SCRIPPS
FLORIDA
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Biochemistry
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Scott Simanski, Research Assistant, and
Nagi G. Ayad, Ph.D., Assistant Professor,
Department of Biochemistry
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
BIOCHEMISTRY 2005
S TA F F S C I E N T I S T S
Anthony D. Smith, Ph.D.
Michael J. Chalmers, Ph.D.
Achim M. Sorg, Ph.D.
Qitao Yu, Ph.D.
Sahba Tabrizifard, Ph.D.
Steve A. Kay*
Professor and Chairman
Jiu Zhao, Ph.D.
Mariola Tortosa, Ph.D.
Nagi G. Ayad, Ph.D.
Assistant Professor
R E S E A R C H A S S O C I AT E S
DEPAR TMENT OF
BIOCHEMISTRY
S TA F F
Alessandra Cervino, Ph.D.**
Assistant Professor
Michael D. Conkright, Ph.D.
Assistant Professor
Patrick R. Griffin, Ph.D.**
Professor
John B. Hogenesch, Ph.D.***
Professor
Malcolm A. Leissring, Ph.D.
Assistant Professor
Phillip LoGrasso, Ph.D.**
Associate Professor
Kendall W. Nettles, Ph.D.**
Assistant Professor
Howard T. Petrie, Ph.D.
Professor
Mathew T. Pletcher, Ph.D.**
Assistant Professor
Teresa M. Reyes, Ph.D.
Assistant Professor
William R. Roush, Ph.D.****
Professor
Associate Dean, Kellogg
School of Science and
Technology, Florida
Oliver Umland, Ph.D.
Juliette C. Walker, Ph.D.
Julie E. Baggs, Ph.D.
Amie L. Williams, Ph.D.
* Joint appointments in the
Department of Cell Biology and
the Institute for Childhood and
Neglected Diseases
** Joint appointment in the
Translational Research Institute
*** Joint appointments in the
Translational Research Institute
and Molecular and Integrative
Neurosciences Department
**** Joint appointments in the
Translational Research Institute
and the Department of
Chemistry
Kristin Clarke, Ph.D.
Etzer Darout, Ph.D.
S C I E N T I F I C A S S O C I AT E S
Amy C. DeBaillie, Ph.D.
Yi An Lu, Ph.D.
Joshua R. Dunetz, Ph.D.
Qi Tao Yu, Ph.D.
Masaki Handa, Ph.D.
Dympna Harmey, Ph.D.
Kevin R. Hayes, Ph.D.
Tamara Hopkins, Ph.D.
Jelena Janjic, Ph.D.
Rebecca J. Kocerha, Ph.D.
Brooke H. Miller, Ph.D.
William Mwangi, Ph.D.
Hiroshi Nakase, Ph.D.
Michael H. Ober, Ph.D.
Swati Prasad, Ph.D.
Troy D. Ryba, Ph.D.
James Tam, Ph.D.
Professor
S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F B O C H E M I S T R Y : A combination of
Claes Wahlestedt, M.D.,
Ph.D.**
Professor
structural approaches, including x-ray crystallography, in the laboratory of K.W. Nettles, Ph.D., and
amide hydrogen-deuterium exchange mass spectrometry, in the laboratory of P.R. Griffin, Ph.D., were
used to dissect how diverse ligands control the allosteric function of the nuclear receptor ligandbinding domain. The ligand-binding domain of the estrogen receptor is shown bound to an estrogen
derivative, trifluoromethylphenylvinyl-estradiol.
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347
348 BIOCHEMISTRY 2005
INVESTIGATORS’ R EPORTS
Role of Ubiquitin-Mediated
Proteolysis in Irreversible
Transitions During the Cell Cycle
N.G. Ayad, A. Smith, D. Harmey, S. Simanski
e are interested in the cell biological basis of
irreversible transitions that occur during the
eukaryotic cell cycle. Ubiquitin-mediated proteolytic pathways ensure that irreversibility is achieved by
targeting specific inhibitors of these transitions for proteasomal degradation. One of the most important ubiquitn E3 ligases is the anaphase-promoting complex (APC).
The complex is active during G1 and in fully differentiated cells. Furthermore, our recent studies indicate that
the APC is required to initiate differentiation of neuronal
precursor cells. This finding is especially important
because it suggests that the complex is controlling an
essential step in cell-cycle exit or differentiation, a control that is both biologically and medically relevant.
Although we understand a great deal about the role
of the APC during the cell cycle, its role in initiating exit
from the cell cycle is virtually unknown. We are uncovering the mechanism of APC activation and the proteins
turned over via the APC during differentiation. For these
studies, we are using both PC12 cells and the primary
cerebellar granule cell system to probe the role of the
APC during differentiation. In addition, in collaboration
with J. Hogenesch, Scripps Florida, we are using cellbased screening technologies to identify novel activators
and inhibitors of the APC. For this research, we have
developed a high-throughput luciferase-based measure
of APC activity. In this method, the N terminus of the
APC substrate cyclin B is fused with luciferase, so an
increase in luciferase corresponds to increased levels of
cyclin B, a finding that indicates lower APC activity. We
recently began screening 15,000 human cDNAs to identify novel APC regulators. Identification of these regulators most likely will illuminate both temporal and spatial
control of APC activity during development.
In addition to identifying novel regulators and substrates of the APC, we have concentrated on one of the
known APC substrates, the cytosolic protein trigger of
mitotic entry 1. This protein is required for degradation
of the mitosis-inhibitory kinase wee1 and entry into
W
Steve A. Kay, Ph.D.
Chairman’s Overview
he Department of Biochemistry at Scripps Research
was recently created to span both the La Jolla and
Florida campuses. The overall theme of the department centers on the need to understand physiologic processes from the molecular level through the whole
organism. Our faculty members are generally multidisciplinary biologists who wield cutting-edge tools of
structural biology, protein dynamics, biological chemistry, genetics and genomics, pathway analysis, and
computational approaches to understand how organisms
maintain homeostasis and respond to stress. We have
broad interests; we seek to answer contemporary questions in neurobiology, metabolic control, immunology,
and cancer biology. By taking integrative approaches to
substantial problems in modern biology, we will affect
the understanding of a wide variety of diseases such as
diabetes, cancer and CNS disorders.
T
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BIOCHEMISTRY 2005
mitosis in both frogs and humans. We are determining
the intracellular location of wee1 degradation in humans
and the role of phosphorylation in wee1 degradation.
In collaboration with J. Busby, Scripps Florida, we are
using mass spectrometry to identify wee1 phosphorylation sites. This multidisciplinary approach will provide greater insight into proteolysis, the cell cycle, and
neurogenesis and may be useful in cancer research and
nerve regeneration studies.
PUBLICATIONS
Ayad, N.G. CDKs give Cdc6 a license to drive into S phase. Cell 122:815, 2005.
