrera,
Y. Chen, B. Clapham, A. Coyle, T. Dickerson, Y. Ding,
L. Eubanks, T. Fujimori, R. Galve, C. Gambs, C. Gao,
T. Hoffman, L. Hom, G. Kaufmann, Y.-S. Kim, E. Laxman,
B.-S. Lee, S.-H. Lee, S.-J. Lee, M. Lillo, S. Mahajan,
H. Matsushita, M. Matsushita, J. McDunn, G. McElhaney,
J. Mee, M. Meijler, J. Moss, J.-I. Park, N. Reed, A. Shafton,
C. Sun, R. Troseth, A.D. Wentworth, P. Wentworth, Jr.,
P. Wirsching, Y. Xu, N. Yamamoto, K. Yoshida, B. Zhou
IN VITRO DESIGN OF HUMAN
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
bioterrorism. We are investigating the scope and advantages of this new antibody display technology as an
adjunct and alternative to existing methods.
In parallel with our work on the basic science of
antibody phage display technology, we continue to
explore new biomedical applications for in vitro antibody libraries. For example, we used an in vitro human
scFv antibody library to select for antibodies that not
only specifically bound to but were internalized into
cancer cells. This finding is clinically relevant, because
many of the side effects of cancer chemotherapy stem
from the inherent nonspecificity of the cytotoxic drugs
used to kill cancer cells. By attaching such drugs to
cancer cell–specific, internalizing antibodies, the deleterious side effects of many of these chemotherapeutic
agents could be attenuated, improving the prognoses
for cancer patients.
Although attaching a single drug molecule to a cancer cell–specific, internalizing antibody clearly is of
therapeutic interest, we prepared dendritic carriers for
attaching multiple drug copies to a single scFv antibody (Fig. 1). Through the use of these hybrid macromolecules, which harness the exquisite specificity and
drug delivery potential of these antibodies to the potent
toxicity of synthetic chemotherapeutic dendrimers, the
outlook for cancer chemotherapy in the coming years
could be all the brighter.
F i g . 1 . Preparation of a site specifically modified scFv antibody–
drug immunoconjugate by using a multifunctional dendritic drug carrier.
VA C C I N AT I O N F O R S E P T I C S H O C K
Septic shock is 1 of the 10 leading causes of infant
and adult mortality in the United States. According to
the Centers for Disease Control and Prevention, this
condition was directly linked to more than 30,000
deaths in 1999 alone. The prime pathogenic determinant of septic shock is lipopolysaccharide, a class of
oligosaccharides liberated by lysis of bacteria that have
multiple O-linked fatty acyl substituents. The Escherichia
coli isoform of lipid A is the most well-studied form of
lipopolysaccharide and is responsible for the toxic effects
associated with most documented cases of septic shock.
31
One promising therapeutic tactic is use of a passive
vaccine, that is, monoclonal antibodies that could, in
principle, be used to bind lipid A and mediate its
clearance from the body during instances of infection.
Although this strategy showed promise at an early stage
of research, it was not effective in clinical trials, underscoring the need for alternative therapeutic strategies.
We developed an immunotherapeutic approach for
treatment of septic shock that involves the use of an
active vaccine, a designed synthetic variant of lipid A
(compound 1 in Fig. 2), that could induce an innate
immune response to future exposure to lipopolysaccharide, thereby abrogating the toxic effects. We designed
F i g . 2 . Bisphosphonate TSAs 2a and 2b were synthesized as
immunogens for active immunization to treat lipid A–mediated
endotoxicosis. The native E coli lipid A structure is depicted in 1.
a structural homolog of E coli lipid A with 2 key features. First, a bisphosphonate transition-state analog
(TSA) motif, a mimic for hydroxide-catalyzed ester
hydrolysis, was incorporated in place of the 2 acyloxyacyl motifs present in the native lipid A structure. This
modified structure was used to elicit antibodies that
not only bound lipid A but also catalytically deactivated
it through catalysis of base-catalyzed fatty ester hydrolysis. Second, 2 different immunostimulatory linkers were
used to attach the final TSA structure to a carrier protein for immunization. These linkers were included to
circumvent the notoriously low immunogenicity of carbohydrate-based antigens, which would otherwise have
severely hampered the selection of a lipid A–specific
antibody binder or catalyst.
Immunization of 3 different mouse strains with compounds 2a and 2b (Fig. 2), as conjugates to the carrier
protein keyhole limpet hemocyanin (KLH), led to quantitatively different immune responses, suggesting that
the choice of linker directly affected the immunogenicity
of the TSA structure. These differences notwithstanding,
the obtained antibodies were qualitatively similar, as
evinced by significant cross-reactivity of the antibodies
elicited to immunogens 2a and 2b.
To investigate the protective effect of the TSA conjugates, we gave the immunized mice sublethal bolus
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
doses of lipid A. We measured the efficacy of the active
vaccine by using the known upregulatory effect of lipid
A on the production of TNF-α. Normalizing to TNF-α
production in control mice not immunized with KLH
conjugates of 2a or 2b, we observed between a 4- to
20-fold reduction in TNF-α production (depending on
the mouse strain) after lipid A challenge in mice protected by previous immunization with KLH conjugates
of 2a or 2b.
It is unclear as yet whether this immunoprotection
against the toxic effects of E coli lipid A is due to the
activity of antibody catalysts or binders. Nonetheless,
these results indicate the therapeutic potential of
rationally designed phosphonate TSAs for harnessing
the native immune system for immunotherapy of septic shock in an active vaccine program. We are isolating and characterizing the responsible antibodies for
mechanistic characterization as quick-acting therapeutic entities themselves in a complementary passive
vaccine approach.
IMMUNOPHARMACOTHERAPY FOR NICOTINE ABUSE
Cigarette smoking is still the leading cause of avoidable death due to cancer worldwide; as such, it has the
unique distinction of being an elective malady of pandemic proportions. Increasingly, evidence suggests that
chronic use of cigarettes and other tobacco products is
due in large part to the addictive psychoactive and reinforcing effects of nicotine on the brain. Although socalled nicotine replacement therapies have grown in
popularity in recent years, these are not true therapies
per se; rather they are merely alternative forms of nicotine delivery. Because these strategies do not attack
the underlying problem, the neurostimulatory effects
of nicotine and concomitant user dependence on it,
they have done little to decrease tobacco dependence
on the whole.
In recent years, we and other researchers have
explored immunopharmacotherapy as an alternative
route to tackle the problem of chronic nicotine dependence. Several different approaches have been used to
elicit antibodies that bind and thereby block nicotine
from exerting its neurostimulatory effects on the brain.
Although the structure of nicotine does not suggest that
it poses any significant difficulties in the generation of
antibody binders, all synthetic nicotine haptens prepared thus far have unexplainably fallen short of their
promise. This situation led us to explore the possibility
of some fundamental, yet hidden, chemical foundation
for this problem.
Recently, new data indicated that in aqueous solution, nicotine exists in multiple conformations arising
from rotation about the pyrrolidine-pyridine linkage axis.
This finding is of key relevance for our research because
it suggests a possible rationale for the unvaryingly low
affinity of nicotine-binding antibodies, irrespective of
protein conjugation chemistry. Whereas we and others
viewed nicotine as a static, torsionally constrained
2-dimensional molecule, the immune system constantly
samples all accessible solution-phase conformations.
In exchange for high specificity and affinity, immunization with nicotine conjugates prepared to date—by us
as well as by others—inevitably leads to a heterogenous antibody response. The isolated antibodies are
characterized by low conformational specificity and an
affinity that is averaged over a broad sample space of
nicotine conformers.
We surmised that the solution-phase conformations
of nicotine might be partly responsible for our otherwise unexplainably low success in generating nicotinebinding antibodies with our first-generation nicotine
hapten (compound 3 in Fig. 3). Therefore, we prepared
conformationally constrained nicotine analogs 4 and 5
(Fig. 3) for immunization to elicit improved antibody
binders. The haptens were attached to KLH by using
the same linkage chemistry previously used for 3 to
allow meaningful side-by-side comparison. We were
gratified to find that the serum antibody titers resulting from immunization with haptens 4 and 5 were 7to 8-fold higher than those obtained with the conformationally free hapten 3.
F i g . 3 . First-generation nicotine hapten 3 and constrained trans
analogs 4 and 5 used to elicit improved nicotine-binding antibodies.
We are isolating and characterizing the affinity of
monoclonal antibodies generated in response to these
conformationally constrained nicotine haptens. We envision that these antibodies could be useful in improving
the efficacy of immunopharmacotherapy strategies as
treatments for nicotine addiction.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
PUBLICATIONS
Ahn, J.-M., Wentworth, P., Jr., Janda, K.D. Soluble polymer-supported convergent
parallel library synthesis. Chem. Commun. (Camb.) 4:480, 2003.
Cannizzaro, C.E., Ashley, J.A., Janda, K.D., Houk, K.N. Experimental determination of the absolute enantioselectivity of an antibody-catalyzed Diels-Alder reaction
and theoretical explorations of the origins of stereoselectivity. J. Am. Chem. Soc.
125:2489, 2003.
Clapham, B., Lee, S.-H., Koch, G., Zimmermann, J., Janda, K.D. The preparation
of polymer bound β-ketoesters and their conversion into an array of oxazoles. Tetrahedron Lett. 43:5407, 2002.
33
Wentworth, P., Jr., McDunn, J.E., Wentworth, A.D., Takeuchi, C., Nieva, J.,
Jones, T., Bautista, C., Ruedi, J.M., Gutierrez, A., Janda, K.D., Babior, B.M.,
Eschenmoser, A., Lerner, R.A. Evidence for antibody-catalyzed ozone formation in
bacterial killing and inflammation. Science 298:2195, 2002.
Wentworth, P., Jr., Wentworth, A.D., Zhu, X., Wilson, I.A., Janda, K.D., Eschenmoser, A., Lerner, R.A. Evidence for the production of trioxygen species during
antibody-catalyzed chemical modification of antigens. Proc. Natl. Acad. Sci.
U. S. A. 100:1490, 2003.
Zhu, X.Y., Heine, A., Monnat, F., Houk, K.N., Janda, K.D., Wilson, I.A. Structural basis for antibody catalysis of a cationic cyclization reaction. J. Mol. Biol.
329:69, 2003.
Delgado, M., Janda, K.D. Polymeric supports for solid phase organic synthesis.
Curr. Org. Chem. 6:1031, 2002.
Dickerson, T.J., Janda, K.D. A previously undescribed chemical link between smoking and metabolic disease. Proc. Natl. Acad. Sci. U. S. A. 99:15084, 2002.
Dickerson, T.J., Janda, K.D. Glycation of the amyloid β-protein by a nicotine metabolite: a fortuitous chemical dynamic between smoking and Alzheimer’s disease.
Proc. Natl. Acad. Sci. U. S. A. 100:8182, 2003.
Dickerson, T.J., Reed, N.N., Janda, K.D. Soluble polymers as scaffolds for recoverable catalysts and reagents. Chem. Rev. 102:3325, 2002.
Gambs, C., Dickerson, T.J., Mahajan, S., Pasternack, L.B., Janda, K.D. High-resolution diffusion-ordered spectroscopy to probe the microenvironment of JandaJel
and Merrifield resins. J. Org. Chem. 68:3673, 2003.
Gao, C., Mao, S., Ditzel, H.J., Farnaes, L., Wirsching, P., Lerner, R.A., Janda, K.D. A
cell-penetrating peptide from a novel pVII-pIX phage-displayed random peptide library.
Bioorg. Med. Chem. 10:4057, 2002.
Gao, C., Mao, S., Kaufmann, G., Wirsching, P., Lerner, R.A., Janda, K.D. A method
for the generation of combinatorial antibody libraries using pIX phage display. Proc.
Natl. Acad. Sci. U. S. A. 99:12612, 2002.
Gao, C., Mao, S., Ronca, F., Zhuang, S., Quaranta, V., Wirsching, P., Janda, K.D.
De novo identification of tumor-specific internalizing human antibody-receptor pairs
by phage-display methods. J. Immunol. Methods 274:185, 2003.
Jones, L.H., Altobell, L.J. III, MacDonald, M.T., Boyle, N.A., Wentworth, P., Jr.,
Lerner, R.A., Janda, K.D. Active immunization with a glycolipid transition state
analogue protects against endotoxic shock. Angew. Chem. Int. Ed. 41:4241, 2002.
Lee, K.J., Mao, S., Sun, C., Gao, C., Blixt, O., Arrues, S., Hom, L.G., Kaufmann,
G.F., Hoffman, T.Z., Coyle, A.R., Paulson, J., Felding-Habermann, B., Janda, K.D.
Phage-display selection of a human single-chain Fv antibody highly specific for
melanoma and breast cancer cells using a chemoenzymatically synthesized GM3carbohydrate antigen. J. Am. Chem. Soc. 124:12439, 2002.
Lee, S.-H., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. Rhodium carbenoid N-H insertion reactions of primary ureas: solution and solid-phase synthesis
of imidazolones. Org. Lett. 5:511, 2003.
Liu, C., Sun, C., Huang, H., Janda, K., Edgington, T. Overexpression of legumain
in tumors is significant for invasion/metastasis and a candidate enzymatic target for
prodrug therapy. Cancer Res. 63:2957, 2003.
Meijler, M.M., Matsushita, M., Altobell, L.J. III, Wirsching, P., Janda, K.D. A new
strategy for improved nicotine vaccines using conformationally constrained haptens.
J. Am. Chem. Soc. 125:7164, 2003.
Redwan, el-R.M., Larsen, N.A., Zhou, B., Wirsching, P., Janda, K.D., Wilson, I.A.
Expression and characterization of a humanized cocaine-binding antibody. Biotechnol. Bioeng. 82:612, 2003.
Shimomura, O., Clapham, B., Spanka, C., Mahajan, S., Janda, K.D. Application
of microgels as polymer supports for organic synthesis: preparation of a small phthalide
library, a scavenger, and a borohydride reagent. J. Comb. Chem. 4:436, 2002.
Sun, C., Wirsching, P., Janda, K.D. Enabling scFvs as multi-drug carriers: a dendritic approach. Bioorg. Med. Chem. 11:1761, 2003.
Wang, Q., Raja, K.S., Janda, K.D., Lin, T., Finn, M.G. Blue fluorescent antibodies as
reporters of steric accessibility in virus conjugates. Bioconjug. Chem. 14:38, 2003.
Catalytic Nucleic Acids for
Treating the Molecular
Basis of Disease
G.F. Joyce, J.T. Hillman, R.M. Kumar, N. Paul, W.M. Shih
n recent years, nucleic acid molecules have shown
promise for the treatment of a variety of diseases.
These molecules can be used to downregulate the
expression of disease-related genes by targeting either
the genetic material itself or the corresponding RNA or
protein product. Antisense agents are one type of therapeutic nucleic acid. These agents usually consist of
short, single-stranded DNA molecules that bind to particular cellular RNAs, leading to inactivation of the
RNAs. Small interfering RNAs are another type of potential therapeutic nucleic acid that bind to specific cellular RNAs, resulting in cleavage of those RNAs through
the “RNA interference” pathway.
We are developing catalytic nucleic acids, composed
of either RNA or DNA, that bind to target RNAs and
inactivate them by carrying out a cleavage reaction that
destroys the target. We obtain these catalysts through a
process of test-tube evolution that mimics natural evolution but occurs much more rapidly and according to
selection constraints that we impose.
I
R N A - C L E AV I N G D N A E N Z Y M E S
We have developed DNA enzymes that can be made
to cleave almost any targeted RNA under cellular conditions. Our most widely used DNA enzyme is the
“10-23” motif, which contains approximately 30 deoxynucleotides and operates with high catalytic efficiency
and sequence specificity. We and scientists in many
other laboratories have used this enzyme to cleave
disease-related mRNAs, both in cultured cells and in
whole animals.
In collaboration with R. Smith, a Skaggs Clinical
Scholar, we focused on the DNA-catalyzed cleavage of
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
superoxide dismutase (SOD1) mRNA as a potential
treatment for amyotrophic lateral sclerosis. Recent
studies indicated that amyotrophic lateral sclerosis is
caused by mutation or inappropriate expression of SOD1,
suggesting that decreased expression of SOD1 might
have therapeutic benefit. We tested the ability of the
10-23 DNA enzyme to inhibit expression of mutant
human SOD1 mRNA in transgenic rats, seeking to
delay the rapid onset of disease in these animals.
Two different DNA enzymes were constructed and
formulated for delivery to the cerebral ventricles by continuous infusion. The compounds were delivered at a
dose of 0.1 mg/kg per day for 30 days. These animals
were compared with control animals that received the
same volume of isotonic sodium chloride solution. Both
the treated and the untreated animals had severe muscle wasting by the end of the treatment period, indicating that the DNA enzymes had no therapeutic benefit.