Ayad, N.G., Rankin, S., Ooi, D., Rape, M., Kirschner, M.W. Identification of ubiquitin ligase substrates by in vitro expression cloning. Methods Enzymol., in press.
Rankin, S., Ayad, N.G., Kirschner, M.W. Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates [published
correction appears in Mol. Cell 18:609, 2005]. Mol. Cell 18:185, 2005.
Song, M.S., Song, S.J., Ayad, N.G., Chang, J.S., Lee, J.H., Hong, H.K., Choi, N.,
Kim, J., Kim, H., Kim, J.W., Choi, E.J., Kirschner, M.W., Lim, D.S. The tumour
suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex. Nat.
Cell Biol. 6:129, 2004.
Wei, W., Ayad, N.G., Wan, Y., Zhang, G., Kirschner, M.W., Kaelin, W.G., Jr.
Destruction of the SCF component Skp2 in G1 by the anaphase-promoting complex. Nature 428:194, 2004.
349
molecule libraries, to identify all of the proteins that
make up and regulate the cAMP pathway. Using this
technology, we successfully identified proteins called
transducers of regulated CREB activity, a novel family
of CREB coactivators. Ascertaining the factors involved
in the cAMP signaling pathway will be paramount in
delineating why the biological function of CREB differs
so drastically between tissues.
PUBLICATIONS
Conkright, M.D., Montminy, M. CREB: the unindicted cancer co-conspirator.
Trends Cell Biol. 15:457, 2005.
Screaton, R.A., Conkright, M.D., Katoh, Y., Best, J.L., Canettieri, G., Jeffries, S.,
Guzman, E., Niessen, S., Yates, J.R. III, Takemori, H., Okamoto, M., Montminy, M.
The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector. Cell 119:61, 2004.
Zhang, X., Odom, D.T., Koo, S.H., Conkright, M.D., Canettieri, G., Best, J., Chen,
H., Jenner, R., Herbolsheimer, E., Jacobsen, E., Kadam, S., Ecker, J.R., Emerson, B., Hogenesch, J.B., Unterman, T., Young, R.A., Montminy, M. Genome-wide
analysis of cAMP-response element binding protein occupancy, phosphorylation,
and target gene activation in human tissues. Proc. Natl. Acad. Sci. U. S. A.
102:4459, 2005.
Molecular Mechanisms of
cAMP-Mediated Transcription
Ligand Interactions and Allosteric
Control in Nuclear Receptors:
Use of Hydrogen-Deuterium
Exchange Mass Spectrometry
M.D. Conkright, B.A. Mercer
P.R. Griffin, S.A. Busby, M.J. Chalmers, S. Prasad
gamut of biological functions depend on the
cAMP signaling cascade, including long-term
memory, survival of beta cells, glucose metabolism, cardiomyopathy, and proliferation of chondrocytes.
We study the molecular mechanisms involved in the
conversion of these signals to transcriptional events.
Increases in cellular levels of cAMP stimulate the expression of numerous genes by liberating the catalytic subunits of protein kinase A, which phosphorylates the
transcription factor cAMP-responsive element binding
protein (CREB). Phosphorylation of CREB promotes
the recruitment of the coactivator CREB-binding protein/p300 and the initiation of transcription.
The diversity of biological functions associated with
CREB and the cAMP pathway will be impossible to
fully understand until all of the components involved
in the pathway have been identified and characterized.
Currently, we are using high-throughput cell-based
screening technologies, including cDNA expression
libraries, small interfering RNA libraries, and small-
eroxisome proliferator-activated receptor γ (PPARγ),
a ligand-dependent transcription factor and member of the nuclear hormone superfamily, is the
molecular target of the drug class known as the glitazones. These compounds are used to treat muscle
insulin resistance, a sign or cause of type 2 diabetes,
and are referred to as insulin sensitizers. The glitazones
are widely prescribed for treatment of type 2 diabetes.
However, they have limited usefulness in patients with
mild insulin resistance or a history of cardiovascular
disease because the drugs are associated with specific
receptor-mediated side affects, such as weight gain,
fluid retention, and plasma volume expansion. Unfortunately, the incidence of cardiovascular disease is high
in patients with type 2 diabetes. In addition, a clear
link exists between body mass index and the incidence
of insulin resistance and type 2 diabetes. Thus, the
search continues for a PPARγ modulator that increases
muscle insulin sensitivity without causing weight gain
and plasma volume expansion.
A
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P
350 BIOCHEMISTRY 2005
Recent studies with animal models of insulin resistance indicated that weight gain and plasma volume
expansion can be minimized without loss of insulin
sensitization by using partial agonists of PPARγ, although
the mechanism of this dissociation of efficacy from
unwanted events is unclear. Studies with preadipocytes
revealed that these partial agonists do not induce adipogenesis as the full agonists do, and expression profiling has shown that the gene expression patterns are
different for the different functional classes of activators. Currently, we are studying the mechanism of
activation of PPARγ by partial agonists and the role of
conformation-mediated recruitment of coactivators in
adipogenesis. In addition, we are developing a rapid,
sensitive structure-based approach to aid in selecting
conformational-specific modulators of PPARγ.
Amide hydrogen-deuterium exchange coupled with
proteolysis and mass spectrometry has become a powerful technique for studying protein structure and dynamics, protein-ligand interactions, and protein-protein
interactions. With this method, we can measure changes
in the solvent accessibility and stability of a protein in
the presence and absence of ligands. We are using
hydrogen-deuterium exchange mass spectrometry to
detect compound-specific conformational stabilization
within the ligand-binding domain of PPARγ. Ligand
binding to the domain alters amide hydrogen-deuterium
exchange rates in specific regions of the protein with
differential magnitude and direction, depending on the
chemical structure of the ligand.
We have shown that perturbations in hydrogendeuterium exchange can be used to classify agonists,
partial agonists, and antagonists of PPARγ. More importantly, these hydrogen-deuterium exchange profiles indicate that the mechanism of activation of PPARγ by full
agonists, such as the glitazones, which involves recruitment of coactivators via stabilization of helix-12 and
the AF2 surface of the ligand-binding domain, is different for certain classes of partial agonists. We are
continuing our investigations of the mechanism of
activation of PPARγ by diverse chemotypes of PPARγ
partial agonists. Our overall goal is to determine the
relationship between ligand-induced receptor conformation and pharmacologic response in rodent models of
type 2 diabetes. To date, we have identified localized
regions of PPARγ that provide sensors specific for binding and conformational induction for specific chemotypes.
Differential recruitment of coactivators by various
chemotypes of PPARγ modulators could be integral to
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the mechanism of activation of PPARγ and its associated physiologic response. Using 2-hybrid assays, we
are screening libraries of nuclear receptor modulators
against a library of coactivator genes to identify the
molecular determinants of coactivator selectivity for
these compounds.