Unlike the situation in humans with amyotrophic
lateral sclerosis, the transgenic rats express high concentrations of mutant human SOD1 mRNA and succumb to the disease by 4 months of age. This model
of the disease may be an overly aggressive one and
one that provides little opportunity for validation of
treatments that depend on downregulation of the overexpressed SOD1 mRNA. Currently, using both normal
and transgenic rats, we are examining whether the
DNA enzymes can significantly reduce mRNA levels.
length. The other 5 struts are based on double crossovers, which have the structure of 2 adjacent double
helices that are joined at 2 crossover points. Each of
these motifs is about twice as rigid as a simple DNA
double helix, lending stiffness to the overall octahedron.
The structure of the DNA octahedron was confirmed
by using electron cryomicroscopy. Approximately 1000
raw images were obtained and averaged to produce the
3-dimensional reconstruction shown in Figure 1. Even
though the overall structure has a regular appearance
at this resolution (~30 Å), each strut has a different
sequence and therefore is independently addressable
by other molecules that bind DNA in a sequence-specific
manner. The interior of the octahedron has a diameter of
about 14 nm, and the 8 triangular “windows” have an
opening of about 8 nm. Thus, it should be possible to
trap macromolecules that are between 8 and 14 nm
in size within the cavity of the octahedron. Furthermore, because it can be constructed from a long, single-stranded DNA molecule, the DNA octahedron can
be cloned into bacteria or other cells, where it can be
amplified and produced in large quantity.
DNA-BASED NANOTECHOLOGY
Reader, J.S., Joyce, G.F. A ribozyme composed of only two different nucleotides.
Nature 420:841, 2002.
We recently began a project to construct rigid DNAbased nanostructures that could be used to encapsulate
macromolecules or to direct the assembly of materials
on a submicrometer scale. We designed and synthesized
a 1.7-kb, single-stranded DNA molecule that folds into
a regular octahedron structure upon heating and cooling. The octahedron consists of 12 struts, each 14 nm
long, that are joined together at 6 four-way junctions
(Fig. 1). Seven of the struts are based on paranemic
crossovers, which have the structure of 2 adjacent double helices that exchange strands 6 times along their
PUBLICATIONS
Kuhns, S.T., Joyce, G.F. Perfectly complementary nucleic acid enzymes. J. Mol.
Evol. 56:711, 2003.
Kumar, R.M., Joyce, G.F. A modular, bifunctional RNA that integrates itself into a
target RNA. Proc. Natl. Acad. Sci. U. S. A. 100:9738, 2003.
Shih, W.M., Quispe, J.D., Joyce, G.F. A 1.7-kilobase single-stranded DNA that
folds into a nanoscale octahedron. Nature, in press.
Biomolecular Computing,
Catalytic Antibodies, Organic
Synthesis, and Synthetic Enzymes
E. Keinan, H.C. Lo, H. Han, S. Sasmal, M. Arifuddin, A. Brik,
S. Saphier, G. Sklute, N. Metanis, M. Soreni, D. Vebenov,
L. Kosoy, M.K. Sinha, A. Alt, I. Ben-Shir, R. Piran, P.E. Dawson
BIOMOLECULAR COMPUTING
F i g . 1 . Structure of a DNA octahedron based on 3-dimensional
reconstruction of images obtained by electron cryomicroscopy. The
single-stranded DNA folds into a regular octahedron upon heat
denaturation and gradual cooling.
lectronic computers can only process data
encoded electronically, yet data for many valuable computations occur naturally as biomolecules. For example, modern medicine often involves
procedures that can be viewed as computational processes, with molecules obtained from a patient as input
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
and drug molecules to be administered as output. In
collaboration with E. Shapiro and Z. Livneh, Weizmann
Institute of Science, and Y. Shoham, Technion, Haifa,
Israel, we developed a biomolecular computing machine
that is a task-oriented, nanoscale, autonomous programmable computer made of molecules (Fig. 1). It
can process biomolecules as data and produce biomolecules as output. We constructed in vitro biomolecular finite automatons that compute autonomously
when their “hardware,” “software,” and “input” are
mixed in solution. The hardware consists of a restriction nuclease and a ligase. Programming the device
amounts to choosing the transition molecules (encoded
by double-stranded DNA) to be mixed.
F i g . 1 . A biomolecular computing machine made of molecules. The hardware consists of a restriction nuclease and a ligase;
the input, transition molecules (software), and detection molecules
are all made of double-stranded DNA.
Each automaton processes an input double-stranded
DNA molecule via a cascade of hybridization, ligation,
and restriction cycles, producing an output doublestranded DNA molecule that encodes the final state of
the computation. The computation runs at a combined
rate of 109 transitions per second with accuracy greater
than 99.8% per transition.
C ATA LY T I C A N T I B O D I E S
In our studies on catalytic antibodies, we focus on
antibody-catalyzed photochemical reactions, oxidation
reactions, and organometallic reactions, as illustrated
in the following examples.
Although the solution photochemical reaction of
ketone 1 (Fig. 2) yields only the cleavage products 2
and 3, in the presence of 20F10, an antibody to 5a
35
F i g . 2 . The photochemical Norrish type II reaction of ketone 1
produces in solution the cleavage products 2 and 3. Antibody
20F10, which was elicited against a mixture of 5a and 5b, catalyzes enantioselective formation of cis-cyclobutanol (4).
and 5b, a Norrish type II reaction occurred. leading to
the selective formation of cis-cyclobutanol (compound
4 in Fig. 2). Furthermore, the fact that compound 4,
which consists of 2 asymmetric centers, is obtained
as a single diastereomer, makes this photoproduct a
valuable building block for the synthesis of natural products. Other photochemical reactions catalyzed by 20F10
afford products of cyclopropanol derivatives from 1,3diaryl-1-propanones and formation of phenanthrenes
from substituted stilbenes.
In addition, an aldolase antibody, 24H6, obtained
by immunization with large 1,3 diketone haptens, has
an active-site lysine residue with a perturbed pK a of
7.0. This antibody catalyzes both the aldol addition and
the retrograde aldol fragmentation with a broad range
of substrates that differ structurally from the haptens.
This antibody also catalyzes the oxidation of α-hydroxyketones to α-diketones and deuterium exchange at the
α position of many ketones and aldehydes.
During our ongoing efforts to develop catalytic antibodies for organometallic reactions, we discovered a
new chemistry of platinum. A novel, air- and moisturestable dihydrido(methyl)platinum(IV) complex, TpPtH2Me
(compound 7 in Fig. 3), was formed as the sole product by the reaction of TpPtMeCO (compound 6 in Fig. 3)
with water. We found that although TpPtH 2Me has a
high energy barrier for the liberation of methane, it
F i g . 3 . Isotopic scrambling of the methylplatinum complex 7
proceeds via a σ-methane intermediate.
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THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
readily forms a σ-methane platinum(II) complex. This
complex is the first example of a hydridoalkyl metal
complex that undergoes isotopic scrambling at elevated
temperatures without concomitant liberation of either
alkane or dihydrogen.
Later, we discovered that the methyl protons in
compound 6 underwent proton exchange with the hydrogen atoms in water molecules. The proton-exchange
reaction was proposed to proceed via a pathway similar to the catalytic triad found in natural enzymes.
ORGANIC SYNTHESIS
The single-step, tandem oxidative polycyclization
reaction with rhenium(VII) reagents is a powerful
method by which polyene alcohols can be converted
into polytetrahydrofuran products in a single step. A
set of rules has been deduced to predict the product’s
configuration in this reaction.
Annonaceous acetogenins, particularly those with
adjacent bis-tetrahydrofuran rings, have remarkable
cytotoxic, antitumor, antimalarial, immunosuppressive,
pesticidal, and antifeedant activities. We developed
synthetic approaches that can be used to generate
chemical libraries of stereoisomeric acetogenins. These
efforts resulted in the total synthesis of several naturally occurring acetogenins, including asimicin, bullatacin, trilobacin, rolliniastatin, solamin, reticulatacin,
rollidecins C and D, goniocin, cyclogoniodenin, and
mucocin, and many nonnatural stereoisomers.
The 34-hydroxyasimicin and its derivatives were
synthesized for photoaffinity labeling studies of bovine
mitochondrial NADH-ubiquinone oxidoreductase (complex I). These studies may shed light on the structure
and function of this intricate enzyme and on the origin
of the high antitumor activity of the annonaceous acetogenins. This work is being done in collaboration with
S.C. Sinha, the Skaggs Institute.
SYNTHETIC ENZYMES
Efforts 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. We
profoundly changed the catalytic activity and mechanism of 4-oxalocrotonate tautomerase via a rationally
designed single amino acid substitution that corresponds
to a mutation in a single base pair. Although the wildtype enzyme catalyzes only the tautomerization of
oxalocrotonate to 2-oxo-3E-hexenedioate, the P1A
mutant catalyzes 2 reactions: the original tautomerization reaction via a general acid-base mechanism and
the decarboxylation of oxaloacetate via a nucleophilic
mechanism. The observation that a single catalytic
group in an enzyme can catalyze 2 reactions by 2 different mechanisms supports the theory that new enzymatic activity can evolve in a continuous manner.
Highly evolved enzymes are optimized not only to
catalyze a desired reaction but also to avoid undesired
processes. We showed that P1A, which catalyzes the
isomerization of 4-oxalocrotonate tautomerase to 2-oxo3E-hexenedioate, also undergoes specific 1,4-addition
to the tautomerization product to form a stable covalent adduct. This research on synthetic enzymes is
being done in collaboration with P.E. Dawson, the
Skaggs Institute.
PUBLICATIONS
Han, H., Sinha, M.K., D’Souza, L., Keinan, E., Sinha S.C. Total synthesis of 34hydroxy asimicin and its photoactive derivative for affinity labeling of the mitochondrial complex I. Chemistry, in press.
Keinan, E., Sinha, S.C. Oxidative polycyclizations with rhenium(VII) oxides. Pure
Appl. Chem. 74:93, 2002.
Lo, H.C., Iron, M.A., Martin, J.M.L., Keinan, E. Organometallic catalytic triad: proton
walk in aqueous platinum complex via a sticky methane. J. Am. Chem. Soc., in press.
Saphier, S., Sinha, S.C., Keinan, E. Antibody-catalyzed enantioselective Norrish
type II cyclization. Angew. Chem. Int. Ed. 42:1378, 2003.
Sklute, G., Oiserowitz, R., Shulman, H., Keinan, E. Antibody-catalyzed benzoin
oxidation as a mechanistic probe for nucleophilic catalysis by an active-site lysine.
Chemistry, in press.
Metabolite-Initiated
Protein Misfolding
J.W. Kelly, S. Deechongkit, M.A. Dendle, T. Foss, D. Fowler,
K. Frankenfeld, N. Green, A. Hurshman, M.B. Huff,
S. Johnson, H.-J. Lim, E. Powers, A. Sawkar, Y. Sekijima,
S. Werner, L. Wiseman, S.-L. You, Q. Zhang
he central theme of our research under the auspices of The Skaggs Institute for Chemical Biology during the past year was the initiation of
new projects related to human health that if successful would generate sufficient preliminary results to make
the projects competitive for funding from the National
Institutes of Health. Two of the projects proposed 2
years ago resulted in highly relevant discoveries. One
of the discoveries was patented and should be translatable to the clinic.
In 1972, C.B. Anfinsen won the Nobel Prize for
showing that the fold of a protein is specified by the
protein’s primary amino acid sequence. This tenet
T
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
makes it difficult to rationalize how the same amino acid
sequence can adopt a native structure in most persons
and amyloidogenic structures in patients with Alzheimer’s
disease in what appears to be a common extracellular
environment. One possibility is that a unique posttranslational modification makes the β-amyloid peptide amyloidogenic, but evidence for this possibility is sparse.
In work supported by the Skaggs Institute, and in
collaboration with R.A. Lerner, The Scripps Research
Institute, we found that the newly discovered abnormal
metabolites associated with chronic inflammation modify β-amyloid and other amyloidogenic polypeptides in
a covalent fashion, initiating the amyloidogenesis of
the peptides under physiologic concentrations. A subset of oxidative metabolites containing an aldehyde
group is unique in that the metabolites enable the
attachment of a large hydrophobic structure to polypeptides by formation of a Schiff base (a covalent bond),
very rapidly initiating aggregation, a process that typically requires the formation of an energetically unfavorable nucleus.
This research provides evidence that the amyloid
diseases associated with β-amyloid and transthyretin,
which do not involve any known mutations—the socalled sporadic amyloid diseases—can be initiated by
the covalent attachment of a small number of aberrant
metabolites. Metabolite-initiated misfolding may also
explain the onset of familial amyloidosis of the Finnish
type caused by gelsolin amyloidogenesis. We are optimistic that this finding could be the long-sought explanation for most human amyloid diseases in which the
cause of the disease is not linked to a mutation.
In a collaboration with D. Burton and A. Williamson,
The Scripps Research Institute, we are studying the
mechanism of how an antibody they produced cures
systemic prion disease, that is, how it prevents the
conversion of the prion protein from its normal functional conformation into the disease-associated scrapie
conformation. Although much remains to be accomplished, these results are encouraging and provide hope
that small molecules may be discovered that function
similarly, allowing the brain component of prion diseases
to be ameliorated.
PUBLICATIONS
Calarese, D.A., Scanlan, C.N., Zwick, M.B., Deechongkit, S., Mimura, Y., Kunert, R.,
Zhu, P., Wormald, M.R., Stanfield, R.L., Roux, K.H., Kelly, J.W., Rudd, P.M., Dwek,
R.A., Katinger, H., Burton, D.R., Wilson, I.A. Antibody domain exchange is an
immunological solution to carbohydrate cluster recognition. Science 300:2065, 2003.
Cohen, F., Kelly, J.W. Therapeutic approaches to protein-misfolding diseases.
Nature 426:905, 2003.
37
Colfer, S., Kelly, J.W., Powers E.T. Factors governing the self-assembly of a
β-hairpin peptide at the air-water interface. Langmuir 19:1312, 2003.
DiDonato, M., Craig, L., Huff; M.E., Thayer, M.M., Cardosa, R.M.F., Kassmann,
C.J., Lo, T.P., Bruns, C.K., Powers, E.T., Kelly, J.W., Getzoff, E.D., Tainer, J.A.
ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization [published correction in J. Mol. Biol. 334:175, 2003]. J. Mol.
Biol. 332:601, 2003.
Ferrao-Gonzales, A.D., Palmieri, L., Valory, M., Silva, J.L., Lashuel, H., Kelly,
J.W., Foguel, D. Hydration and packing are crucial to amyloidogenesis as revealed
by pressure studies on transthyretin variants that either protect or worsen amyloid
disease. J. Mol. Biol. 328:963, 2003.
Foguel, D., Suarez, M.C., Ferrao-Gonzales, A.D., Porto, T.C.R., Palmieri, L., Einsiedler, C.M., Andrade, L.R., Lashuel, H.A., Lansbury, P.T., Kelly, J.W., Silva, J.L.
Dissociation of amyloid fibrils of α-synuclein and transthyretin by pressure reveals
their reversible nature and the formation of water-excluded cavities. Proc. Natl.
Acad. Sci. U. S. A. 100:9831, 2003.
Green, N.S., Palaninathan, S.K., Sacchettini, J.C., Kelly, J.W. Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin
tetramerization. J. Am. Chem. Soc. 125:13404, 2003.
Hammarström, P., Sekijima, Y., White, J.T., Wiseman, R.L., Lim, A., Costello,
C.E., Atland, K., Garzuly, F., Budka, H., Kelly, J.W. D18G transthyretin is
monomeric, aggregation prone, and not detectable in plasma and cerebrospinal
fluid: a prescription for central nervous system amyloidosis? Biochemistry
42:6656, 2003.
Hammarström, P., Wiseman, R.L., Powers, E.T., Kelly, J.W. Prevention of
transthyretin amyloid disease by changing protein misfolding energetics. Science
299:713, 2003.
Huff, M.E., Page, L., Balch, W.E., Kelly, J.W. Gelsolin domain 2 Ca2+ affinity
determines susceptibility to furin proteolysis and familial amyloidosis of Finnish
type. J. Mol. Biol. 334:119, 2003.
Kelly, J.W. Towards an understanding of amyloidogenesis. Nat. Struct. Biol.
9:323, 2002.
Kelly, J.W., Balch, W.E. Amyloid as a natural product. J. Cell Biol. 161:461, 2003.
Lu, J.R., Perumal, S., Powers, E.T., Kelly, J.W., Webster, J.R.P., Penfold, J.
Adsorption of β-hairpin peptides on the surface of water: a neutron refection study.
J. Am. Chem. Soc. 125:3751, 2003.
Nguyen, H., Jager, M., Moretto, A., Gruebele, M., Kelly, J.W. Tuning the freeenergy landscape of a WW domain by temperature, mutation, and truncation. Proc.
Natl. Acad. Sci. U. S. A. 100:3948, 2003.
Razavi, H., Palaninathan, S.K., Powers, E.T., Wiseman, R.L., Purkey, H.E.,
Mohamedmohaideen, N.N., Deechongkit, S., Chiang, K.P., Dendle, M.T.A., Sacchettini, J.C., Kelly, J.W. Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis,
evaluation and mechanism of action. Angew. Chem. Int. Ed. 42:2758, 2003.
Sekijimi, Y., Hammarström, P., Matsumura, M., Shimizu, Y., Iwata, M., Tokuda,
T., Ikeda, S., Kelly, J.W. Energetic characteristics of the new transthyretin variant
A25T may explain its atypical central nervous system pathology. Lab. Invest.
83:409, 2003.
Zhang, Q., Kelly, J.W. Cys 10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions. Biochemistry 42:8756, 2003.
Zhang, Q., Powers, E.T.. Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Metabolite
initiated protein misfolding in Alzheimer’s disease. Proc. Natl. Acad. Sci., in press.
38
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Expression of Therapeutic
Proteins in Eukaryotic Algae
S.P. Mayfield, E. Brown, M. Beligni, A. Manuell,
K. Yamaguchi, S.E. Franklin
herapeutic proteins, including hormones, enzymes,
and monoclonal antibodies, account for approximately 10% of the total number of pharmaceutical agents on the market and are the fastest growing
area of drug development. With the advent of genomics
and proteomics technologies, the rate of discovery of
new protein therapeutic agents is predicted to increase
in the years ahead. Although this prediction bodes well
for our ability to treat a myriad of diseases, many of
the relevant diseases are chronic and require several
grams of therapeutic protein per patient annually. This
requirement presents a challenge to the pharmaceutical industry because existing technologies for producing these therapeutic proteins are extremely expensive.
Thus, we appear to be at a crossroads where the
ability of medical science to treat human disease may
outpace the ability of industry to produce therapeutic
agents at a reasonable cost and in sufficient amounts.
In addition, healthcare providers, both public and private, are increasingly reticent to pay for therapies that
although efficacious are cost prohibitive. Hence, an
ever-increasing demand exists for systems that can be
used to produce therapeutic proteins economically and
at scales not previously achieved.
Our current research is focused on developing just
such a system for the expression of monoclonal antibodies in the eukaryotic alga Chlamydomonas reinhardtii.
Currently, monoclonal antibodies are produced primarily
by culture of mammalian cells, and capital costs for the
production facilities can run into the hundreds of millions
of dollars. Because of these high capital costs and the
complexity of mammalian production systems, the production capacity for monoclonal antibodies is expected
to fall substantially short of demand during the next 5
years. In addition to capital costs, media and other costs
for mammalian cell culture are also quite expensive, making monoclonal antibodies some of the most expensive
drugs on the market. Clearly, systems with reduced costs
of capitalization that allow rapid scale-up and economical production can greatly affect the way in which protein therapeutic agents are manufactured.
We constructed strains of C reinhardtii that express
a number of variants of a human antibody directed
against herpes simplex virus. We showed that these
T
antibodies assemble in the algae to form fully functional
molecules that bind herpes simplex proteins. Antibody
binding to the coat protein of herpes simplex virus
blocks viral propagation, making these algally expressed
antibodies potential therapeutic agents.
In a related project, we are assessing the ability of
an antibody Fab fragment that has catalytic activity to
assemble into a functional molecule in vivo. The ability to produce catalytic antibodies in a cost-effective
manner and on a large scale could revolutionize the
pharmaceutical and chemical industries.
We are also exploring the ability of this system to
produce therapeutic proteins such as IL-10. Evidence
suggests that IL-10 may be an effective oral therapy
for irritable bowel syndrome. Here again, current production strategies make oral delivery of IL-10 cost prohibitive, whereas production and delivery in algae might
be quite cost effective.
Finally, we developed 2 reporter gene systems for
eukaryotic algae, 1 based on green fluorescent protein
and 1 on bacterial luciferase, that will allow us to better define the transcriptional and translational elements
required to obtain even higher levels of expression of
therapeutic proteins.
PUBLICATIONS
Franklin, S., Ngo, B., Efuet, E., Mayfield, S.P. Development of a GFP reporter
gene for Chlamydomonas reinhardtii chloroplast. Plant J. 30:733, 2002.
Mayfield, S.P., Franklin, S.E., Lerner, R.A. Expression and assembly of a fully
active antibody in algae. Proc. Natl. Acad. Sci. U. S. A. 100:438, 2003.
Mayfield, S.P., Schultz, J. Development of a luciferase reporter gene, luxCt, for
Chlamydomonas reinhardtii chloroplast. Plant J., in press.
Chemical Synthesis and
Chemical Biology
K.C. Nicolaou, R. Baati, T. Bando, M. Bella, F. Bernal,
W. Brenzovich, S. Bulat, D. Chen, J. Chen, E. Couladouros,
P. Dagneau, P. Diamandis, A. Estrada, M. Follmann,
T. Francis, M. Frederick, L. Gomez Paloma, M. Govindasamy,
D. Gray, P. Guntupalli, S. Harrison, J. Hao, X. Huang,
R. Hughes, M. Jennings, F. Kaiser, D. Kim, T. Koftis, S. Lee,
Y. Li, T. Ling, D. Longbottom, F. Marr, C. Mathison,
T. Montagnon, M. Nevalainen, A. Nalbandian, R. Noronha,
B. Paraselli, B. Pratt, W. Qian, G. Rassias, M. Reddy,
M. Rodriguez, A. Roecker, B. Safina, P. Sasmal, D. Seigel,
S. Snyder, X. Sun, G. Vasilikogiannakis, S. Vyskocil, H. Xu,
Y. Yamada, M. Zak
O
ne of the most vigorous research frontiers in
chemistry is total synthesis. Considered by
many the engine that drives organic synthesis
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
forward, this scientific endeavor combines elements of
art, science, and technology and provides a strong
foundation for drug discovery. The field is fueled by the
pharmaceutical and biotechnology industries because
of potential applications in medicine and by the discovery of naturally occurring novel molecules with biological properties relevant to disease. The examples of
aspirin, penicillin, and paclitaxel come to mind, but
these are only a few of the myriad of naturally occurring lead compounds that provided direct or indirect
medical breakthroughs. With the advances of molecular and cell biology, the science of chemical synthesis
promises to become an even sharper tool in the hands
of the medicinal chemists who are poised to design
and synthesize the next generation of medicines.
Our research efforts under the funding auspices of
The Skaggs Institute for Chemical Biology are directed
at the total synthesis of biologically active natural products and the application of the gained knowledge to
the design, chemical synthesis, and biological evaluation of new molecules. Thus, our aim is to enable and
facilitate studies in biology and drug discovery and
development, and our programs are designed to optimize the educational and training opportunities for
young men and women.
In the past year, we made marked progress in the
areas of molecular design, chemical synthesis, and
chemical biology. Specifically, we designed and synthesized a group of new and potent epothilone analogs,
one of which is now in clinical trials as an anticancer
drug. Our efforts in the anticancer field also included
a new total synthesis of the marine-derived antitumor
agent diazonamide A and of 1-O-methyllalteriflorone,
a derivative of the plant-derived antitumor agent lateriflorone. Our studies on the potent antitumor lomaiviticins
and our efforts to synthesize the halipeptins, a new
class of marine-derived antitumor agents, are continuing (Fig. 1).
In the area of antibiotics, we extended our progress toward the highly complex antibiotic thiostrepton
with the synthesis of a number of truncated analogs.
In the field of cardiovascular disease, in collaboration
with R.M. Evans, the Salk Institute, La Jolla, California, we designed, synthesized, and tested several
high-affinity ligands to FXR, a steroid receptor related
to bile acid metabolism.
Our work on the environmentally dangerous marine
neurotoxin azaspiracid-1 and the powerful insecticide
azadirachtin resulted in the first synthesis of the proposed structure of the former (proving the proposed
39
F i g . 1 . Selected target molecules.
structure was incorrect) and considerable progress
toward reaching synthesis of the latter. We also made
further advances in new synthetic technologies that are
expected to enable drug discovery and development.
PUBLICATIONS
Bocci, G., Nicolaou, K.C., Kerbel, R.S. Protracted low-dose effects on human
endothelial cell proliferation and survival in vitro reveal a selective antiangiogenic
window for various chemotherapeutic drugs. Cancer Res. 62:6938, 2002.
Downes, M., Verdecia, M., Roecker, A.J., Hughes, R., Hogenesch, J.B., KastWoelbern, H.R., Bowman, M.E., Ferrer, J.L., Anisfeld, A.M., Edwards, P.A.,
Rosenfeld, J.M., Alvarez, J.G.A., Noel, J.P., Nicolaou, K.C., Evans, R.M. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol. Cell
11:1079, 2003.
Giannakakou, P., Nakano, M., Nicolaou, K.C., O’Brate, A., Yu, J., Blagosklonny,
M.V., Greber, U.R., Fojo, T. Enhanced microtubule-dependent trafficking and p53
nuclear accumulation by suppression of microtubule dynamics. Proc. Natl. Acad.
Sci. U. S. A. 99:10855, 2002.
Nicholas, G.M., Eckman, L.L., Ray, S., Hughes, R.O., Pfefferkorn, J.A., Barluenga, S., Nicolaou, K.C., Bewley, C.A. Bromotyrosine-derived natural and synthetic products as inhibitors of mycothiol-S-conjugate amidase. Bioorg. Med.
Chem. Lett. 12:2487, 2002.
Nicolaou, K.C. Perspectives in total synthesis: a personal account. Tetrahedron
59:6683, 2003.
40
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Nicolaou, K.C., Chen, D.Y.-K., Li, Y., Qian, W., Ling, T., Vyskocil, S., Koftis, T.V.,
Govindasamy, M., Uesaka, N. Total synthesis of the proposed azaspiracid-1 structure, 2: coupling of C1-C20, C21-C27 and C28-C40 fragments and completion of the
synthesis. Angew. Chem. Int. Ed. 42:3649, 2003.
Molecular Recognition
and Encapsulation
Nicolaou, K.C., Evans, R.M., Roecker, A.J., Hughes, R., Downes, M., Pfefferkorn,
J.A. Discovery and optimization of non-steroidal FXR agonists from natural product-like libraries. Org. Biomol. Chem. 1:908, 2003.
J. Rebek, Jr., T. Amaya, P. Ballester,* S. Biros, W.-D. Cho,
Nicolaou, K.C., Jennings, M.P., Dagneau, P. An expedient entry into the fused
polycyclic skeleton of vannusal A. Chem. Commun. (Camb.) 2480, 2002, Issue 21.
L. Kröck, Y. Kim, H. Onagi, L. Palmer, B. Purse,
Nicolaou, K.C., Kim, D.W., Baati, R., O’Brate, A., Giannakakou, P. Total synthesis
and biological evaluation of (–)-apicularen A and analogues thereof. Chemistry
9:6177, 2003.
E. Ullrich, M. Yamanaka
Nicolaou, K.C., Koumbis, A.E. Chemistry, biology and medicine in the 21st century. In: Science, Frontier Technology, and Orthodoxy: Proceedings of the Biomedical Ethics Symposium. Holy Church of Greece, Athens, 2002, p. 177.
ALKANES WRUNG OUT TO DRY
Nicolaou, K.C., Li, Y., Uesaka, N., Koftis, T.V., Vyskocil, S., Ling, T., Govindasamy, M., Qian, W., Bernal, F., Chen, D.Y.-K. Total synthesis of the proposed
azaspiracid-1 structure, 1: construction of the enantiomerically pure C1-C20, C21C27 and C28-C40 fragments. Angew. Chem. Int. Ed. 42:3643, 2003.
Nicolaou, K.C., Mathison, C.J.N., Montagnon, T. New reactions of IBX: oxidation
of nitrogen- and sulfur-containing substrates to afford useful synthetic intermediates. Angew. Chem. Int. Ed. 42:4077, 2003.
Nicolaou, K.C., Mitchell, H.J., Snyder, S.A. Synthesis of complex carbohydrates:
everninomicin 13,384-1. In: Carbohydrate-Based Drug Discovery. Wong, C.-H.
(Ed.). Wiley-VCH, New York, 2003, p. 948.
Nicolaou, K.C., Montagnon, T., Snyder, S.A. Tandem reactions, cascade sequences,
and biomimetic strategies in total synthesis. Chem. Commun. (Camb.) 551, 2003,
Issue 5.
Nicolaou, K.C., Montangon, T., Vassilikogiannakis, G. Natural products and chemical synthesis at the forefront of the fight against cancer [in Greek]. Bio 22, 2003,
Issue 5.
Nicolaou, K.C., Nevalainen, M., Zak, M., Bulat, S., Bella, M., Safina, B.S. Synthetic studies on thiostrepton: construction of thiostrepton analogues with the thiazoline-containing macrocycle. Angew. Chem. Int. Ed. 42:3418, 2003.
Nicolaou, K.C., Rao, P.B., Hao, J., Reddy, M.V., Rassias, G., Huang, X., Chen,
D.Y.-K., Snyder S.A. The second total synthesis of diazonamide A. Angew. Chem.
Int. Ed. 42:1753, 2003.
T. Dale, J. Friese, A. Gissot, S. Gu, F. Hof, D.W. Johnson,
D. Rachavi-Robinson, A. Scarso, A. Shivanyuk, L. Trembleau,
* Universitat de les Illes Balears, Palma de Mallorca, Spain
lkanes are the simplest structures made up of
repeating units, and their apolar surfaces are
the ultimate hydrophobic molecules. Akanes
typically adopt extended, straight-chain conformations
in solution that minimize strain but maximize surface
area. Reduction of the hydrophobic surface in water
can be achieved by folding to more compact forms,
but unfavorable interactions result.
We found that the long alkyl chains of common
surfactants such as sodium dodecyl sulfate adopt a
coiled conformation when bound within a synthetic
receptor in aqueous solution. The coiled alkane (Fig. 1)
complements the size, shape, and chemical surface of
the receptor better than the extended conformation does,
even though strain is introduced by twisting the backbone. The proper filling of space and burial of hydrophobic surface are thought to drive the molecular
recognition between receptor and the alkane: the alkane
experiences a dry environment inside the receptor. The
A
Nicolaou, K.C., Ritzén, A., Namoto, K., Buey, R.M., Fernando Díaz, J., Andreu,
J.M., Wartmann, M., Altmann, K.-H., O’Brate, A., Giannakakou, P. Chemical synthesis and biological evaluation of novel epothilone B and trans-12,13-cyclopropyl
epothilone B analogues. Tetrahedron 58:6413, 2002.
Nicolaou, K.C., Roecker, A.J., Hughes, R., van Summeren, R., Pfefferkorn, J.A.,
Winssinger, N. Novel strategies for the solid phase synthesis of substituted indolines and indoles. Bioorg. Med. Chem. 11:465, 2003.
Nicolaou, K.C., Roecker, A.J., Monenschein, H., Guntupalli, P., Follmann, M.
Studies towards the synthesis of azadirachtin: enantioselective entry into the
azadirachtin framework through cascade reactions. Angew. Chem. Int. Ed.
42:3637, 2003.
Nicolaou, K.C., Sasmal, P.K., Rassias, G., Reddy, M.V., Altmann, K.-H., Wartmann,
M., O’Brate, A., Giannakakou, P. Design, synthesis and biological properties of highly
potent epothilone B analogues. Angew. Chem. Int. Ed. 42:3515, 2003.
Nicolaou, K.C., Sasmal, P.K., Xu, H., Namoto, K., Ritzén, A. Total synthesis of 1O-methyllateriflorone. Angew. Chem. Int. Ed. 42:4225, 2003.
Nicolaou, K.C., Snyder, S.A. Cascade reactions in total synthesis: recent advances.
Actual. Chim. 81:April-May, 2003.
F i g . 1 . Model of an alkane (dodecane) coiled inside a vase-shaped
synthetic receptor.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
helical conformation adopted by the alkane shows that
even the simplest molecules have enough information
built into their structures to fold properly in response
to their surroundings.
T H E N AT U R E O F E N C L O S E D S PA C E S
We have long been interested in the synthesis and
characterization of chemical containers, large molecular hosts that can completely surround smaller guests.
We use these molecules to explore “inner space,” the
inside of a molecule. This environment appears be very
different from that of bulk solvent. One host forms by
spontaneous assembly of 2 identical but self-complementary modules and is held together by hydrogen
bonds (Fig. 2, left) The interior cavity of this assembly
provides enough space to surround 1, 2, or even 3
guest molecules. Using nuclear magnetic resonance
techniques, we examined the behavior of the molecules
inside and discovered the response of the guest to the
cramped quarters: chemical reactions between guests
are accelerated, weak interactions between guests are
isolated and magnified, and the arrangements of guests
in the space create new forms of stereochemistry.
41
Large proteins, such as enzymes or receptors, often
recognize smaller proteins through a well-defined binding site, and the interaction between the large and
small proteins is often compared with that of a lock
and its key: only keys containing the correct notches
and grooves will fit and function. Regulation of these
recognition events via small, synthetic molecules is
desirable because protein-protein interactions elicit
specific responses within the cell. The ability to block
certain pathways and enhance others lies at the center of medicinal chemistry (Fig. 3).
F i g . 3 . Left, Model structure of a small peptide adopting an α-
helix conformation. The functional groups used to recognize other
proteins are shown as spheres. Right, An example of a small molecule that displays groups in a similar orientation to that found in
peptides and can interfere with protein-protein interactions.