We have also used hydrogen-deuterium exchange
mass spectrometry to study other biological systems.
In collaboration with D. Silverman, University of
Florida, Gainesville, Florida, we are examining differences in exchange rates between the dimer and tetramer interfaces of manganese superoxide dismutase.
Our goal is to understand how this protein regulates
the conversion of superoxide to oxygen and hydrogen
peroxide to protect cells against oxidative damage. In
other studies with G. Fields, Florida Atlantic University, Boca Raton, Florida, we are using hydrogen-deuterium exchange to examine the mechanisms of both
unwinding and cleaving of triple helical peptides by
matrix metalloprotease 1, an enzyme involved in the
metastasis of breast cancer and the progression of
arthritis. Finally, in collaboration with W. Cance at the
University of Florida, we are using hydrogen-deuterium
exchange to characterize interactions for peptides that
bind to focal adhesion kinase and have anticancer activity
in cell cultures.
Research is also under way to improve the technical aspects of the hydrogen-deuterium exchange method.
We have developed a fully automated system for hydrogen-deuterium exchange experiments. An autosampler
is used to mix solutions and control the online enzymatic
digestion of the protein being analyzed and the subsequent separation of the products. Importantly, we have
written unique software that enables us to measure
subsecond hydrogen-deuterium exchange rates.
To increase the dynamic range of the proteins and
complexes that we can study by using hydrogen-deuterium exchange, we are collaborating with A.G. Marshall
at the National High Magnetic Field Laboratory at Florida
State University, Tallahassee, Florida. Dr. Marshall and
his colleagues are widely acknowledged as the world
leaders in the development of Fourier transform ion
cyclotron resonance mass spectrometry (FT-ICR MS).
FT-ICR MS provides the highest resolving power and
mass accuracy of any mass analyzer (100-fold greater
than our current ion-trap mass spectrometer). The resolution of FT-ICR MS enables us to follow the deuterium
uptake of more proteolytic peptides during hydrogendeuterium exchange, and the improved mass accuracy
BIOCHEMISTRY 2005 351
increases the likelihood that the peptide-sequence assignments are correct.
The Scripps Florida robot used for automated preparation of samples for hydrogen-deuterium exchange was
interfaced to a 14.5-T FT-ICR mass spectrometer at the
National High Magnetic Field Laboratory. The custombuilt 14.5-T magnet is the highest field magnet ever constructed for use in FT-ICR MS. Using this system, we
determined the hydrogen-deuterium exchange of a complex consisting of the ligand-binding domains of PPARγ
and retinoic X receptor α in the presence and absence
of a PPARγ full agonist and retinoic acid. The results
are the first known hydrogen-deuterium exchange analysis of how drug binding affects the structure of an
entire protein complex, rather than a single protein.
The automation of the hydrogen-deuterium exchange
method creates an absolute requirement for an integrated
data management and analysis system. A single study
may generate up to 40 Gb of raw data, which must be
archived and processed. No such commercial solution
exists. In collaboration with N. Tsinoremas, C. Mader,
B. Pascal, and J. Zysmann, Scripps Florida, we are
developing integrated data analysis software and computing infrastructure to control workflow, storage, and
analysis of data obtained in hydrogen-deuterium exchange
experiments. This system is fully integrated with our
laboratory information management system. When completed, the software, named HD Desktop, will be offered
to academic laboratories as an open-source solution.
Cell-Based Screening and the
Mammalian Circadian Clock
J.B. Hogenesch, T.K. Sato, J.E. Baggs, K.R. Hayes,
J.M. Geskes, J.N. Harada
ompletion of the sequencing of several mammalian
genomes has revealed a surprisingly sparse cohort
of genes that encode proteins. Despite this unexpectedly limited repertoire, most of these genes have
never been reported in the literature. To bridge this gap
between gene identification and an understanding of
their functions, we developed high-throughput methods
to study the function of genes in cellular pathways. These
methods work on the basis of 2 principles: gain of function and loss of function. For gain-of-function screens, we
overexpress 16,000+ distinct mouse and human cDNAs
individually in cells and study the consequential pheno-
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type of interest. For loss-of-function screens, we use
short interfering RNAs to suppress expression of specific
mRNAs and then analyze the resultant cellular phenotypes. We currently have a library of 28,000 short interfering RNAs designed to reduce transcript levels of 7000
human genes that can be targeted by pharmacologic
drugs or protein therapeutic agents. Because of the
generic nature of the screens, many different types of
cell-based screens can be performed, including cell proliferation, use of easily measured output of light, and even
high-content imaging, in which individual cells are photographed and scored for distinct phenotypes. Thus, we
can study the function of a substantial part of the mammalian genome by using powerful cell-based assays and
can determine the cellular roles for many previously poorly
or unannotated genes.
We are using these methods to study the circadian
clock in mammals. Circadian rhythms are conserved
from cyanobacteria to Neurospora to Drosophila to mice
to humans; each organism regulates aspects of its physiology to be in tune with the environment. In mammals,
complex behaviors such as locomotor activity and physiologic events such as heme biosynthesis, hormonal
signaling, and temperature rhythms are regulated by the
clock. Interestingly, in all of the organisms mentioned,
the clock itself is regulated at the level of transcription.
Several transcription factors conserved between flies,
mice, and humans have been identified as clock components. Along with some accessory factors, the genes
for these components work in concert to generate molecular rhythms of transcription with a period length of 24
hours. The entire pathway exists not only in the brain,
the site of the master circadian oscillator, but also in
peripheral tissues and even individual cells.
We have adapted a cell line model of clock function
in order to use molecular genetic tools long applied in
research on bacteria and yeasts. We are using gain- and
loss-of-function alleles of the proteins Clock, Bmal, and
cryptochrome in screens to examine clock function.
Evaluation of the alleles in cellular assays revealed the
absolute requirement of transcriptional repression for
clock function.
The power of mammalian cellular genetics has rapidly
matured during the past several years. We have used
these tools and techniques not only to study the circadian clock in mammals but also to understand the function of noncoding RNAs, numerous cellular signaling
pathways, and basic cellular processes such as proliferation and differentiation. Potentially, these tools can be
352 BIOCHEMISTRY 2005
used to uncover the functions of many important and
understudied genes and can lead the way to the next
generation of therapeutic drug targets. As such, the
tools are an important addition to the existing repertoire used in studies of gene function.
PUBLICATIONS
Harada, J.N., Bower, K.E., Orth, A.P., Callaway, S., Nelson, C.G., Laris, C.,
Hogenesch, J.B., Vogt, P.K., Chanda, S.K. Identification of novel mammalian growth
regulatory factors by genome-scale quantitative image analysis. Genome Res.
15:1136, 2005.