F i g . 2 . Left, a cylindrical, dimeric capsule encapsulates 2 molecules of toluene. Right, a large hexameric assembly surrounds 8
molecules of benzene.
Recent advances with even larger hosts such as
hexameric assemblies enabled us to study ever larger
inner spaces (Fig. 2, right). In this system, we can
observe simultaneous encapsulation of many small
guests (up to 8 or more). Many of these larger molecules
have unique optical and electrochemical properties
that can be used to probe the molecular environment
of the capsule interior. The encapsulation of small- and
medium-sized guests revealed never before seen trends
in the packing of molecules into confined spaces.
PROTEOMIMETICS
Recognition events between molecules in biology
are often mediated by many weak attractive forces.
The aim of our research is to manipulate specific
protein-protein interactions with carefully crafted mimics. These molecules contain structural features similar to those of natural protein surfaces but lack the
undesirable aspects of proteins, that is, vulnerability
to enzymes and poor adsorption. The synthetic structures can display chemical groups identical to those
found on protein surfaces. We are building a collection
of synthetic molecules that act as scaffolds and that
can be used repeatedly to imitate protein surfaces.
The molecules are prepared in modular form through
standard organic synthetic methods enhanced by combinatorial chemistry techniques.
DYNAMICS
We are also blending molecular recognition with
catalysis to recruit weak, noncovalent forces that
cause changes in covalent bonds. The concave, inner
surface of the host must be outfitted with functional
groups that can be in contact with the guests held
inside. We synthesized a vase-shaped host molecule
with a methyl ester group directed toward an interior
42
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
cavity. With certain amine guests, transfer of the carbon atom of the methyl ester takes place from host to
guest (Fig. 4). The size of the cavity allows selectivity
for different amine guests, and for ideally suited amines,
the reaction rate is at least 105 times faster than the
comparable reaction in solution.
Potential Medical Consequences
of Ambiguity in the Genetic Code
P. Schimmel, J. Bacher, A. Bates, K. Beebe,
V. de Crécy-Lagard, K. Ewalt, J. Liu, M. Lovato,
D. Metzgar, C. Myers, L. Nangle, B. Nordin, F. Otero,
J. Reader, L. Ribas de Pouplana, B. Slike, M. Swairjo,
K. Tamura, W. Waas, X. Yang
mbiguity in the genetic code may have a significant impact on human health and disease. The
code is an algorithm that relates each amino acid
to specific nucleotide triplets that are found in genes
and in mRNAs. The code is established in reactions
catalyzed by a group of enzymes known as aminoacyl
tRNA synthetases. In these reactions, each of the 20
natural amino acids is matched with its cognate tRNA.
The amino acid is linked to its cognate tRNA through
an ester linkage. Each specific tRNA harbors the triplet
of the genetic code for a specific amino acid. Thus, by
matching each amino acid with its cognate tRNA, the
aminoacyl tRNA synthetases relate each amino acid to
the appropriate triplet.
Each of the 20 amino acids has a distinct, separate
aminoacyl tRNA synthetase. If any of these enzymes
makes an error, that is, attaches the wrong amino acid
to a given tRNA, then the algorithm of the code is
broken. As a consequence of the error, the wrong amino
acid is incorporated at a specific codon during translation of an mRNA. The result is the synthesis of proteins
that have small imperfections, for example, a change
of just a single amino acid of the many (hundreds to
even thousands) that the protein is made up of. These
imperfections can accumulate and be detrimental to cell
viability. The effect on viability has been shown in bacterial cells, but limited information is available on this
effect in mammalian cell systems.
Ambiguity in the genetic code occurs when, as
stated, a mismatch occurs between a given amino acid
and a tRNA. Normally, mismatches are prevented by a
specialized editing activity that is embedded in many of
the aminoacyl tRNA synthetases. This activity corrects
errors of mismatching amino acids with their tRNAs by
hydrolyzing the ester linkage that joins the 2 components, amino acid and tRNA. The activity is powerful
and can rapidly clear mistakes that lead to ambiguity.
However, simple point mutations in the active site
for editing are sufficient to disrupt editing and cause
ambiguity in the genetic code. In bacterial cells, the
A
F i g . 4 . Model of a guest quinuclidine molecule inside a host
cavitand. The ester group at the top of the host is poised to transfer its methyl group to the amine of the guest.
PUBLICATIONS
Amrhein, P., Wash, P.L., Shivanyuk, A., Rebek, J., Jr. Metal ligation regulates conformational equilibria and binding properties of cavitands. Org. Lett. 4:319, 2002.
Bartfai, T., Behrens, M.M., Gaidarova, S., Pemberton, J., Shivanyuk, A., Rebek,
J., Jr. A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain
inhibits IL-1 receptor-mediated responses. Proc. Natl. Acad. Sci. U. S. A.
100:7971, 2003.
Johnson, D.W., Hof, F., Palmer, L.C., Martin, T., Obst, U., Rebek, J., Jr. Glycoluril
ribbons tethered by complementary hydrogen bonds. Chem. Commun. (Camb.)
14:1638, 2003.
Purse, B.W., Ballester, P., Rebek, J., Jr. Reactivity and molecular recognition:
amine methylation by an introverted ester. J. Am. Chem. Soc. 125:14682, 2003.
Shivanyuk, A., Friese, J.C., Döring, S., Rebek, J., Jr. Solvent-stabilized molecular
capsules. J. Org. Chem. 68:6489, 2003.
Shivanyuk, A., Rebek, J., Jr. Assembly of resorcinarene capsules in wet solvents.
J. Am. Chem. Soc. 125:3432, 2003.
Trembleau, L., Rebek, J., Jr. Helical conformation of alkanes in a hydrophobic cavitand. Science 301:1219, 2003.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
ambiguity can be so great that cell death occurs. Lethality happens in part because a single mutation in an
aminoacyl tRNA synthetase can lead to imperfections
in all of the cellular proteins. As these imperfections
build up, among others, the enzymes, signaling peptides, structural proteins, and replication and transcription factors needed for cell growth are made less and
less effective (Fig. 1).
43
PUBLICATIONS
Beebe, K., Merriman, E., Schimmel, P. Structure-specific tRNA determinants for
editing a mischarged amino acid. J. Biol. Chem. 278:45056, 2003.
Beebe, K., Ribas de Pouplana, L., Schimmel, P. Elucidation of tRNA-dependent
editing by a class II tRNA synthetase and significance for cell viability. EMBO J.
22:668, 2003.
Bishop, A.C., Beebe, K., Schimmel, P. Interstice mutations that block site-to-site
translocation of a misactivated amino acid bound to a class I tRNA synthetase.
Proc. Natl. Acad. Sci. U. S. A. 100:490, 2003.
Cen, S., Javanbakht, H., Kim, S., Shiba, K., Craven, R., Rein, A., Ewalt, K.,
Schimmel, P., Musier-Forsyth, K., Kleiman, L. Retrovirus-specific packaging of
aminoacyl tRNA synthetases with cognate primer tRNAs. J. Virol. 76:13111, 2002.
Ewalt, K.L., Schimmel, P. Activation of angiogenic signaling pathways by two human
tRNA synthetases. Biochemistry 41:13344, 2002.
Frugier, M., Giegé, R., Schimmel, P. RNA recognition by designed peptide creates
“artificial” tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 100:7471, 2003.
Hendrickson, T.L., Schimmel, P. Transfer RNA-dependent amino acid discrimination by aminoacyl-tRNA synthetases. In: Translation Mechanisms. LaPointe, J.,
Brakier-Gringas, L. (Eds.). Kluwer Academic, New York, and Landes Bioscience,
Georgetown, TX, 2003, p. 34. A volume in the series Molecular Biology Intelligence Unit. Also available at Eurekah.com.
F i g . 1 . Mischarged tRNAs generated by an editing-deficient
aminoacyl tRNA synthetase lead to incorrect incorporation of amino
acids. The proteins generated would carry small imperfections, resulting in a statistical pool of proteins. Possible fates for these statistical proteins include altered specificity (or activity) and inability to
fold correctly. Such imperfections would have diverse ramifications
relevant to disease. ER indicates endoplasmic reticulum.
To date, no human disease has been associated with
an editing-defective aminoacyl tRNA synthetase. However, a concerted effort to make this sort of connection
has not occurred. Thus, our objective is to establish
editing-defective aminoacyl tRNA synthetases in an
animal model such as the mouse. In preliminary experiments, we altered the editing site of a human aminoacyl tRNA synthetase by mutation. When the gene for
the altered mutant enzyme was introduced into mammalian cells, it affected the phenotype of these cells,
even though the native, nonmutant enzyme was also
present. (Thus, the phenotypic changes are due to a
“trans-dominant” effect of the mutant protein that overrides the native, wild-type enzyme.) In future experiments, a similar construction will be placed in mice.
Analysis of phenotypes associated with specific diseases can then be undertaken. These phenotypes would
be the result of imperfections introduced into specific
proteins that, for example, affect pathways related to
insulin signaling (diabetes), oncogenesis, and autoimmunity. Once a connection can be made between an
editing defect and a specific disease, therapeutic
approaches can be explored.
Kushiro, T., Schimmel, P. Trbp111 selectively binds a noncovalently assembled
tRNA-like structure. Proc. Natl. Acad. Sci. U. S. A. 99:16631, 2002.
Liu, J., Yang, X.-L., Ewalt, K.L., Schimmel, P. Mutational switching of a yeast
tRNA synthetase into a mammalian-like synthetase cytokine. Biochemistry
41:14232, 2002.
Nordin, B.E., Schimmel, P. Isoleucyl tRNA synthetase. In: Aminoacyl tRNA Synthetases. Francklyn, C. (Ed.). Kluwer Academic, New York, and Eurekah.com,
in press.
Nordin, B.E., Schimmel, P. Transiently misacylated tRNA is a primer for editing of
misactivated adenylates by class I aminoacyl-tRNA synthetases. Biochemistry
42:12989, 2003.
Ribas de Pouplana, Schimmel, P. Alanyl tRNA synthetases. In: Aminoacyl tRNA
Synthetases. Francklyn, C. (Ed.). Kluwer Academic, New York, and Eurekah.com,
in press.
Schimmel, P., Tamura, K. tRNA structure goes from L to λ. Cell 113:276, 2003.
Tamura, K., Schimmel, P. Peptide synthesis with a template-like RNA guide and
aminoacyl phosphate adaptors. Proc. Natl. Acad. Sci. U. S. A. 100:8666, 2003.
Tamura, K., Schimmel, P. Ribozyme programming extends the protein code. Nat.
Biotechnol. 20:669, 2002.
Wang, C.-C., Chang, K.J., Tang, H.-L., Hsieh, C.-J., Schimmel, P. Mitochondrial
form of a tRNA synthetase can be made bifunctional by manipulating its leader
peptide. Biochemistry 42:1646, 2003.
Yang, X.-L., Liu, J., Skene, R.J., McRee, D.E., Schimmel, P. Crystal structure of
an EMAP-II-like cytokine released from a human tRNA synthetase. Helv. Chim.
Acta 86:1246, 2003.
Yang, X.-L., Skene, R.J., McRee, D.E., Schimmel, P. Crystal structure of an aminoacyl tRNA synthetase cytokine. Proc. Natl. Acad. Sci. U. S. A. 99:15369, 2002.
44
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Expanding the Genetic Code
P.G. Schultz, J.C. Anderson, M. Bose, C. Cho, J. Gildersleeve,
J. Hong, Q. Huang, J. Liu, K.H. Min, M. Mukherji,
S. Santoro, F. Tian, L. Wang, P. Yang, J. Yin, C. Yu,
Z. Zhang, L.X. Zheng
he genetic codes of every known organism encode
20 amino acid building blocks with triplet codons
generated from 4 nucleotides. These 20 amino
acids contain a limited number of functional groups,
including acids, amides, alcohols, basic amines, and
thiols. Clearly, additional functional groups are needed
in proteins as indicated by the large number of posttranslational modifications and cofactors and by the rare
occurrence of the unnatural amino acids selenocysteine
and pyrrololysine. Why then are there only 20 genetically
encoded amino acids? Is this number the ideal one, or
would additional amino acids allow the generation of proteins or even entire organisms with enhanced functions?
To address this fundamental question, we developed
a method that allows us to genetically encode, in bacteria, novel amino acids beyond the common 20. This
method involves the generation of an orthogonal tRNA
that is not a substrate for any natural aminoacyl synthetases and that inserts its cognate amino acid in
response to the amber nonsense codon. An orthogonal
synthetase is then generated that recognizes this unique
tRNA and no other. The substrate specificity of this
synthetase is then evolved to recognize a desired “21st”
amino acid and no endogenous amino acid. We showed
that this method can be used to efficiently incorporate
a large number of amino acids into proteins in Escherichia coli with fidelity rivaling that of the common
amino acids.
In the past year, we added glycosylated amino acids,
redox-active amino acids, and keto-containing amino
acids to the growing list of novel genetically encoded
amino acids. In addition, we showed that acetylenecontaining amino acids can be used to selectively modify proteins with fluorophores and polyethylene glycol
methods that may facilitate cellular studies of protein
function and the development of therapeutic proteins.
We also generalized our method to yeast. Using
an E coli–derived orthogonal tRNA synthetase pair and
a 2-step positive and negative selection based on suppression of a nonsense codon in the gene gal4, we
added 5 novel amino acids independently to the genetic
code of Saccharomyces cerevisiae. We are using these
amino acids to probe signal transduction in yeast. The
T
yeast system also should allow us to apply this method
to higher eukaryotes. Indeed, we showed that 6-hydroxytryptophan can be selectively incorporated into proteins in mammalian cells in response to the opal codon,
and we are attempting to genetically encode novel
amino acids in the multicellular organism Caenorhabditis elegans.
We also developed a consensus-based strategy that
allows us to generate additional orthogonal tRNA synthetase pairs for incorporating multiple distinct amino
acids into a single protein, including lysine, tyrosine,
and glutamate pairs. Importantly, we showed that the
lysine pair can be used to genetically encode an unnatural amino acid (homoglutamine) in response to the
4-base codon AGGA.
In collaboration with F. Romesberg and colleagues,
The Scripps Research Institute, we are also involved in
efforts to expand the genetic lexicon to include a third
base pair in addition to the Watson-Crick adenine-thymine and guanine-cytosine base pairs. Such bases
would allow us to encode additional genetic information
and to evolve nucleic acids with novel properties. We
have focused on the use of hydrophobic and metalmediated base-pairing interactions. We synthesized
more than 50 such bases and identified a number of
homo and hetero base pairs with thermodynamic stability and selectivity rivaling or exceeding those of the
Watson-Crick base pairs.
In the past year, we focused largely on the development of simple methylated and fluorinated 5- and
6-membered ring “bases.” Pairs derived from these
nucleosides are quite selective and stable, and a number are good substrates for DNA polymerases. Currently, we are attempting to crystallize duplex DNAs
containing these unnatural base pairs and to evolve in
vitro polymerases that can incorporate these bases into
DNA with high fidelity.
PUBLICATIONS
Alfonta, L., Zhang, Z., Uryu, S., Loo, J.A., Schultz, P.G. Site-specific incorporation
of a redox-active amino acid into proteins. J. Am. Chem. Soc. 125:14662, 2003.
Chin, J.W., Cropp, T.A., Anderson, J.C., Mukherji, M., Zhang, Z., Schultz, P.G. An
expanded eukaryotic genetic code. Science 301:964, 2003.
Liu, H., Wang, L., Brock, A., Wong, C.-H., Schultz, P.G. A method for the generation of glycoprotein mimetics. J. Am. Chem. Soc. 125:1702, 2003.
Mehl, R.A., Anderson, J.C., Santoro, S.W., Wang, L., Martin, A.B., King, D.S.,
Horn, D.M., Schultz, P.G. Generation of a bacterium with a 21 amino acid genetic
code. J. Am. Chem. Soc. 125:935, 2003.
Wang, L., Zhang, Z., Brock, A., Schultz, P.G. Addition of the keto functional group to
the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 100:56, 2003.
Zhang, Z., Smith, B.A., Wang, L., Brock, A., Cho, C., Schultz, P.G. A new strategy for site-specific modification of proteins in vivo. Biochemistry 42:6735, 2003.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Click Chemistry and the Search
for Biological Activity
K.B. Sharpless, Y. Baba, S. Boral, J. Cappiello, T. Chan,
A. Converso, M. David, A. Feldman, V. Fokin, R. Hilgraf,
P. Holzer, C. Kappe, T. Kitayama, A. Krasinski, I. Lewis,
V. Litosh, R. Manetsch, A. McPherson, J. Muldoon,
S. Narayan, V. Rostovtsev, S. Silverman, N. Stuhr-Hansen,
E. Van der Eycken, P. Wu, Y. Xie
he primary goals of our research are the discovery and development of selective, powerful, and
reliable transformations that allow rapid preparation of compounds with desired biological function from
readily available starting materials. During the past year,
we developed several such reactions and applied them
to targets of chemical diversity and biological activity.
Additionally, we focused on performing these and related
transformations in water, the lingua franca of life on
Earth. In addition to this pursuit of developing novel
synthetic transformations, we refined a “Trojan horse”
approach to identification of drug candidates in which
a biologically active substance is selectively synthesized
by the target enzyme itself. The highlights of our research
are described here.
Water is rarely used or even considered as a solvent
for organic reactions. The 2 foremost reasons are the
lack of solubility of most organic compounds in this
medium and concerns that the high “acid-base” reactivity will interfere with the desired reaction. However,
it is hard to ignore water as a solvent. Beyond being
the most abundant natural solvent, it has extraordinary
physical properties, and although the traditional concerns seem well founded, many examples indicate that
reactions commonly performed with organic solvents
work equally well, if not better, in water.
During the past several years, we encountered
numerous examples of water as the “perfect” solvent.
These include (1) osmium-catalyzed dihydroxylation and
aminohydroxylation, (2) nucleophilic opening of epoxides and aziridines, (3) cycloaddition reactions of all
kinds, (4) most oxime ether, hydrazone, and aromatic
heterocycle condensation processes, and (5) the formation of an amide from a primary amine and an acid
chloride when aqueous Schotten-Baumann conditions
are used. In many of these instances, the starting material, product, or both are relatively insoluble in water;
in others, the reagents are water sensitive, yet the reac-
T
45
tions proceed with high yield and fewer side products
than when organic solvents are used. Thus encouraged,
we envision a special style of organic synthesis, one
based on an entire family of reactions for which water
is the best solvent.
In our continuing quest for new, supremely reliable
reactivity, we uncovered several convenient approaches
to 1H-tetrazole functionality, which is commonly found
in a variety of drugs and biologically active compounds.
The most convenient route to 5-substituted-1H-tetrazoles is the addition of azide ion to nitriles. Although
many methods can be used for this transformation,
appealing synthetic routes are scarce. All of the reported
syntheses have one or more of the following drawbacks:
a requirement for expensive and toxic metals, severe
water sensitivity, or the presence of hydrazoic acid,
which is highly toxic and explosive as well as volatile.
In addition, all of the known methods require the use
of organic solvents, which are expensive and environmentally unfriendly. Therefore, we were pleased to find
a safer and exceptionally efficient process for transforming nitriles into tetrazoles in water; the only other
reagents are sodium azide and a zinc salt (Fig. 1).
F i g . 1 . Zinc-catalyzed synthesis of 1H-tetrazoles from nitriles
and sodium azide.
Thus, formation of tetrazoles proceeds with excellent yields and scope in refluxing water. Thanks to the
low pK a of 1H-tetrazoles and their highly crystalline
nature, a simple acidification is usually sufficient to
provide the pure products. Thus, we prepared a wide
variety of tetrazoles by using an exceedingly simple
procedure, often involving little more than mixing the
starting materials in water, heating, and filtering off
pure products.
To further expand the usefulness of this transformation, we applied this method to the synthesis of
tetrazole analogs of amino acids. Such compounds are
important in peptide chemistry as mimics for terminal
carboxylic acid residues and for cis-amide bonds. In
many cases, these analogs are as active as the native
46
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
compounds but are more metabolically stable, making
them attractive medicinal targets. Simple heating of the
α-amino nitrile in a mixture of water and isopropanol at
reflux (about 80°C) in the presence of sodium azide and
zinc bromide effects clean conversion to the α-amino
tetrazole. These solvents are both environmentally
benign and easily isolated; acidification and extraction
are usually sufficient to provide the product as a pure
solid (Fig. 2).
F i g . 2 . Synthesis of tetrazole analogs of amino acids.
As a result of collaborations with colleagues at The
Scripps Research Institute, many of our synthetic products are currently being tested for a wide range of activities. These include screening for antibacterial agents,
in our laboratory; inhibition of HIV protease, in collaboration with J. Elder, The Scripps Research Institute,
and C.-H. Wong, the Skaggs Institute; amyloid binding, with J. Kelly, the Skaggs Institute; RNA binding,
with J. Williamson, the Skaggs Institute; and inhibition of anthrax lethal factor, with P. Vogt, The Scripps
Research Institute.
PUBLICATIONS
Himo, F., Demko, Z.P., Noodleman, L., Sharpless, K.B. Why is tetrazole formation
by addition of azide to organic nitriles catalyzed by zinc (II) salts? J. Am. Chem.
Soc. 125:9983, 2003.
Lee, L.V., Mitchell, M.L., Huang, S.-J., Fokin, V.V., Sharpless, K.B., Wong, C.-H.
A potent and highly sensitive inhibitor of human α-1,3-fucosyltransferase via click
chemistry. J. Am. Chem. Soc. 125:9588, 2003.
Ripka, A.S., Diaz, D.D., Sharpless, K.B., Finn, M.G. First practical synthesis of
formamidine ureas and derivatives. Org. Lett. 5:1531, 2003.
Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V.V., Sharpless, K.B., Finn, M.G. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am.
Chem. Soc. 125:3192, 2003.
Antibody Catalysis, Organic
Synthesis, and Prodrug and
Targeting Therapies
S.C. Sinha, R.A. Lerner, L.-S. Li, S. Das, S. Dutta
ur research interests involve synthetic methods,
total synthesis of biologically important natural
products, antibody catalysis, and development
of prodrug and targeting therapies. In the past year, we
mainly focused on the development of new synthetic
strategies, including the stereoselective synthesis of
the substituted oxacyclic ketones by the intramolecular aldol-type reaction. We also reported a new method
for the intermolecular aldol-type reaction between a
ketone and an acetal or a ketal. Using the aldol-type
reaction, in combination with aldolase monoclonal
antibodies, we are developing chemoabzymatic syntheses of a number of macrocyclic compounds, including
epothilones and sorangiolides.
We are collaborating with E. Keinan, the Skaggs
Institute, in the synthesis of annonaceous acetogenins
and in reactions mediated by rhemium(VII) oxides. In
the joint effort, we synthesized an analog of asimicin,
an annonaceous acetogenin, for the photoaffinity labeling of the corresponding receptor.
In the prodrug therapy project, we synthesized and
evaluated a number of prodrugs from doxorubicin and
paclitaxel. In the targeting approach, we used SCS-873,
a small-molecule dual antagonist of the integrins αvβ3
and α v β 5 to transform the function of the catalytic
aldolase monoclonal antibody 38C2 into a targeting
antibody. The conjugate composed of SCS-873 and
38C2 targets cancer cells that overexpress the integrins
α v β 3 and α v β 5 and inhibits the growth of the cells.
The prodrug and targeting therapies are done in collaboration with C.F. Barbas and C. Rader, The Scripps
Research Institute.
O
SYNTHETIC METHODS AND THE SYNTHESIS OF
N AT U R A L P R O D U C T S
Our intramolecular and intermolecular aldol-type
reaction is an alternative 1-step Mukaiyama reaction.
In the classic Mukaiyama reaction, a carbonyl compound is first transformed into the corresponding silyl
enol ether, which then reacts with an acetal to form
β-alkoxy carbonyl compounds. In our 1-step process, a
boron enolate of a ketone, generated in situ by reacting a ketone with dibutylboron triflate and diisopropyl-
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
ethyl amine, reacts with an acetal or a ketal to provide the corresponding β-alkoxy carbonyl compounds.
The intramolecular reaction is highly stereoselective
although limited in scope. Thus, only substrates containing methyl ketone react effectively to provide the
4-tetrahydropyrone derivatives with very high cis-selectivity. The intermolecular reaction is, on the other hand,
versatile but less stereoselective (Fig. 1).
F i g . 1 . Dibutylboron triflate/i-Pr 2NEt–mediated aldol-type reaction of ketones with acetals.
In the past year, we also focused on the synthesis
of macrolide natural products, such as sorangiolides A
and B (Fig. 2). Although, these macrolides have a weak
antibiotic activity against gram-positive bacteria, the
synthesis will provide access to their analogs.
47
CBI analogs, and epothilones. We are developing new
linkers for the production of the prodrugs. We are also
synthesizing new haptens to elicit aldolase antibodies.
Both monoclonal antibodies and small-molecule antagonists will be used to direct the aldolase monoclonal
antibodies to cancer cells.
We synthesized a number of integrin-targeting compounds, including SCS-563 and SCS-572. These compounds are based on the SKB compounds. SCS-563
and SCS-572 are equipped with a linker, which allowed
us to prepare the corresponding biotin and diketone
derivatives. All of these compounds bound to cells
expressing the integrins αvβ3 and αvβ5. The diketone
compounds SCS-864, SCS-873, and SCS-913 and
many other diketone-containing integrin-targeting compounds that we synthesized, react covalently with the
reactive lysine residues in the binding site of an aldolase monoclonal antibody. The resultant conjugates have
several merits, including prolongation of the half-life of
the antagonist and in vivo assembly of the conjugate.
We used the conjugate composed of SCS-873 and
the antibody 38C2 to study the effect of the conjugate on the growth of the Kaposi sarcoma and melanoma, which express the integrin αvβ3. In mice with
human cancer xenografts, compared with SCS-873
alone, the SCS-873–38C2 conjugate significantly
inhibited tumor growth.
PUBLICATIONS
Das, S., Li, L.S., Sinha, S.C. Stereoselective aldol-type cyclization reaction mediated by dibutylboron triflate/diisopropylethylamine. Org. Lett. 6:123, 2004.
Han H., Sinha, M.K., D’Souza, L., Keinan, E., Sinha, S.C. Total synthesis of 34hydroxyasimicin and its photoactive derivative for affinity labeling of the mitochondrial complex I. Chemistry, in press.
Li, L.S., Das, S., Sinha, S.C. Efficient one-step aldol-type reaction of ketones with
acetals and ketals mediated by dibutylboron triflate/diisopropylethyl amine. Org.
Lett. 6:127, 2004.
Rader, C., Sinha, S.C., Popkov, M., Lerner, R.A., Barbas, C.F. III. Chemically programmed monoclonal antibodies for cancer therapy: adaptor immunotherapy based
on a covalent antibody catalyst. Proc. Natl. Acad. Sci. U. S. A. 100:5396, 2003.
Rader, C., Turner, J.M., Heine, A., Shabat, D., Sinha, S.C., Wilson, I.A., Lerner,
R.A,, Barbas, C.F. A humanized aldolase antibody for selective chemotherapy and
adaptor immunotherapy. J. Mol. Biol. 332:889, 2003.
F i g . 2 . Structures of sorangiolides A and B.
Saphier, S., Sinha, S.C., Keinan, E. Antibody-catalyzed enantioselective Norrish
type II cyclization. Angew. Chem. Int. Ed. 42:1378, 2003.
P R O D R U G A N D TA R G E T I N G T H E R A P I E S
Prodrug therapy provides a unique approach that
can be used to minimize the toxic effects of a drug.
Ideally, a nontoxic prodrug selectively releases the
cytotoxic form of the drug at an appropriate site. We
are developing new prodrugs of cytotoxic molecules,
including paclitaxel, doxorubicin analogs, enediynes,
48
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Macromolecular Machines as
Master Keys in Genome Integrity,
the Cell Cycle, Control of Reactive
Oxygen Species, and Pathogenesis
J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras, R. Brudler,
R.M. Cardoso, J.M. Castagnetto, B.R. Chapados, C. Chahwan,
L. Craig, D.S. Daniels, M. DiDonato, G. Divita, L. Fan, S. Han,
C. Hitomi, K. Hitomi, J.L. Huffman, K.-P. Hopfner, D. Hosfield,
C.D. Mol, G. Moncalian, S.S. Parikh, C.D. Putnam, D.S. Shin,
M.E. Stroupe, O. Sundheim, T.I. Wood, A. Yamagata
he goal of our research funded by the Skaggs
Institute is to bridge the gaps from molecular
structure to cell biology to therapeutics. We proceed by providing insights on the structure and function of macromolecular machines, by designing novel
drugs and proteins, and by creating a molecular-based
understanding of and intervention into human diseases.
Importantly, the Skaggs funding appropriately leverages
our Computational Center for Macromolecular Structure
at The Scripps Research Institute and our synchrotron
beamline, Structurally Integrated Biology for Life Sciences, at the University of California/Lawrence Berkeley National Laboratory, Berkeley, California, which is
designed to characterize macromolecular machines and
conformational changes.
This year we published results of research on
machines relevant to cellular protection via control
of reactive oxygen species, pathogenesis, and cancerrelated aspects of genome maintenance. We made
substantial progress in understanding the molecular
machinery that acts in all 3 major processes related
to cancer susceptibility: DNA repair, DNA replication,
and DNA recombination.
Our work on control of reactive oxygen species by
the protein superoxide dismutase addresses the central
paradox of how mutations that are spread over the protein sequence cause a single disease phenotype. We
found that mutations in superoxide dismutase that cause
the fatal neurodegenerative disease familial amyotrophic
lateral sclerosis are positioned at sites expected to affect
formation of the dimer and the integrity of the subunits.
This instability promotes the formation of amyloid-like
filamentous aggregates that resemble those present in
the neurons of patients who have the disease (Fig. 1).
As a result of our studies on pathogenesis, we published x-ray crystal structures of type IV pilins from Vib-
T
F i g . 1 . Mutations that destabilize the structural architecture of
Cu,Zn superoxide dismutase promote formation of amyloid-like fibrils.
A, Stereo pair shows structural changes at the H43 mutation site
and differences in the molecular surface (brick texture). B, Electron
micrograph of negatively stained mutant superoxide dismutase shows
formation of amyloid-like fibrils after 22 days’ incubation at 37°C,
pH 3.5, in the presence of EDTA. No fibrils were observed under
these conditions for the wild-type enzyme. Scale bar = 40 nm.
rio cholerae and Pseudomonas aeruginosa and a model
for their assembly based on our additional electron
microscopy and image analysis. Because type IV pilins
are virulence factors, understanding their structure and
function is critical to controlling cholera, pneumonia,
gonorrhea, meningitis, and severe diarrhea. We showed
that the conserved N termini of type IV pilins provide
a hydrophobic surface for self-assembly, allowing each
pilin subunit to interact with multiple neighboring subunits along an extensive surface area to form a thin
flexible filament with remarkable tensile strength. Variable regions in the C terminus form specific subunit
interaction sites and expose a variable surface that
imparts highly specialized pilus functions of colonization, self-aggregation, twitching motility, DNA uptake,
and immune escape.
In our studies on the pathogenesis of schistosomiasis, we characterized the mechanism by which glutathione S-transferase from the parasite Schistosoma
japonicum catalyzes the conjugation of glutathione with
toxic secondary products of membrane lipid peroxidation.
In DNA repair, we analyzed the complex evolutionary, functional, and structural relationships between
light-dependent DNA repair photolyase enzymes and
cryptochromes. We calculated a comprehensive phylogenetic tree for the photolyase/cryptochrome protein
family, determined the first crystal structure of a cryptochrome (Synechocystis cryptochrome), and performed
microarray analyses on the whole Synechocystis genome.
Our results suggest a new cryptochrome protein class
(cryptochrome DASH) in plants and bacteria and indicate its involvement in transcriptional control. The cryptochrome structure reveals commonalities with DNA
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
repair photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features.
Cytidine deamination to uracil is crucial for antibody
hypervariation as part of the normal immune response,
but outside lymphocytes uracil is mutagenic in replicating DNA. Ubiquitous enzymes called dUTPases remove
deoxyuridine triphosphate (dUTP) from the nucleotide
pool to prevent its misincorporation, and another ubiquitous pathway exists to remove uracil from DNA upon
spontaneous deamination of cytidine. We determined
crystal structures of the bifunctional deoxycytidine triphosphate (dCTP) deaminase and dUTPase from Methanococcus jannaschii, which converts dCTP or dUTP
to deoxyuridine monophosphate (dUMP) and has the
added metabolic role of initiating the conversion of dCTP
to deoxythymidine triphosphate (dTTP) to adjust intracellular levels. These structures revealed the specificity for both dCTP and dUTP, the likely mechanism for
deaminase activity, and how the protein might be subject to feedback inhibition by dTTP.
DNA recombination is responsible for repair of the
most serious form of genome damage: breaks in double-stranded DNA. We successfully used comparative
structural genomics, mutational analyses, and cell biology to understand the molecular basis of the role of the
breast cancer susceptibility protein BRCA2 in the control of Rad51-mediated DNA recombination. The Rad51
protein forms a polymeric filament around broken DNA
ends as part of its mechanism to catalyze DNA strand
exchange, the precursor reaction of DNA recombination.
By solving the full-length polymeric structure of the
Pyrococcus furiosus Rad51 homolog (Fig. 2), we identified the key Rad51 polymerization motif (Rad51-PM)
that forms the basis for intermolecular stabilization of
active polymeric Rad51 nucleoprotein filaments and
oligomeric Rad51 inactive storage rings. Moreover, this
motif is found in 8 repetitive sequences within BRCA2,
which we found mimic and disassemble inactive Rad51
rings and also chaperone Rad51 subunits to sites of
double-stranded breaks to initiate formation of Rad51
polymers on DNA. Further support of our hypothesis
that Rad51-mediated DNA recombination is controlled
by interface exchange via Rad51-PM is supported by
the identification of the motif in Rad52, a mediator
protein also involved in the formation of Rad51-DNA
polymers (Fig. 2).