Sato, T.K., Panda, S., Miraglia, L.J., Reyes, T.M., Rudic, R.D., McNamara, P.,
Naik, K.A., FitzGerald, G.A., Kay, S.A., Hogenesch, J.B. A functional genomics
strategy reveals Rora as a component of the mammalian circadian clock. Neuron
43:527, 2004.
Su, A.I., Wiltshire, T., Batalov, S., Lapp, H., Ching, K.A., Block, D., Zhang, J.,
Soden, R., Hayakawa, M., Kreiman, G., Cooke, M.P., Walker, J.R., Hogenesch, J.B.
A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl.
Acad. Sci. U. S. A. 101:6062, 2004.
Willingham, A.T., Orth, A.P., Batalov, S., Peters, E.C., Wen, B.G., Aza-Blanc, P.,
Hogenesch, J.B., Schultz, P.G. A strategy for probing the function of noncoding
RNAs finds a repressor of NFAT. Science 309:1570, 2005.
Genetic Determinants of the
Efficacy of Selective Serotonin
Reuptake Inhibitors
M.T. Pletcher, B.H. Miller, B.M. Young, G.M. Zastrow
linical depression is a mood disorder of high morbidity and mortality that is estimated to occur in
more than 15% of the adult population in the
United States. Depression has a wide range of symptoms, including loss of energy, changes in weight, diminished interest or pleasure in daily activities, insomnia or
excessive sleep, anxiety, slowness of movement, feelings
of worthlessness, difficulty concentrating, and thoughts
of death.
Depression can be a one-time occurrence but is often
an ongoing problem throughout a person’s lifetime. A
total 15% of patients with major depressive disorders
die of suicide. The onset of depression is often linked
to environmental factors such as life events that greatly
increase stress. Despite this association, depression has
a strong genetic component. Genetics accounts for
40%–50% of the risk for of depression in a person’s
lifetime, and the risk for members of a family does not
change if they are raised separately.
We are using cell-based screening technology and a
mouse model to identify the genes and pathways that
contribute to depressive behavior. Currently, we are con-
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ducting a survey in 30 strains of mice for differences in
molecular, biochemical, and behavioral traits that represent endophenotypes (i.e., single well-defined symptoms
or traits of a much more complex disorder) associated with
depression. In cataloging the variations in quantities
of neurotransmitters and stress hormones in the blood,
gene expression in regions of the brain such as the hippocampus and hypothalamus, and performance in behavioral models such as the tail suspension and open field
tests, we are producing the data necessary to identify
the genetic controls for each of these traits. Using the
in silico genetic mapping method, we can correlate the
natural variation for these measurements in the inbred
lines of mice with the underlying haplotype structure of
the mice, thereby pinpointing biologically important genes.
In parallel, we are developing a cell-based assay to
monitor the function of the serotonin transporter, the
gene target of the most common pharmaceutical agents
for depression: selective serotonin reuptake inhibitors.
Using this assay, we can screen the 18,000 full-length
clone cDNA library of Scripps Florida for genes that modify the function of the transporter. Additionally, introducing a selective serotonin reuptake inhibitor into the
screen will enable us to identify genes that modulate
the efficacy of this important class of drugs. Cumulatively, we hope to provide a better understanding of the
molecular basis of depression and its treatment to allow
improvements in diagnosis and therapies.
PUBLICATIONS
Bystrykh, L., Weersing, E., Dontje, B., Sutton, S., Pletcher, M.T., Wiltshire, T., Su, A.I.,
Vellenga, E., Wang, J., Manly, K.F., Lu, L., Chesler, E.J., Alberts, R., Jansen, R.C.,
Williams, R.W., Cooke, M.P., de Haan, G. Uncovering regulatory pathways that affect
hematopoietic stem cell function using “genetical genomics.” Nat. Genet. 37:225, 2005.
Kahlem, P., Sultan, M., Herwig, R., Steinfath, M., Balzereit, D., Eppens, B., Saran,
N.G., Pletcher, M.T., South, S.T., Stetten, G., Lehrach, H., Reeves, R.H., Yaspo,
M.L. Transcript level alterations reflect gene dosage effects across multiple tissues in
a mouse model of Down syndrome. Genome Res. 14:1258, 2004.
Pletcher, M., Wiltshire, T. Can we find the genes involved in complex traits? Genome
Biol. 5:347, 2004.
Pletcher, M.T., McClurg, P., Batalov, S., Su, A.I., Barnes, S.W., Lagler, E., Korstanje, R.,
Wang, X., Nusskern, D., Bogue, M.A., Mural, R.J., Paigen, B., Wiltshire, T. Use of a
dense single nucleotide polymorphism map for in silico mapping in the mouse. PLoS Biol.
2:e393, 2004.
Sandberg, M.L., Sutton, S.E., Pletcher, M.T., Wiltshire, T., Tarantino, L.M., Hogenesch, J.B.,
Cooke, M.P. c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation. Dev. Cell 8:153, 2005.
Sussan, T.E., Pletcher, M.T., Murakami, Y., Reeves, R.H. Tumor suppressor in lung
cancer 1 (TSLC1) alters tumorigenic growth properties and gene expression. Mol.
Cancer 4:28, 2005.
Wen, B.G., Pletcher, M.T., Warashina, M., Choe, S.H., Ziaee, N., Wiltshire, T.,
Sauer, K., Cooke, M.P. Inositol (1,4,5) trisphosphate 3 kinase B controls positive
selection of T cells and modulates Erk activity. Proc. Natl. Acad. Sci. U. S. A.
101:5604, 2004.
BIOCHEMISTRY 2005
353
Functional Neuroanatomy of
Appetite and Metabolism
PUBLICATIONS
Sato, T.K., Panda, S., Miraglia, L.J., Reyes, T.M., Rudic, R.D., McNamara, P.,
Naik, K.A., FitzGerald, G.A., Kay, S.A., Hogenesch, J.B. A functional genomics
strategy reveals Rora as a component of the mammalian circadian clock. Neuron
43:527, 2004.
T.M. Reyes, K.J. Clarke, C.D. Easson
Sawchenko, P.E., Yuan, Z.F., Reyes, T.M. The corticotropin-releasing factor family
of signaling molecules and their roles in adaptive responses to stress. J. Histochem. Cytochem. 52:S14, 2004.
besity is an important risk factor in the development of heart disease, diabetes, stroke, and
metabolic syndrome, and the prevalence of obesity is increasing in all segments of the population.
Development and resolution of obesity involve an interaction between genetic, environmental, and behavioral factors, and the role of the CNS in this process is the focus
of our research program. We are defining the CNS pathways that regulate appetite and metabolism. Our central
goals are to define CNS molecules that respond to and
drive alterations in appetite and metabolism and to characterize the expression and regulation of these molecules.
To this end, we couple functional neuroanatomic
techniques with genomics and high-throughput screening tools in genetic and disease models. One focus is
the loss of appetite and alterations in metabolism that
occur in acute and chronic infection or inflammation.