PUBLICATIONS
Abdalla, A.M., Bruns, C.M., Tainer, J.A., Mannervik, B., Stenberg, G. Design of a
monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein. Protein Eng. 15:827, 2002.
49
F i g . 2 . Breast cancer and the repair of DNA damage. The Rad51-
PM consists of an intermolecular β-sheet formed by β0 and β3 of
adjacent Rad51 subunits and serves as a flexible hinge to stabilize
Rad51 heptameric rings as revealed by the x-ray crystal structure
(A) of full-length Rad51 and the crystal structure of DNA-bound
helical filaments docked into an electron microscopy 3-dimensional
reconstruction (B). C, Rad51-PM also contains conserved phenylalanine and alanine residues on one subunit and hydrophobic pockets on the adjacent subunit. This motif is mimicked by BRCA2 for
Rad51 disassembly and DNA loading. D, By identifying a second
critical Rad51-BRCA2 binding element composed of an α-helical
clamp, we were able to design an archaeal Rad51 mutant that
could be targeted to damaged DNA by human BRCA2 within human
cells upon γ-irradiation. Fluorescent foci in human cell nuclei formed
by green fluorescent protein fused to the designed archaeal Rad51
mutant are shown.
Adak, S., Bilwes, A.M., Panda, K., Hosfield, D., Aulak, K.S., McDonald, J.F.,
Tainer, J.A., Getzoff, E.D., Crane, B.R., Stuehr, D.J. Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans. Proc.
Natl. Acad. Sci. U. S. A. 99:107, 2002.
Aoyagi, M., Arvai, A.S., Tainer, J.A., Getzoff, E.D. Structural basis for endothelial
nitric oxide synthase binding to calmodulin [published correction appears in EMBO
J. 22:1234, 2003]. EMBO J. 22:766, 2003.
Brudler, R., Hitomi, K., Daiyasu, H., Toh, H., Kucho, K., Ishiura, M., Kanehisa,
M., Roberts, V.A., Todo, T., Tainer, J.A., Getzoff, E.D. Identification of a new cryptochrome class: structure, function, and evolution. Mol. Cell 11:59, 2003.
Cardoso, R.M., Daniels, D.S., Bruns, C.M., Tainer, J.A. Characterization of the
electrophile binding site and substrate binding mode of the 26-kDa glutathione Stransferase from Schistosoma japonicum. Proteins 51:137, 2003.
Cardoso, R.M., Thayer, M.M., DiDonato, M., Lo, T.P., Bruns, C.K., Getzoff, E.D.,
Tainer, J.A. Insights into Lou Gehrig’s disease from the structure and instability of the
A4V mutant of human Cu,Zn superoxide dismutase. J. Mol. Biol. 324:247, 2002.
Chapados, B.R., Hosfield, D.J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J.A.
Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in
DNA replication and repair. Cell 116:39, 2004.
50
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Craig, L., Taylor, R.K., Pique, M.E., Adair, B.D., Arvai, A.S., Singh, M., Lloyd,
S.J., Shin, D.S., Getzoff, E.D., Yeager, M., Forest, K.T., Tainer, J.A. Type IV pilin
structure and assembly: x-ray and EM analyses of Vibrio cholerae toxin-coregulated
pilus and Pseudomonas aeruginosa PAK pilin. Mol. Cell 11:1139, 2003.
DiDonato, M., Craig, L., Huff, M.E., Thayer, M.M., Cardoso, R.M.F., Kassmann,
C.J., Lo, T.P., Bruns, C.K., Powers, E.T., Kelly, J.W., Getzoff, E.D., Tainer, J.A.
ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization [published correction appears in J. Mol. Biol. 334:175,
2003]. J. Mol. Biol. 332:601, 2003.
of HIV that is involved in transport of viral mRNAs from
the cell nucleus, where mRNA is made, into the cytoplasm, where the mRNA is used to make the viral proteins (Fig. 1). The RRE is the binding site for the HIV
Rev protein, and the Rev-RRE interaction is critical for
viral replication. If we can find small molecules that
block this interaction, they would impair HIV replication.
Hearn, A.S., Stroupe, M.E., Cabelli, D.E., Ramilo, C.A., Luba, J.P., Tainer, J.A.,
Nick, H.S., Silverman, D.N. Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine
C gamma into the substrate access channel. Biochemistry 42:2781, 2003.
Hopfner, K.P., Tainer, J.A. Rad50/SMC proteins and ABC transporters: unifying
concepts from high-resolution structures. Curr. Opin. Struct. Biol. 13:249, 2003.
Huffman, J.L., Li, H., White, R.H., Tainer, J.A. Structural basis for recognition and
catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus
jannaschii. J. Mol. Biol. 331:885, 2003.
McMurray, C.T., Tainer, J.A. Cancer, cadmium and genome integrity. Nat. Genet.
34:239, 2003.
Shin, D.S., Pellegrini, L., Daniels, D.S., Yelent, B., Craig, L., Bates, D., Yu, D.S.,
Shivji, M.K., Hitomi, C., Arvai, A.S., Volkmann, N., Tsuruta, H., Blundell, T.L., Venkitaraman, A.R., Tainer, J.A. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J. 22:4566, 2003.
Wang, M., Howell, J.M., Libbey, J.E., Tainer, J.A., Fujinami, R.S. Manganese
superoxide dismutase induction during measles virus infection. J. Med. Virol.
70:470, 2003.
Targeting HIV RNA
J.R. Williamson, A. Bunner, J. Chao, S. Edgcomb,
P.M. Funke, M. Hennig, E. Johnson, K.A. Lehmann,
P.K. Radha, M. Recht, S.P. Ryder, L.G. Scott, E. Sperling,
M.W. Trevathan
lthough a number of widely used therapeutic
strategies exist to combat HIV infection and
AIDS, HIV still has a strong propensity to mutate,
becoming resistant to drug therapies. The 2 main classes
of anti-HIV drugs are protease inhibitors and reverse
transcriptase inhibitors. These drugs target 2 of the
critical viral enzymes required for HIV replication.
Most known drugs target proteins, as is the case
for the currently prescribed anti-HIV drugs. HIV is a
retrovirus, and its genetic material is composed of RNA,
in contrast to the DNA of humans. In addition to serving as the genetic material, HIV RNA contains several
important regions critical for programmed reproduction
if the virus inside the host cell. If these key RNA regulatory elements could be blocked by drugs, we would have
a novel therapeutic approach to combat HIV infection.
We are working toward developing small molecules
as drug candidates for blocking a particular RNA element in HIV. The Rev response element (RRE) is a part
A
F i g . 1 . Function of the HIV Rev protein and the RRE in HIV repli-
cation. Binding of Rev to the RRE in the nucleus facilitates transport of viral mRNA into the cytoplasm. Early in infection, a set of
regulatory proteins is synthesized, whereas late in infection, structural proteins are synthesized. Binding of Rev to RRE in the nucleus
is thus a critical switch in the viral life cycle and an important
therapeutic opportunity.
We are developing libraries of chemical compounds
that are more appropriate for binding RNA targets than
are most known drugs, which bind to protein targets.
Most proteins have hydrophobic pockets, whereas RNA
molecules are very polar and bear charged residues. The
types of molecules that will preferentially bind RNAs are
expected to be somewhat different from the druglike molecules developed on the basis of medicinal chemistry.
The libraries of compounds for our first screening
effort were made from a collection of commercially
available compounds, selected for both their druglike
character and the diversity of their chemical structures.
Each compound in the library consists of a different
molecular framework that corresponds to frameworks
typically found in known drug molecules. A representative set of molecules from the library is shown in
Figure 2A.
We are using these compound libraries and nuclear
magnetic resonance spectroscopy to identify compounds
that bind to the RRE. We record a nuclear magnetic
resonance spectrum of a potential RRE-binding compound in the presence and absence of the RRE target
(Fig. 2B), and from the changes in the spectrum, we
can determine if the molecule is binding to the RNA.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
51
Gonsalvez, G.B., Lehmann, K.A., Ho, D.K., Stanitsa, E.S., Williamson, J.R.,
Long, R.M. RNA-protein interactions promote asymmetric sorting of the ASH1
mRNA ribonucleoprotein complex. RNA 9:1383, 2003.
Hoggan, D.B., Chao, J.A., Prasad, G.S., Stout, C.D., Williamson, J.R. Combinatorial crystallization of an RNA-protein complex. Acta Crystallogr. D Biol. Crystallogr.
59(Pt. 3):466, 2003.
Crystallographic Studies of
Immune Recognition and
Therapeutic Targets
I.A. Wilson, D.A. Calarese, R.M.F. Cardoso, C.-G. Cheong,
A.L. Corper, M.D.M. Crispin, M.-A. Elsliger, S. Ferguson,
A. Gakhal, P.A. Horton, S. Ito, N.A. Larsen, J.G. Luz,
E. Ollmann Saphire, J.B. Reiser, R. Sanders, D.A. Shore,
R.L. Stanfield, R.S. Stefanko, J. Stevens, P. Verdino,
D.W. Wolan, D. Wu, L. Xu, D.M. Zajonc, Y. Zhang, X. Zhu
main focus of our research is determining the
crystal structure of proteins involved in human
diseases; our goal is to design structure-based
therapies. The proteins of interest include antibodies
that neutralize HIV type 1 (HIV-1), antibodies that catalyze the degradation of cocaine and heroin, anticancer
target enzymes in the purine biosynthesis pathway, and
molecules involved in the innate and adaptive immune
responses to microbial pathogens.
A
F i g . 2 . A, Small molecules for screening against RNA targets.
These molecules were selected by using a chemical informatics procedure to be small soluble druglike molecules with chemical frameworks different from the frameworks typically found in drug molecules. B, Nuclear magnetic resonance spectroscopy screening for a
compound that specifically binds to the RRE. The experiment performed is termed water-LOGSY. The bottom tracing is a reference
spectrum of the compound alone. The inverted signals in the top
tracing indicate that the compound tested is binding to the RRE.
In the middle tracing, binding to simple double-stranded RNA (the
control) is much less, indicating that the compound binds specifically to the RRE.
We obtained several initial hits from our first round
of screening against the RRE. These compounds bind
too weakly to be inhibitors of viral replication, so we
will need to identify analogs of our initial hits that have
increased potency. In addition, we should be able to
obtain structural information on how these first-generation inhibitors bind to the RRE RNA and use this
information to guide synthesis of the next generation
of compounds.
PUBLICATIONS
Chao, J.A., Prasad, G.S., White, S.A., Stout, C.D., Williamson, J.R. Inherent protein
structural flexibility at the RNA-binding interface of L30e. J. Mol. Biol. 326:999, 2003.
Cilley, C.D., Williamson, J.R. Structural mimicry in the phage ϕ21 N peptide-boxB
RNA complex. RNA 9:663, 2003.
H I V VA C C I N E D E S I G N
The HIV-1 envelope glycoproteins gp120 and gp41
are the targets of neutralizing antibodies and are of
interest as potential components of an HIV-1 vaccine.
However, only a handful of antibodies against these
proteins have been identified that can potently neutralize a wide array of primary HIV-1 isolates. We carried out x-ray structural studies for several of these
broadly neutralizing human antibodies to understand
their mechanism of action. We are examining b12,
2G12, and 447-52D, which are antibodies to gp120,
and 4E10, an antibody to gp41.
The human antibody 2G12 neutralizes a broad
range of HIV-1 isolates by binding an unusually dense
cluster of carbohydrate moieties on the gp120 envelope
glycoprotein. Crystal structures of the Fab fragment of
2G12 in complex with the dimannose and the oligomannose revealed that 2 Fabs assemble into an interlocked V H domain-swapped dimer. The dimerized Fabs
provide an extended surface for multivalent interaction
with a conserved cluster of oligomannose sugars on the
52
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
surface of gp120. This extraordinary domain-swapped
configuration was not observed previously in more than
200 antibody structures and therefore can provide a
novel template for engineering antibodies with higher
affinities against molecular clusters.
Crystal structures were also determined for human
antibodies 447-52D (Fig. 1) and 4E10 in complex with
peptides corresponding to the antibodies’ linear epitopes.
These structures not only help explain how these potent
antibodies recognize their antigens but also provide
templates for the design of structurally constrained
immunogens. The HIV-1 studies are done in collabora-
is unknown. In collaboration with N.C. Bruce, University of York, York, England, we determined the crystal
structures of heroin esterase alone and in complex with
the inhibitor dimethylarsinic acid. The findings provide
a structural framework for improving the specificity and
sensitivity of a recently reported heroin biosensor.
In collaboration with R. Lerner and K. Janda, the
Skaggs Institute, and P. Wirsching, The Scripps Research
Institute, we are investigating a novel approach to treat
cocaine abuse by administering catalytic antibodies
that metabolize cocaine. The crystal structures of these
antibodies (3A6, 7A1, and 4D3) bound to the substrate
cocaine, to transition-state analogs, or to product complexes will be crucial in elucidating the catalytic mechanism and for redesigning haptens and engineering
proteins to increase the efficiency and specificity of
the antibodies.
THE ADAPTIVE IMMUNE RESPONSE AGAINST
M I C R O B I A L PAT H O G E N S
F i g . 1 . 447-52D is a human antibody to HIV-1 that recognizes
the V3 loop region of gp120. The antibody light and heavy chains
are shown as light and dark gray ribbons; the V3 peptide, as a
ball-and-stick representation. The V3 peptide binds primarily via
the H3 complementarity-determining region loop, forming a 3stranded β-sheet, with most of the V3-Fab interactions through
main chain–main chain hydrogen bonds. This mode of binding is
restricted in sequence solely at the crown region of the V3 loop,
one reason why 447-52D may be unusually broadly neutralizing.
tion with D. Burton, The Scripps Research Institute;
H. Katinger, University of Agriculture, Vienna, Austria;
and S. Zolla-Pazner, New York University Medical Center, New York, New York.
COCAINE AND HEROIN ANTIBODIES AND ENZYMES
Heroin esterase hydrolyzes the acetyl groups from
heroin and phenyl acetate to yield morphine and phenol,
respectively, although the natural substrate for the enzyme
Binding of the murine T-cell receptor (TCR) 2C to
the non-self H-2K bm3 MHC molecule presenting the
self-peptide dEV8 elicits an alloresponse, which in tissue graft recipients can lead to graft rejection and organ
failure. Comparison of the structure of this complex
(2C–H-2K bm3 –dEV8) with that of the corresponding
TCR–self-antigen complex revealed subtle rearrangements
in the binding interface that mediate the alloreactivity.
These rearrangements alter the surface complementarity and number of atomic contacts between the TCR
and the MHC-peptide antigen. In collaboration with
D. Kranz, University of Illinois, Urbana, Illinois, we are
investigating the T-cell selection process by studying
the interaction of engineered, high-affinity TCRs with
MHC molecules. We crystallized one of these TCRs, M40,
in complex with the H-2K b–SIYR superagonist antigen.
We are also investigating the structural basis of
recognition of glycosylated and nonglycosylated peptides by MHC class I molecules for cancer immunotherapy. A successful anticancer vaccine is an important
goal, and currently MUC1 (a tumor-associated antigen)
is a favored target antigen. Low-affinity MUC1 peptides,
which do not contain canonical anchor residues, induce
strong T-cell responses and protect mice against a
tumor challenge. In collaboration with V. Apostolopoulos, Austin Research Institute, Melbourne, Australia,
we previously found that a MUC1 peptide, SAPDTRPA,
without the standard anchor residues, still bound to the
MHC class I molecule H-2K b . This research is being
extended to include the crystal structures of low-affin-
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
ity peptides, short peptides (5-mer), and glycopeptides,
all of which induce T-cell responses in mice.
An inflammatory joint disease with many similarities
to human rheumatoid arthritis develops spontaneously in
KRN TCR transgenic mice. Presentation of the class II
MHC molecule I-A g7 in complex with a self-peptide
derived from glucose-6-phosphate isomerase to the
KRN TCR of these mice is a critical step in the initiation of the disease. The crystal structures of I-A g7 in
complex with the isomerase and of the TCR KRN will
enhance our understanding of how autoimmune disease is mediated at the molecular level.
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
Members of the CD1 family of nonpolymorphic, nonclassical MHC molecules bind lipids and glycolipids for
presentation to specialized T cells. We recently determined the structure of the human CD1a isoform in complex with a sulfatide self-antigen (Fig. 2). Compared with
healthy subjects, patients with multiple sclerosis have
greater numbers of T cells that react with these selfantigens, a condition that contributes to the pathogen-
F i g . 2 . View into the binding groove of CD1a. The glycosphingolipid sulfatide is shown in the binding pocket of CD1a between the
α1-α2 helices. The tail end of the fatty acid and the sulfate group
of the galactose sugar moiety are exposed for T-cell recognition.
esis of this autoimmune disease. We are testing an
array of TCRs, against both self-antigens and foreign
antigens, for expression and crystallization in complex
with CD1-glycolipids. Analysis of the presentation of
self-antigens and foreign antigens by various CD1 isotypes will greatly enhance our understanding of the
initiation of the cellular immune response and the triggering of autoimmune diseases. Lipid and glycolipid
ligands are provided by our collaborators, D.B. Moody
and M.B. Brenner, Harvard Medical School, Boston,
53
Massachusetts, and C.H. Wong, the Skaggs Institute.