Plasma cytokines are elevated during infection and inflammation, and these endocrine signaling molecules act on
the brain and are associated with a loss of appetite and
changes in metabolism. Studies are under way to identify regions of the brain that drive the primary response
to cytokines and to identify the neuropeptides and neurotransmitters recruited to effect these changes.
The ability to site specifically alter gene expression
in the brain is an important tool for these studies. We
are developing animals in which the genes for various
neuropeptides and adipokines are expressed conditionally in a Cre-LoxP system. Cre recombinase is introduced
either through crosses with mice that express Cre or
through stereotaxic delivery of lentiviruses that express
Cre recombinase.
In a related series of studies, we are investigating
the link between systemic infection and obesity. Adverse
conditions in utero can increase the risk for obesity.
Little is known about the mechanisms that underlie this
developmental programming. We are using RNA profiling studies to determine whether epigenetic modifications as a result of events in utero (either nutritional or
immune) can exacerbate the development of obesity.
Our research combines the power of whole-genome
profiling with detailed functional neuroanatomic analyses of single molecules. Collectively, these studies
should contribute to understanding how the CNS and
inflammation interact in the development of obesity.
O
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Synthesis of Natural Products,
Development of Synthetic Methods,
and Medicinal Chemistry
W.R. Roush, R. Bates, Y.-T. Chen, T. Dineen,* E. Flamme,*
G. Halvorsen, M. Handa, C. Heitzman,* C.-W. Huh,
L. Julian,* W. Lambert,* R. Lira, C. Liu,* D. Mergott,*
E. Mertz,* J. Methot,* J. Neitz,* C. Nguyen, M. Ober,
P. Orahovats,* R. Owen,* R. Pragani, J. Qi,* J.B. Shotwell,*
A. Sorg, L. Steffans, K. Takao,** J. Tinsley,* M. Tortosa,
T. Trullinger,* P. Va, A. Williams, S. Winbush, N. Zheng*
* University of Michigan, Ann Arbor, Michigan
** Keio University, Yokohama, Japan
ur research has 2 major themes. One is the
synthesis of structurally complex, biologically
active natural products (Fig. 1). These efforts
in total synthesis are pursued in parallel with stereochemical studies and the development of new synthetic
methods. We have been particularly interested in stereochemical aspects of intramolecular and transannular
Diels-Alder reactions, in the development of methods
for the diastereoselective and enantioselective reactions of allylmetal reagents with carbonyl compounds,
and in the development of highly stereoselective methods for synthesis of 2-deoxyglycosides. Recent efforts
to develop new synthetic methods included studies on
the Diels-Alder reactions of (Z)-dienes, double allylboration reactions of aldehydes with γ-boryl-substituted
allylboranes for stereocontrolled synthesis of 1,5-endiols, synthesis of highly substituted tetrahydrofurans via
[3 + 2]-annulation reactions of highly functionalized
allylsilanes, and use of 2-deoxy-2-iodoglycosyl imidates
and 2-deoxy-2-iodoglycosyl fluorides for the highly
stereocontrolled synthesis of 2-deoxy-β-glycosides.
Natural products of current interest to us include
amphidinolides C and F, amphidinol 3, angelmicin B,
annonaceous acetogenin analogs, durhamycin A and
analogs, integramicin, lomaiviticin A, peloruside A,
psymberin, quartromicin D1, reidispongiolide A, spin-
O
354 BIOCHEMISTRY 2005
agent of Chagas’ disease, and Plasmodium falciparum,
the most virulent of the malaria parasites. This research
is performed in collaboration with colleagues at the
University of California, San Francisco. We have also
participated in the development of proapoptotic benzodiazepines, in collaboration with G.D. Glick, University of Michigan, and the design of novel heterocycles
targeting HIV. New projects at Scripps Florida involve
discovery of small molecules that affect cancer and
other validated targets, studies of the structure-activity
relationship of certain natural products, and optimization of the pharmacologic profiles of natural products.
PUBLICATIONS
Dineen, T.A., Roush, W.R. Stereoselective synthesis of the octahydronaphthalene unit
of integramycin via an intramolecular Diels-Alder reaction. Org. Lett. 7:1355, 2005.
Dineen, T.A., Roush, W.R. Total synthesis of cochleamycin A. Org. Lett. 6:2043, 2004.
Durham, T.B., Blanchard, N., Savall, B.M., Powell, N.A., Roush, W.R. Total synthesis of formamicin. J. Am. Chem. Soc. 126:9307, 2004.
Flamme, E.M., Roush, W.R. Synthesis of the C(1)-C(25) fragment of amphidinol 3:
application of the double-allylboration reaction for synthesis of 1,5-diols. Org. Lett.
7:1411, 2005.
Flamme, E.M., Roush, W.R. Synthesis of 2,6-trans-disubstituted 5,6-dihydropyrans from (Z)-1,5-syn-endiols. Beilstein J. Org. Chem. [serial online] 1:7, 2005.
Available at: http://bjoc.beilstein-journals.org/.
Heitzman, C.L., Lambert, W.T., Mertz, E., Shotwell, J.B. Tinsley, J.M., Va, P.,
Roush, W.R. Efficient protiodesilylation of unactivated C(sp3)-SiMe2Ph bonds using
tetrabutylammonium fluoride. Org. Lett. 7:2405, 2005.
Julian, L.D. Newcom, J.S., Roush, W.R. Total synthesis of (+)-13-deoxytedanolide. J. Am. Chem. Soc. 127:6186, 2005.
Liu, C.L. Smith, W.J. III, Gustin, D.J., Roush, W.R. Experimental evidence for
chair-like transition states in aldol reactions of methyl ketone lithium enolates:
stereoselective synthesis and utilization of a deuterium-labeled enolate as a probe
of reaction stereochemistry. J. Am. Chem. Soc. 127:5770, 2005.
Mergott, D.J., Frank, S.A., Roush, W.R. Total synthesis of (–)-spinosyn A. Proc.
Natl. Acad. Sci. U. S. A. 101:11955, 2004.
Mertz, E., Tinsley, J.M., Roush, W.R. [3 + 2]-Annulation reactions of chiral allylsilanes and chiral aldehydes: studies on the synthesis of bis-tetrahydrofuran substructures of annonaceous acetogenins. J. Org. Chem. 70:8035, 2005.
F i g . 1 . Structures of recently synthesized natural products.
osyn A biosynthetic intermediates, scytophycin C, superstolide A, tedanolide, and tetrafibricin. We selected these
molecules as targets because of their biological properties and their interesting and complex structures. We
place a significant emphasis on the discovery, development, and/or illustration of new reactions and synthetic
methods for achieving high levels of stereochemical control in each of these synthesis efforts.
Our second major area of interest focuses on problems in bioorganic chemistry and medicinal chemistry.
One long-term project involves the design and synthesis of inhibitors of cysteine proteases isolated from tropical parasites, such as Trypanosoma cruzi, the causative
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Methot, J.L., Roush, W.R. Nucleophilic phosphine organocatalysis. Adv. Synth.
Catal. 346:1035, 2004.
Narayan, S., Roush, W.R. Studies toward the total synthesis of angelmicin B (hibarimicin B): synthesis of a model CD-D′ arylnaphthoquinone. Org. Lett. 6:3789, 2004.
Owen, R.M., Roush, W.R. Stereoselective synthesis of the C(1)-C(11) fragment of
peloruside A. Org. Lett. 7:3941, 2005.
Roush, W.R., Neitz, R.J. Studies on the synthesis of landomycin A: synthesis of
the originally assigned structure of the aglycone, landomycinone, and revision of
structure. J. Org. Chem. 69:4906, 2004.
Shotwell, J.B., Roush, W.R. Synthesis of the C11-C29 fragment of amphidinolide F.
Org. Lett. 6:3865, 2004.
Tinsley, J.M., Mertz, E., Chong, P.Y., Rarig, R.-A.F., Roush, W.R. Synthesis of (+)bullatacin via the highly diastereoselective [3 + 2] annulation reaction of a racemic
aldehyde and a nonracemic allylsilane. Org. Lett. 7:4245, 2005.
Tinsley, J.M., Roush, W.R. Total synthesis of asimicin via highly stereoselective [3 + 2]
annulation reactions of substituted allylsilanes. J. Am. Chem. Soc. 127:10818, 2005.
BIOCHEMISTRY 2005
Genome Science and
CNS Drug Discovery
C. Wahlestedt, P. Kenny, P. McDonald, M.A. Faghihi,
J. Kocerha, M. Przydzial, H. Thonberg,* H.-Y. Zhang,*
T. Andersson,* O. Larsson,* L. Huminiecki,* C. Schéele,*
C. Dahlgren,* P. Georgii-Hemming,* K. Wennmalm,*
Z. Liang,* J.A. Timmons*
* Karolinska Institutet, Stockholm, Sweden
I D E N T I F I C AT I O N A N D F U N C T I O N A L A N A LY S I S O F
NONCODING AND ANTISENSE TRANSCRIPTS
ost likely conventional protein-coding genes
account for only a minority of human RNA
transcripts. A substantial component of the
full-length mouse and human cDNA sets that we and
others have analyzed does not contain an annotated
protein-coding sequence and most likely corresponds
to noncoding RNA. Many of the noncoding sequences
constitute natural antisense RNA transcripts. We have
shown that the majority of noncoding RNAs identified
to date have substantial conservation across species.
Moreover, we have shown that many noncoding RNA
and antisense transcripts have differential expression
under various conditions and can affect conventional
gene expression.
M
RNA INTERFERENCE AND DEVELOPMENT OF HIGHTHROUGHPUT GENOMICS TECHNOLOGY
RNA interference has become one of the most important gene manipulation technologies. Short interfering
RNA, the inducer of RNA interference in mammals, can
be used to elucidate gene functions by rapidly silencing
expression of a target gene. Today, short interfering RNAs
are widely used as research tools and have potential
for becoming therapeutic agents. We have built a portfolio of short interfering RNA technology. In this package we have a powerful short interfering RNA vector
system, a validation system, and a design system, all of
which are unique. Combining these technologies with
the high-throughput chemistry for on-chip DNA synthesis, we have set up a system for constructing short interfering RNAs. Finally, we have also introduced the use of
locked nucleic acids in short interfering RNAs and have
shown a range of beneficial properties of these agents.
G P R O T E I N – C O U P L E D R E C E P T O R S A S D R U G TA R G E T S
More than half of known drugs bind to G protein–
coupled receptors (GPCRs). We have continued our
long-standing work on GPCRs, particularly certain
neuropeptide receptors. At Scripps Florida, these efforts
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
355
are forming part of the drug discovery program. Moreover, during the past year, we published on our human
GPCR database, which contains polymorphism data,
that is, data that point to interindividual differences in
GPCR sequences. Information on polymorphisms may
be increasingly important in drug discovery and development efforts.
HUMAN GENETICS AND PHARMACOGENOMICS
We are pursuing drug discovery related to several
human disorders that affect the brain. Our goal is to
identify biomarkers that are associated with such common disorders. We wish to understand what makes certain individuals susceptible and how their responses to
drug treatment may differ (pharmacogenomics). We are
involved in genotyping DNA from patients with major
depression, alcoholism, diabetes, obesity, Alzheimer’s
disease, Parkinson’s disease, and attention deficit/hyperactivity disorder.
PUBLICATIONS
Carninci, P., Kasukawa, T., Katayama, S., et al. The transcriptional landscape of
the mammalian genome. Science 309:1559, 2005.
Chalk, A.M., Wahlestedt C., Sonnhammer E.L.L. Improved and automated prediction of effective siRNA. Biochem. Biophys. Res. Commun. 319:264, 2004.
Chen, M., Zhang, L., Zhang, H.Y., Xiong, X., Wang, B., Du, Q., Lu, B., Wahlestedt, C.,
Liang, Z. A universal plasmid library encoding all permutations of small interfering
RNA. Proc. Natl. Acad. Sci. U. S. A. 102:2356, 2005.
Ding, B., Kull, B., Liu, Z., Mottagui-Tabar, S., Thonberg, H., Gu, H.F., Brookes,
A.J., Grundemar, L., Karlsson, C., Hamsten, A., Arner, P., Ostenson, C.G., Efendic, S., Monne, M., von Heijne, G., Eriksson, P., Wahlestedt, C. Human neuropeptide Y signal peptide gain-of-function polymorphism is associated with increased body
mass index: possible mode of function. Regul Pept. 127:45, 2005.
Du, Q., Thonberg, H., Wang, J., Wahlestedt, C., Liang, Z. A systematic analysis of
the silencing effects of an active siRNA at all single-nucleotide mismatched target
sites [published correction appears in Nucleic Acids Res. 33:3698, 2005]. Nucleic
Acids Res. 33:1671, 2005.
Du, Q., Thonberg, H., Zhang, H.-Y., Wahlestedt, C., Liang, Z. Validating siRNA
using a reporter made from synthetic DNA oligonucleotides. Biochem. Biophys.
Res. Commun. 325:243, 2004.
Elmén, J., Thonberg, H., Ljungberg, K., Frieden, M., Westergaard, M., Xu, Y.,
Wahren, B., Liang, Z., Ørum, H., Koch, T., Wahlestedt, C. Locked nucleic acid
(LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids
Res. 33:439, 2005.
Elmén J., Wahlestedt, C., Brytting, M., Wahren, B., Ljungberg, K. SARS virus
inhibited by siRNA. Preclinica 2:135, 2004.