Collaborators in our studies on CD1 and TCRs include
L. Teyton, The Scripps Research Institute; M. Kronenberg, La Jolla Institute for Allergy and Immunology,
San Diego, California; and R. Dwek and P. Rudd, University of Oxford, Oxford, England.
A N T I F O L AT E S F O R C H E M O T H E R A P Y
Glycinamide ribonucleotide transformylase is a key
folate-dependent enzyme in the de novo purine biosynthesis pathway and is a target for the design of antitumor drugs. We determined structures for both apo and
complexed forms of the human transformlyase at various pH values. The 1.7-Å structure of the enzyme in
complex with an epoxide-based folate analog provided
by D. Boger, The Scripps Research Institute, provided
important new insights into the mechanism of inhibition of human gycinamide ribonucleotide transformylase
and for the design of inhibitors of the enzyme.
Aminoimidazole carboxamide ribonucleotide (AICAR)
transformylase inosine monophosphate cyclohydrolase
(ATIC) is a homodimeric folate-dependent enzyme that
encompasses both AICAR transformylase and inosine
monophosphate cyclohydrolase activities responsible
for the catalysis of the penultimate and final steps of
the purine de novo synthesis pathway. ATIC is also a
potential target for the development of anticancer drugs.
Structure-based development of novel inhibitors aimed
at reducing nonspecific interactions with other folatedependent enzymes within the cell are being designed
in collaboration with D. Boger, The Scripps Research
Institute; G. Beardsley, Yale University, New Haven,
Connecticut; and S. Benkovic, Pennsylvania State University, University Park, Pennsylvania. Complexes of
both human and avian ATIC with substrates and inhibitors
(Fig. 3) are being studied to elucidate the mechanistic
capabilities of the complexes and advance the development of inhibitors for both of the activities in ATIC.
In collaboration with C. Li and A. Olson, The Scripps
Research Institute, we are using virtual ligand screening via protein docking to identify potential small-molecule ligands for ATIC. Using this approach, combined
with enzymatic assays, we identified several novel inhibitors that have micromolar binding affinities. A crystal
structure of ATIC in complex with one of the inhibitors
allowed the compound to be unambiguously identified
from the electron density. The structure provided an
important initial template for iterations of design and
optimization of the inhibitors as novel chemotherapeutic agents.
54
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Royle, L., Roos, A., Harvey, D.J., Wormald, M.R., Van Gijlswijk-Janssen, D.J.,
Redwan, el-R.M., Wilson, I.A., Daha, M.R., Dwek, R.A., Rudd, P.M. Secretory IgA
N- and O-glycans provide a link between the innate and adaptive immune systems.
J. Biol. Chem. 278:20140, 2003.
Rudolph, M.G., Kelker, M.S., Schneider, T.R., Yeates, T.O., Oseroff, V., Heidary,
D.K., Jennings, P.A., Wilson, I.A. Use of multiple anomalous dispersion to phase
highly merohedrally twinned crystals of interleukin-1β. Acta Crystallogr. D Biol.
Crystallogr. 59(Pt. 2):290, 2003.
Rudolph, M.G., Stevens, J., Speir, J.A., Trowsdale, J., Butcher, G.W., Joly, E.,
Wilson, I.A. Crystal structures of two rat MHC class Ia (RT1-A) molecules that are
associated differentially with peptide transporter alleles TAP-A and TAP-B. J. Mol.
Biol. 324:975, 2002.
F i g . 3 . Solvent-accessible surface of the multisubstrate adduct
inhibitor bound to the active site of AICAR transformylase. The
structure is significant because the location of the folate moiety
corroborates the formyl transfer mechanism that was previously
proposed on the basis of the substrate-bound ATIC complex. This
structural template can aid in future development of inhibitors to
AICAR transformylase with more optimal pharmacokinetic properties.
PUBLICATIONS
An, Y., Shao, Y., Alory, C., Matteson, J., Sakisaka, T., Chen, W., Gibbs, R.A., Wilson, I.A., Balch, W.E. GDI-Rab GTPase recycling. Structure (Camb.) 11:347, 2003.
Brinen, L.S., Canaves, J.M., Dai, X., Deacon, A.M., Elsliger, M.A., Eshaghi, S.,
Floyd, R., Godzik, A., Grittini, C., Grzechnik, S.K., Guda, C., Jaroszewski, L.,
Karlak, C., Klock, H.E., Koesema, E., Kovarik, J.S., Kreusch, A., Kuhn, P., Lesley,
S.A., McMullan, D., McPhillips, T.M., Miller, M.A., Miller, M.D., Morse, A., Moy,
K., Ouyang, J., Robb, A., Rodrigues, K., Selby, T.L., Spraggon, G., Stevens, R.C.,
van den Bedem, H., Velasquez, J., Vincent, J., Wang, X., West, B., Wolf, G., Taylor, S.S., Hodgson, K.O., Wooley, J., Wilson, I.A. Crystal structure of a zinc-containing glycerol dehydrogenase (TM0423) from Thermotoga maritima at 1.5 Å
resolution. Proteins 50:371, 2003.
Calarese, D.A., Scanlan, C.N., Zwick, M.B., Deechongkit, S., Mimura, Y., Kunert, R.,
Zhu, P., Wormald, M.R., Stanfield, R.L., Roux, K.H., Kelly, J.W., Rudd, P.M., Dwek,
R.A., Katinger, H., Burton, D.R., Wilson, I.A. Antibody domain exchange is an
immunological solution to carbohydrate cluster recognition. Science 300:2065, 2003.
DeSantis, G., Liu, J., Clark, D.P., Heine, A., Wilson, I.A., Wong, C.H. Structurebased mutagenesis approaches toward expanding the substrate specificity of D-2deoxyribose-5-phosphate aldolase. Bioorg. Med. Chem. 11:43, 2003.
Diaz, M., Stanfield, R.L., Greenberg, A.S., Flajnik, M.F. Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR): identification of a new
locus preferentially expressed in early development. Immunogenetics 54:501, 2002.
Larsen, N.A., Heine, A., de Prada, P., Redwan, el-R., Yeates, T.O., Landry, D.W.,
Wilson, I.A. Structure determination of a cocaine hydrolytic antibody from a
pseudomerohedrally twinned crystal. Acta Crystallogr. D Biol. Crystallogr. 58(Pt.
12):2055, 2002.
Luz, J.G., Hassig, C.A., Pickle, C., Godzik, A., Meyer, B.J., Wilson, I.A. XOL-1,
primary determinant of sexual fate in C elegans, is a GHMP kinase and a structural
prototype for a class of developmental regulators. Genes Dev. 17:977, 2003.
Pantophlet, R., Ollmann Saphire, E., Poignard, P., Parren, P.W., Wilson, I.A.,
Burton, D.R. Fine mapping of the interaction of neutralizing and nonneutralizing
monoclonal antibodies with the CD4 binding site of human immunodeficiency virus
type 1 gp120. J. Virol. 77:642, 2003.
Pantophlet, R., Wilson, I.A., Burton, D.R. Hyperglycosylated mutants of human
immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV
vaccine design. J. Virol. 77:5889, 2003.
Redwan, el-R.M., Larsen, N.A., Zhou, B., Wirsching, P., Janda, K.D., Wilson, I.A.
Expression and characterization of a humanized cocaine-binding antibody. Biotechnol. Bioeng. 82:612, 2003.
Schwarzenbacher, R., Canaves, J.M., Brinen, L.S., Dai, X., Deacon, A.M., Elsliger,
M.A., Eshaghi, S., Floyd, R., Godzik, A., Grittini, C., Grzechnik, S.K., Guda, C.,
Jaroszewski, L., Karlak, C., Klock, H.E., Koesema, E., Kovarik, J.S., Kreusch, A.,
Kuhn, P., Lesley, S.A., McMullan, D., McPhillips, T.M., Miller, M.A., Miller, M.D.,
Morse, A., Moy, K., Ouyang, J., Robb, A., Rodrigues, K., Selby, T.L., Spraggon, G.,
Stevens, R.C., van den Bedem, H., Velasquez, J., Vincent, J., Wang, X., West, B.,
Wolf, G., Hodgson, K.O., Wooley, J., Wilson, I.A. Crystal structure of uronate isomerase
(TM0064) from Thermotoga maritima at 2.85 Å resolution. Proteins 53:142, 2003.
Stanfield, R.L., Ghiara, J.G., Ollmann Saphire, E., Profy, A.T., Wilson, I.A. Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop.
Virology 315:159, 2003.
Turner, J.M., Larsen, N.A., Basran, A., Barbas, C.F. III, Bruce, N.C., Wilson, I.A.,
Lerner, R.A. Biochemical characterization and structural analysis of a highly proficient cocaine esterase. Biochemistry 41:12297, 2002.
Wada, M., Hsu, C.C., Franke, D., Mitchell, M., Heine, A., Wilson, I., Wong, C.H.
Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric
aldol reactions. Bioorg. Med. Chem. 11:2091, 2003.
Wentworth, P., Jr., Wentworth, A.D., Zhu, X., Wilson, I.A., Janda, K.D., Eschenmoser, A., Lerner, R.A. Evidence for the production of trioxygen species during
antibody-catalyzed chemical modification of antigens. Proc. Natl. Acad. Sci. U. S. A.
100:1490, 2003.
Wolan, D.W., Greasley, S.E., Beardsley, G.P., Wilson, I.A. Structural insights into
the avian AICAR transformylase mechanism. Biochemistry 41:15505, 2002.
Zajonc, D.M., Elsliger, M.A., Teyton, L., Wilson, I.A. Crystal structure of CD1a in
complex with a sulfatide self antigen at a resolution of 2.15 Å. Nat. Immunol.
4:808, 2003.
Zhang, Y., Desharnais, J., Greasley, S.E., Beardsley, G.P., Boger, D.L., Wilson,
I.A. Crystal structures of human GAR Tfase at low and high pH and with substrate
β-GAR. Biochemistry 41:14206, 2002.
Zhang, Y., Desharnais, J., Marsilje, T.H., Li, C., Hedrick, M.P., Gooljarsingh, L.T.,
Tavassoli, A., Benkovic, S.J., Olson, A.J., Boger, D.L., Wilson, I.A. Rational
design, synthesis, evaluation and crystal structure of a potent inhibitor of human
GAR Tfase: 10-(trifluoroacetyl)-5-10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid.
Biochemistry 42:6043, 2003.
Zhu, X., Heine, A., Monnat, F., Houk, K.N., Janda, K.D., Wilson I.A. Structural basis
for antibody catalysis of a cationic cyclization reaction. J. Mol. Biol. 329:69, 2003.
Zhu, X., Larsen, N.A., Basran, A., Bruce, N.C., Wilson, I.A. Observation of an arsenic
adduct in an acetyl esterase crystal structure. J. Biol. Chem. 278:2008, 2003.
Zhu, Y., Rudensky, A.Y., Corper, A.L., Teyton, L., Wilson, I.A. Crystal structure of
MHC class II I-Ab in complex with a human CLIP peptide: prediction of an I-Ab
peptide-binding motif. J. Mol. Biol. 326:1157, 2003.
Zwick, M.B., Parren, P.W., Saphire, E.O., Church, S., Wang, M., Scott, J.K.,
Dawson, P.E., Wilson, I.A., Burton, D.R. Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency
virus type 1 gp120. J. Virol. 77:5863, 2003.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Bioorganic Chemistry and
Drug Discovery
C.-H. Wong, F. Agnelli, C. Behrens, M. Best, A. Brik,
M.C. Bryan, A. Buchnyskyy, G. Cain, A. Chang, W.C. Cheng,
A. Datta, S. Duron, F. Fazio, D. Franke, J.I. Furukawa,
M.C. Galan, S. Hanson, Z.-Y. Hong, J. Hsu, C.-Y. Huang,
H.K. Lee, L. Lee, F.-S. Liang, D. Lin, H. Liu, J. Liu,
55
DeSantis, G., Liu, J., Clark, C.P., Heine, A., Wilson, I.A., Wong, C.-H. Structurebased mutagenesis approaches toward expanding the substrate specificity of D-2deoxy-ribose-5-phosphate aldolase. Bioorg. Med. Chem. 11:43, 2003.
Fazio, F., Bryan, M.C., Blixt, O., Paulson, J.C., Wong, C.-H. Synthesis of sugar
arrays in microtiter plates. J. Am. Chem. Soc. 124:14397, 2003.
Lee, L.V., Mitchell, M.L., Huang, S.-J., Fokin, V.V., Sharpless, K.B., Wong, C.-H.
A potent and highly selective inhibitor of human α-1,3-fucosyltransferase via click
chemistry. J. Am. Chem. Soc. 125:9588, 2003.
Liu, H., Ritter, T.K., Sadamoto, R., Sears, P.S., Wu, M., Wong, C.-H. Acceptor
specificity and inhibition of the bacterial cell-wall glycosyltransferase Mur G. Chembiochem 4:603, 2003.
M. Numa, P. Nyffeler, T. Polat, D. Thayer, S.K. Wang,
G. Watt, D. Wu, Y.Y. Yang, N. Yu, G.-W. Xing, R. Xu
uring the past year, our effort was directed
toward carbohydrate-based drug discovery. In
continuation of our interest in developing new
enzymatic methods for organic synthesis, we developed
new aldolases through directed evolution and structurebased mutagenesis and used the modified enzymes in
the synthesis of iminocyclitol derivatives as fucosyltransferase inhibitors. We also developed a new noncovalent method to assemble saccharide microarrays in
styrene microtiter plates for high-throughput screening.
The saccharides prepared were attached to a C13–C16
hydrocarbon chain at the anomeric center for the noncovalent attachment to the microplate surface. The
sugars were further modified enzymatically by using
glycosyltransferases to expand the diversity of the
array. This array was then used in a high-throughput
assay for discovery of fucosyltransferase inhibitors as
potential drug candidates for the treatment of inflammatory diseases.
We also used the copper(I)-catalyzed triazole formation, in collaboration with K.B. Sharpless, the Skaggs
Institute, to prepare lipidated oligosaccharides for
attachment to styrene plates and to discover new fucosyltransferase inhibitors. The triazole-forming chemistry
was carried out in the plates, and then screening in situ
was used to identify potent inhibitors. This strategy was
effective for discovery of new inhibitors of any enzyme
and was applied to the discovery of new HIV protease
inhibitors and glycosidase inhibitors by using different
reaction and in situ screening methods.
D
PUBLICATIONS
Brik, A., Muldoon, J., Lin, Y.-C., Elder, J.H., Goodsell, D., Olson, A.J., Fokin, V.V.,
Sharpless, K.B., Wong, C.-H. Rapid diversity-oriented synthesis in microtiter plates
for in situ screening of HIV protease inhibitors. Chembiochem 4:1246, 2003.
Chapman, E., Bryan, M.C., Wong, C.-H. Mechanistic studies of β-arylsulfotransferase-IV. Proc. Natl. Acad. Sci. U. S. A. 100:910, 2003.
Chapman, E., Ding, S., Schultz, P.G., Wong, C.-H. A potent and highly selective
sulfotransferase inhibitor. J. Am. Chem. Soc. 124:14524, 2002.
Liu, H., Wang, L., Brock, A., Wong, C.-H., Schultz, P.G. A method for the generation of glycoprotein mimetics. J. Am. Chem. Soc. 125:1702, 2003.
Mak, C.C., Brik, A., Lerner, D.L., Elder, J.H., Morris, G.M., Olson, A.J., Wong,
C.-H. Design and synthesis of broad-based mono- and bicyclic inhibitors of HIV
and FIV proteases. Bioorg. Med. Chem. 11:2025, 2003.
Mong, T.-K., Huang, C.-Y., Wong, C.-H. A new reactivity-based one-pot synthesis
of N-acetyllactosamine oligomers. J. Org. Chem. 68:2135, 2003.
Mong, T.-K., Lee, H.K., Duron, S.G., Wong, C.-H. Reactivity-based one-pot synthesis of fucose GM1 oligosaccharide: a sialylated antigenic epitope of small-cell
lung cancer. Proc. Natl. Acad. Sci. U. S. A. 100:797, 2003.
Ritter, T.K., Mong, K.-K., Liu, H., Nakatani, T., Wong, C.-H. A programmable onepot oligosaccharide synthesis for diversifying the sugar domains of natural products: a case study of vancomycin. Angew. Chem. Int. Ed. 42:4657, 2003.
Sawkar, A.R., Cheng, W.-C., Beutler, E., Wong, C.-H., Balch, W.E., Kelly, J.W.
Chemical chaperones increase the cellular activity of N370S β-glucosidase: a therapeutic strategy for Gaucher disease. Proc. Natl. Acad. Sci. U. S. A. 99:15428, 2002.
Silvestri, M.G., DeSantis, G., Wong, C.-H. Asymmetric aldol reactions using aldolases.
Top. Stereochem. 23:267, 2003.