Elmén, J., Zhang, H.-Y., Zuber, B., Ljungberg, K., Wahren, B., Wahlestedt, C.,
Liang, Z. Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication. FEBS Lett.
578:285, 2004.
Faghihi, M.A., Mottagui-Tabar, S., Wahlestedt, C. Genetics of neurological disorders. Expert Rev. Mol. Diagn. 4:317, 2004.
Gu, H.F., Abulaiti, A., Östenson, C.-G., Humphreys, K., Wahlestedt, C., Brookes,
A.J., Efendic, S. Single nucleotide polymorphisms in the proximal promoter region
of the adiponectin (APM1) gene are associated with type 2 diabetes in Swedish
Caucasians. Diabetes 53(Suppl.1):S31, 2004.
356 BIOCHEMISTRY 2005
Heilig, M., Zachrisson, O., Thorsell, A., Ehnvall, A., Mottagui-Tabar, S., Sjögren, M.,
Åsberg, M., Ekman, R., Wahlestedt, C., Ågren, H. Decreased cerebrospinal fluid
neuropeptide Y (NPY) in patients with treatment refractory unipolar major depression: preliminary evidence for association with preproNPY gene polymorphism. J.
Psychiatr. Res. 38:113, 2004.
Isacson, R., Kull, B., Wahlestedt, C., Salmi, P. A 68930 and dihydrexidine inhibit
locomotor activity and d-amphetamine-induced hyperactivity in rats: a role of inhibitory
dopamine D1/5 receptors in the prefrontal cortex? Neuroscience 124:33, 2004.
Katayama, S., Tomaru, Y., Kasukawa, T., Waki, K., Nakanishi, M., Nakamura, M.,
Nishida, H., Yap, C.C., Suzuki, M., Kawai, J., Suzuki, H., Carninci, P., Hayashizaki, Y.,
Wells, C., Frith, M., Ravasi, T., Pang, K.C., Hallinan, J., Mattick, J., Hume, D.A.,
Lipovich, L., Batalov, S., Engstrom, P.G., Mizuno, Y., Faghihi, M.A., Sandelin, A.,
Chalk, A.M., Mottagui-Tabar, S., Liang, Z., Lenhard, B., Wahlestedt, C.; RIKEN
Genome Exploration Research Group; Genome Science Group (Genome Network
Project Core Group); FANTOM Consortium. Antisense transcription in the mammalian transcriptome. Science 309:1564, 2005.
Kemmer, D., Faxén, M., Hodges, E., Lim, J., Herzog, E., Ljungström, E., Lundmark, A., Olsen, M.K., Podowski, R., Sonnhammer, E.L.L., Nilsson, P., Reimers, M.,
Lenhard, B., Roberds, S.L., Wahlestedt, C., Höög, C., Agarwal, P., Wasserman,
W.W. Exploring the foundation of genomics: comparative analysis of transcript profiling technologies. Comp. Funct. Genomics 5:584, 2004.
Larsson, O., Schéele, C., Liang, Z., Moll, J., Karlsson, C., Wahlestedt, C. Kinetics
of senescence-associated changes of gene expression in an epithelial, temperaturesensitive SV40 large T antigen model. Cancer Res. 64:482, 2004.
Larsson, O., Wahlestedt, C., Timmons, J.A. Considerations when using the significance analysis of microarrays (SAM) algorithm. BMC Bioinformatics 6:129, 2005.
Mottagui-Tabar, S., Faghihi, M.A., Mizuno, Y., Engstrom, P.G., Lenhard, B.,
Wasserman, W.W., Wahlestedt, C. Identification of functional SNPs in the 5-prime
flanking sequences of human genes. BMC Genomics 6:18, 2005.
Mottagui-Tabar, S., McCarthy, S., Reinemund, J., Andersson, B., Wahlestedt, C.,
Heilig, M. Analysis of 5-hydroxytryptamine 2c receptor gene promoter variants as
alcohol-dependence risk factors. Alcohol. 39:380, 2004.
Pang, K.C., Stephen, S., Engström, P.G., Tajul-Arifin, K., Chen, W., Wahlestedt, C.,
Lenhard, B., Hayashizaki, Y., Mattick, J.S. RNAdb: a comprehensive mammalian
noncoding RNA database. Nucleic Acids Res. 33:D125, 2005.
Thonberg, H., Dahlgren, C., Wahlestedt, C. Antisense-induced Fas mRNA degradation produces site-specific stable 3′-mRNA fragment by endonuclease cleavage
at the complementary sequence. Oligonucleotides 14:221, 2004.
Thonberg, H., Schéele, C.C., Dahlgren, C., Wahlestedt, C. Characterization of
RNA interference in rat PC12 cells: requirement of GERp95. Biochem. Biophys.
Res. Commun. 318:927, 2004.
Timmons, J.A., Larsson, O., Jansson, E., Fischer, H., Gustafsson, T., Greenhaff,
P.L., Ridden, J., Rachman, J., Peyrard-Janvid, M., Wahlestedt, C., Sundberg, C.J.
Human muscle gene expression responses to endurance training provide a novel
perspective on Duchenne muscular dystrophy. FASEB J. 19:750, 2005.
Wahlestedt, C., Brookes, A.J., Mottagui-Tabar, S. Lower rate of genomic variation
identified in the trans-membrane domain of monoamine sub-class of human G-protein
coupled receptors: the Human GPCR-DB Database. BMC Genomics 5:91, 2004.
Xu, Y., Linde, A., Larsson, O., Thormeyer, D., Elmén, J., Wahlestedt, C., Liang, Z.
Functional comparison of single- and double-stranded siRNAs in mammalian cells.
Biochem. Biophys. Res. Commun. 316:680, 2004.
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Infectology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Chris Baker, Ph.D., Research Associate, Department of Infectology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
INFECTOLOGY 2005
DEPAR TMENT OF
INFECTOLOGY
SENIOR RESEARCH
A S S O C I AT E S
S TA F F
Carlos Coito, Ph.D.
Charles Weissmann, M.D.,
Ph.D.
Professor and Chairman
Corinne Lasmezas, Ph.D.
Professor
Vittorio Verzillo, Ph.D.
R E S E A R C H A S S O C I AT E S
Chris Baker, Ph.D.
Donny Strosberg, Ph.D.
Professor
Tim Tellinghuisen, Ph.D.
Assistant Professor
Prem Subramaniam, Ph.D.
Shawn Browning, Ph.D.
S E N I O R S TA F F
SCIENTIST
Nicole Sales, Ph.D.
S TA F F S C I E N T I S T
Sukhvir Mahal, Ph.D.