Tolbert, T.J., Wong, C.-H. Glycoprotein synthesis. In: McGraw-Hill 2003 Yearbook
of Science and Technology. McGraw-Hill, New York, 2003, p. 159.
Wada, M., Hsu, C.-C., Franke, D., Mitchell, M., Heine, A., Wilson, I., Wong, C.-H.
Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric
aldol reactions. Bioorg. Med. Chem. 11:2091, 2003.
Wu, C.-Y., Chang, C.-F., Chen, J.S.-Y., Wong, C.-H. Lin, C.-H. Rapid diversity-oriented synthesis in microtiter plates for in situ screening: discovery of potent and
selective α-fucosidase inhibitors. Angew. Chem. Int. Ed. 42:4661, 2003.
Studies of Macromolecular
Recognition by Multidimensional
Nuclear Magnetic Resonance
P.E. Wright, H.J. Dyson, M. Martinez-Yamout, J. Covalt,
R. De Guzman, T. Dunzendorfer-Matt, N. Goto, B. Hudson,
J. Lansing, T. Nishikawa, G. Perez-Alvarado, J. Wojciak,
T. Zor, J. Hosea, M. Landes
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
S
56
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
fundamental molecular events of life. Knowledge of the
3-dimensional structures of biological macromolecules
is key to understanding their interactions and functions
and also forms the basis for rational design of new
drugs. A particularly powerful method for mapping the
3-dimensional structures and interactions of biological
macromolecules in solution is multidimensional nuclear
magnetic resonance spectroscopy. We are applying 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 elucidation of 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 mediates 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 the various domains
of CBP and map their functional interactions with other
components of the transcriptional machinery.
Some years ago, we determined the 3-dimensional
structure of the kinase-inducible activation domain 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.
In the past year, we determined the structure of the
complex between KIX and the transcriptional activation
domain of the proto-oncogene c-Myb (Fig. 1). The Myb
activation domain is intrinsically unstructured but folds
into a helical conformation on binding to KIX; it uses
the same hydrophobic binding groove as the CREB
activation domain but makes more intimate hydrophobic contacts. The structure provides new insights into
the factors that determine constitutive and inducible
F i g . 1 . The c-Myb transcriptional activation domain forms a
helix on binding in a hydrophobic surface groove on the KIX domain
of CBP.
binding to KIX. We also showed that the activation
domain of the mixed lineage leukemia protein binds
cooperatively with Myb and CREB to an allosteric site
on KIX, suggesting a direct mechanism by which CBP
can mediate transcriptional synergy, and we are refining the structure of the ternary complex formed between
the KIX domain of CBP and the activation domains of
Myb and CREB.
We performed a structural and thermodynamic
analysis of the complex formed between CBP and a
p160 nuclear receptor coactivator, the activator for thyroid hormone and retinoid receptors (ACTR). CBP and
the p160 coactivators play an essential role in the
nuclear hormone response and are implicated in cancer
and other diseases. Our structure reveals the mechanism
by which p160 coactivators recruit CBP and forms the
basis for design of novel therapeutic agents that could
modulate the nuclear hormone response. The ACTR and
CBP domains are both intrinsically unstructured, but
they fold synergistically to form a cooperatively folded
intermolecular complex. Mutagenesis experiments indicated that the molecular interaction is finely tuned for
specificity rather than for binding affinity.
We recently determined the structure of the complex formed between the carboxy-terminal activation
domain of the hypoxia-inducible transcription factor
HIF-1 and the Taz 1 zinc finger motif of CBP. HIF-1
activates genes that are crucial for cell survival under
hypoxic conditions; this is accomplished through interactions between its α-subunit (HIF-1α) and CBP/p300.
The hypoxic response, which plays an important role
in tumor progression and metastasis, is tightly regulated
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
in the cell. In particular, the protein CITED2 functions
as a negative feedback regulator that inhibits HIF-1α
by competing for binding to CBP/p300. We recently
determined the 3-dimensional structure of the complex between the activation domain of CITED2 and the
Taz 1 domain of CBP (Fig. 2). The CITED2 domain is
intrinsically unstructured and folds upon binding to CBP.
Our structure shows that CITED2 and HIF-1α bind to
partially overlapping surfaces of the Taz 1 domain and
compete for binding through a highly conserved sequence
57
PUBLICATIONS
De Guzman, R.N., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Interaction of
the TAZ1 domain of CREB-binding protein with the activation domain of CITED2:
regulation by competition between intrinsically unstructured ligands for non-identical binding sites. J. Biol. Chem., in press.
De Guzman, R.N., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Structure
and function of the CBP/p300 TAZ domains. In: Zinc Finger Proteins: From Atomic
Contact to Cellular Function. Iuchi, S., Kuldell, N. (Eds.). Landes Bioscience,
Georgetown, TX, in press.
Gearhart, M.D., Holmbeck, S.M.A., Evans, R.M., Dyson, H.J., Wright, P.E.
Monomeric complex of human orphan estrogen related receptor-2 with DNA: a
pseudo-dimer interface mediates extended half-site recognition. J. Mol. Biol.
327:819, 2003.
Perez-Alvarado, G.C., Martinez-Yamout, M., Allen, M.M., Grosschedl, R., Dyson,
H.J., Wright, P.E. Structure of the nuclear factor ALY: insights into post-transcriptional
regulatory and mRNA nuclear export processes. Biochemistry 42:7348, 2003.
Schnell, J.R., Dyson, H.J., Wright, P.E. Structure, dynamics and catalytic function
of dihydrofolate reductase. Ann. Rev. Biophys. Biomol. Struct., in press.
F i g . 2 . Structure of the activation domain of CITED2 (dark gray
ribbon) bound to the Taz 1 domain of CBP. The zinc atoms are
shown as gray spheres.
motif. This work provides new insights into the mechanism by which intrinsically unstructured proteins can
compete effectively for binding to a common target
within the complex macromolecular assembly that regulates transcription.
Gene expression in eukaryotes is mediated both by
transcriptional regulators and by posttranscriptional
events that control the lifetime of the mRNA. We recently
determined the structure of the zinc-binding domain of
the regulatory protein TIS11d bound to its cognate
AU-rich single-stranded RNA recognition element.
TIS11d is a member of the tristetraprolin family of regulatory proteins that bind the recognition element in
the 3′ untranslated region of the mRNA of TNF-α and
mediate its expression by promoting degradation of the
message. Our structure provides novel insights into
the mechanisms of site-specific recognition of singlestranded RNA.
58
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Staff Awards and Activities
Boger, D.L.—Adrien Albert Medal, Royal Society of
Joyce, G.F.—Member, U.S. National Academy of Sciences;
Chemistry, London, England; Bachmann Lecturer,
University of Michigan, Ann Arbor, Michigan; Ingersoll
Lecturer, Vanderbilt University, Nashville, Tennessee;
Member, Medicinal Chemistry Study Section, National
Institutes of Health; Member, Electorate Nominating
Committee, American Association for the Advancement
of Science; Member, Drug Candidate Evaluation Committee, Institute for the Study of Aging; Editor-in-Chief,
Bioorganic and Medicinal Chemistry Letters; Editorial
Boards, Tetrahedron Publications, Organic Reactions,
Journal of Organic Chemistry, Current Opinion in
Drug Discovery and Development, Current Drugs.
Head of Faculty in Chemical Biology, Faculty 1000,
Biology Reports Ltd.; Associate Editor, BioSystems,
Evolutionary Computation, Origins of Life and Evolution of the Biosphere.
Ernest Beutler— E. Donnall Thomas Lecture and Prize,
American Society of Hematology; Professional Achievement Citation, University of Chicago Alumni Association;
Kent Kiekow Memorial Leukemia Lecture, Northwestern
University Medical School, Chicago, Illinois; Member,
National Academy of Sciences; Member, American
Academy of Arts and Sciences; Scientific Advisory
Boards, Baxter Healthcare, Edwards Lifesciences,
iMetrikus, MPD Foundation, MyOwnMD, Optimer
Pharmaceuticals, Insert Therapeutics; Contributing
Editor, Blood Cells, Molecules, and Diseases; Associate
Editor, Acta Haematologica, Molecular Medicine.
Dawson, P.E.— Research Fellowship in Chemistry, Alfred
P. Sloan Foundation; Member, Faculty in Chemical Biology, Faculty 1000, Biology Reports, Ltd.; Editorial
Board, Letters in Peptide Science.
Janda, K.D.— Fellow, American Association for the
Advancement of Science; Sigma-Aldrich Lecturer,
Milwaukee Section, American Chemical Society,
Milwaukee, Wisconsin; Rayson Huang Visiting Lecturer,
University of Hong Kong, Hong Kong, China; Shire
BioChem Lecturer, Universite de Montreal, Montreal,
Canada; Editorial Boards, Chemical Reviews, Combinatorial Chemistry Research and Applications, Bioorganic
and Medicinal Chemistry Letters, Bioorganic and
Medicinal Chemistry, Combinatorial Chemistry HighThroughput Screening.
Lerner, R.A.—Paul Ehrlich and Ludwig Darmstaedter
Prize, Paul Ehrlich Foundation, Frankfurt, Germany;
Honorary Doctoral Degree, Ben-Gurion University of
the Negev, Beer-Sheva, Israel; Editorial Boards,
Bioorganic and Medicinal Chemistry, Bioorganic and
Medicinal Chemistry Letters, Catalysis Technology,
Chemistry and Biology, Drug Targeting and Delivery,
Journal of Virology, Molecular Biology and Medicine,
Molecular Medicine, Journal of Peptide Research,
Vaccine, Angewandte Chemie.
Nicolaou, K.C.—Tetrahedron Prize for Creativity in
Organic Chemistry, Tetrahedron Publications; Nobel
Laureate Signature Award for Graduate Education in
Chemistry, American Chemical Society; Alder and
Bayer Lecturer, Cologne, Germany; AstraZeneca Lecturer, University of Montreal and AstraZeneca, Montreal,
Canada; Bristol Myers Lecturer, Boston College, Boston,
Massachusetts; Co-Editor-in-Chief, Chemistry and
Biology; Editorial Boards, Tetrahedron Publications,
Synthesis, Carbohydrate Letters, Chemistry—A European Journal, Perspectives in Drug Discovery and
Design, Indian Journal of Chemistr y, Section B,
Combinatorial Chemistry High-Throughput Screening,
Current Opinion in Bioorganic Chemistry, Current
Organic Chemistry, Organic Letters, ChemBioChem.
Rebek, J., Jr.— Chemical Pioneer Award, American
Institute of Chemists; Woodward Scholar, Harvard
University, Boston, Massachusetts; Ronald Breslow
Award for Achievement in Biomimetic Chemistry,
American Chemical Society; Advisory Boards, Physical
Sciences Division, University of Chicago, National
Cancer Institute, and Institute of Chemical Research of
Catalonia, Spain; Editorial Boards, Tetrahedron Publications, Chemistry and Biology, Current Opinion in
Chemical Biology, Journal of the Chemical Society—
Perkin Transactions, Journal of Organic Chemistry,
Bioorganic and Medicinal Chemistry Letters, Progress
in Physical Organic Chemistry, Journal of Supramolecular Chemistry.
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
Schultz, P.G.—Paul Ehrlich and Ludwig Darmstaedter
Award, Paul Ehrlich Foundation, Frankfurt, Germany.
Sharpless, K.B.—Honorary Member, Kitasato Institute,
Tokyo, Japan; Visiting Professor, Kitasato University,
Tokyo, Japan; Danforth Lecturer, Grinnell College,
Grinnell, Iowa; Robbins Lecturer, Pomona College,
Claremont, California; Editorial Boards, Enantiomer,
Advanced Synthesis and Catalysis, Chirality, Current
Opinion in Drug Discovery and Development, Organic
Letters; Honorary Advisory Board, Synlett.
Williamson, J.R.—Associate Editor, Annual Reviews of
Biophysics and Biomolecular Structure; Editorial
Boards, RNA, Molecular Cell, Chemistry and Biology.
Wilson, I.A.— Fellow, Royal Society of London; Fellow,
American Academy of Arts and Sciences; Member,
Burroughs Wellcome Career Awards Advisory Committee; Member, Scientific Advisory Board, Keystone Symposia; Associate Editor, Journal of Molecular Biology,
Immunity; Editorial Boards, Science, Journal of
Experimental Medicine.
Wong, C.-H.—Member, National Research Council on
Chemical Sciences and Technology; Scientific Advisor,
Max-Planck-Institute, Dortmund, Germany; Editor-inChief, Bioorganic and Medicinal Chemistry; Editorial
Boards, Tetrahedron Publications, Current Opinion in
Chemical Biology, Biocatalysis, Advanced Synthesis
and Catalysis.
Wright, P.E.—Honorary Doctor of Science, University of
Sydney, Sydney, Australia; Editor-in-Chief, Journal of
Molecular Biology; Editorial Boards, Biochemistry,
Current Opinion in Structural Biology, Journal of Biomolecular NMR.
59
60
THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2003
SUBJECT INDEX
Acetogenins 36, 46
Adaptive immunity 52
AIDS 13
Alkanes 40
Alzheimer’s disease 37
Amyloid diseases 37
Amyloidogenesis 37
Amyotrophic lateral sclerosis 28,
34, 48
Anandamide 20
Antibiotics 19
Antibodies
expression in algae 38
HIV neutralizing 51
to cocaine 52
to tumors 14
Antibody libraries 30
Anticancer agents 53
Antisense agents 33
Asymmetric synthesis 13
ATIC 53
Bacterial pathogenesis 48
Biomolecular computing 34
Bioorganic chemistry 18, 55
Cancer chemotherapy 31
Cancer 13, 20
Catalysis 45
Catalytic antibodies 13, 34, 46
Cell adhesion molecules 22
Cell cycle 48
control of 16
Cell diagnostics 29
Chemical biology 38
Chemical ligation 21
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.
Chemical physiology 20
Chemical synthesis 38
Click chemistry 45
Cryptochromes 28
DNA enzymes 33
DNA repair 48
DNA-binding proteins 56
Drug design 18, 29, 53
Drug discovery 50, 55
Encapsulation 40
Enzyme inhibitors 55
Enzymes
synthetic 34
Erythropoietin 18
Fatty acid amide hydrolase 20
Fluorescent proteins 28
Genetic code 14, 42, 44
Genome integrity 48
Glycinamide ribonucleotide
transformylase 53
Heroin esterase 52
HIV 50
vaccine to 51
Hypoxia 56
Immune recognition 51
Immunopharmacotherapy 30
Innate immunity 16, 53
Integrins 46
Lipopolysaccharides 31
Macromolecular machines 48
Major histocompatibility complex 52
Medicinal chemistry 18
Membrane proteins 20
Molecular evolution 33
Molecular recognition 40
Multidrug resistance 20
NADPH oxidase 16
Nanotechnology 34
Nanotubes 30
Natural products 19, 39, 46
Nicotine abuse 32
Nitric oxide synthases 27
Nuclear magnetic resonance
spectroscopy 55
Nucleic acids 33
structure of 23
Oleamide 20
Organic synthesis 34, 46
Organocatalysis 13
Pain 20
Peptide synthesis 21
Phage display technology 30
Photoactive yellow protein 28
Platelet receptors 17
Prion diseases 37
Prodrug therapy 46
Protein-protein interactions 18
Proteins
DNA binding 14
folding of 38
structure of 27
synthesis of 21
Proteomics 21
Proteomimetics 41
Reactive oxygen species 16, 48
Receptors 20
on T cells 52
Ribosomes 22
Ribozymes 24
RNA
assembly and catalysis of 24
Saccharides 55
Self-organized networks 29
Septic shock 31
Serine hydrolases 21
Siroheme synthase 27
Sleep 20
Stem cells 23
Sulfite reductases 27
Superoxide dismutases 28, 48
Synapses 22
Synthetic chemistry 18
Targeting therapy 46
Therapeutic proteins expression
in algae 38
Transcription 56
Transfer RNAs 42
Translation 22
Transporters 20
Ubiquitination 16
Vaccines 31, 51
Viral coats 22
Viral infection 16
Viruses
as molecular building blocks 25
cowpea mosaic virus 26
flock house virus 26
hepatitis C virus 17
lymphocytic choriomeningitis
virus 16
nudaurelia capensis ω virus 26
X-ray crystallography 51
Zinc fingers 14
The Skaggs Institute for
Chemical Biology scientific
report is published annually
by The Scripps Research
Institute and is available
on request from
Editor
Barbara L. Halliburton, Ph.D.
D E PA R T M E N TA L
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The Skaggs Institute for
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C O O R D I N AT I O N
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Department of Molecular and
Experimental Medicine
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Department of Neurobiology
Rigid molecular objects, approximately 22 nm in diameter,
constructed from 1.7-kb strands of DNA. Their structure
was determined by using 3-dimensional reconstruction of
images obtained by electron cryomicroscopy. This work was
performed by William M. Shih, Ph.D., in the labora
tory of Gerald F. Joyce, M.D., Ph.D., with assistance from
Joel D. Quispe in the Center for Integrated Molecular
Biosciences. Figure courtesy of Michael Pique.
THE SCRIPPS RESEARCH INSTITUTE
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