S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F I N F E C T O L O G Y : A single, prion-
infected neuroblastoma cell labeled with green fluorescent protein is cultured on a feeder layer of
uninfected cells. The prions secreted by the single infected cell are quantified by using the scrapie
cell assay. The purpose of the project is to determine how prions are propagated in a chronically
infected cell culture. Research and photograph by C.A. Baker, Ph.D., Research Associate, in the
laboratory of C. Weissmann, M.D., Ph.D.
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359
360 INFECTOLOGY 2005
INVESTIGATORS’ R EPORTS
Generation and Transmission
of Prions
C. Weissmann, C.A. Baker, S.P. Mahal, C. Demczyk,
A. Sherman
he agents that cause transmissible spongiform
encephalopathies such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are termed prions.
The unusual resistance of prions to radiation led early
on to the proposal that prions might be devoid of nucleic
acid and consist only of protein. The discovery of PrPSc, a
protease-resistant protein found only in organisms with
transmissible spongiform encephalopathies; the cloning
of PrP cDNA and its gene; the recognition that the gene
encodes a normal host protein, PrPC, from which PrPSc
is derived by conformational rearrangement; and the
linkage between the PrP gene and familial prion disease
supported the suggestion that an abnormal conformer of
PrPC (generically designated as PrP*) is the main or perhaps only constituent of prions. The essential role of PrP
in prion diseases was established by the finding that mice
lacking the PrP gene were resistant to disease and incapable of propagating prions.
The “protein only” hypothesis proposes that the
infectious, abnormal conformer of PrP C is propagated
autocatalytically; a specific mechanism is suggested by
the “seeding hypothesis.” Intriguingly, distinct prion
strains, which generate different disease phenotypes,
may be associated with the same PrP sequence, suggesting that the phenotypes are encoded by different
PrP conformations.
Infectivity of prions is classically measured in a bioassay that takes many months to complete and requires
large numbers of animals. We created a cell-based assay
(scrapie cell assay, SCA) that can be carried out in less
than 2 weeks, is at least as sensitive and accurate as
the animal assay, and allows the simultaneous processing of hundreds of samples by a semiautomated procedure. It has been proposed that prions consist mainly or
entirely of the protease-resistant conformer PrPSc and
that they are resistant to heating at 80°C for 60 minutes. Using the SCA, we found, in collaboration with
F. Properzi, MRC Prion Unit, London, England, that standard protease digestion or heating at 80°C eliminated
T
Charles Weissmann, M.D.,Ph.D.
Chairman’s Overview
he Department of Infectology was founded in 2004,
and the first research group focused on prion diseases. These diseases are of interest not only
because of the emergence of bovine spongiform encephalopathy (mad cow disease) in the United Kingdom, where
transmission to humans has led to more than 150 deaths
due to variant Creutzfeldt-Jakob disease, but also because
of the unusual properties of the infectious agent, which
is unique in lacking a nucleic acid genome. The first
priority was to establish a cell-based assay for prions to
circumvent the slow, expensive, and inaccurate mousebased bioassay. The cell-based assay is being used to
analyze the properties of prions and their propagation,
both in cell culture and in cell-free systems. In a second
phase, the assay will be adapted for high-throughput
screening for drugs capable of blocking prion propagation.
Corinne Lasmezas joined the department in the summer of 2005 to direct a second group working on prion
diseases. A further research effort is directed toward
screening for drugs against leishmaniasis, specifically
against J-binding protein, a putative target, that involves
Scripps scientists in both California and Florida. A third
field of endeavor is hepatitis C; the 2 group leaders in
this area are Donny Strosberg and Tim Tellinghuisen, who
joined the department in November 2005. The growth
of the department is currently restricted by the lack of
space; however, the planned construction of a further
temporary building should alleviate this bottleneck.
T
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The Scripps Research Institute. All rights reserved.
INFECTOLOGY 2005
more than 90% of prion infectivity, indicating that the
essential component is a conformer of PrP other than
PrP Sc . Again using the SCA, in collaboration with
S. Supattapone and N. Deleault, Dartmouth Medical
School, Hanover, New Hampshire, we found that a cellfree system containing PrP C and primed with prioninfected brain homogenate increased prion infectivity
6-fold within 8 hours, showing that infectivity can be
rapidly amplified in a cell-free system.
Only a few cell lines are susceptible to persistent
infection by prions, for as yet unknown reasons. The
high susceptibility of the subclone N2aPK1 developed
for the SCA is unstable, and even after repeated cloning,
susceptible populations consist of both highly susceptible and almost resistant cells; susceptibility appears
to be determined epigenetically. Similarly, populations
of cloned, chronically infected N2aPK1 cells consist of
infected and uninfected cells. Interestingly, a nondividing subpopulation is the most prolific in producing and
secreting prions, while a dividing subpopulation generates very low levels of prions, perhaps because of the
emergence of only a small number of “producer cells”
from the population.
N2aPK1 cells are, surprisingly, susceptible to a
particular murine prion strain (139A) but not to any
others tested so far. We are now searching for cell lines
that are sensitive to other strains and are striving to
identify the features that enable susceptibility to prions in general and to certain strains in particular. A
panel of cell lines with distinct susceptibilities will
greatly facilitate the currently arduous task of typing
of prion strains.
361
maniasis occur in humans: cutaneous, mucocutaneous,
and visceral. According to estimates, 1.5 million to 2
million new cases occur annually, mainly in the tropics and subtropics, including the Middle East. An
effective, well-tolerated, and inexpensive therapy is
greatly needed.
The DNA of Leishmania organisms, as well as
that of other kinetoplastids, contains a modified base,
J (β-D -glucosyl-hydroxymethyluracil), that does not
occur in higher eukaryotes. Leishmania species contain a protein, J-binding protein (JBP), that binds to
the modified base within the context of a nucleotide
sequence and plays a role in the conversion of thymine
to J. Deletion of JBP is lethal for the organisms. Therefore, most likely a compound that interferes with the
binding of JBP to J would be detrimental to the growth
or survival of Leishmania organisms. Because J and
JBP do not occur in the host, such a compound might
lead to a therapeutic drug.
Using a JBP-based approach, members of our broadly
based consortium have set out to search for a lead compound, and if proof of principle is achieved, to develop
a drug. A fluorescence polarization assay for the binding
of a J-containing, fluorescently labeled oligonucleotide to
recombinant JBP has been developed and will be optimized for high-throughput screening of libraries of various compounds at Scripps Florida.
Host-Virus Protein Interactions
in Hepatitis C
A.D. Strosberg, C. Coito, S. Kota, D. Willoughby*
J-Binding Protein of Leishmania
as a Potential Drug Target
C. Weissmann, P. Subramaniam, P. Wentworth,* D. Millar,**
R. Sabatini,*** P. Borst****
*Department of Chemistry, Scripps Research
** Department of Molecular Biology, Scripps Research
*** Marine Biological Laboratory, Woods Hole, Massachusetts
**** Netherlands Cancer Institute, Amsterdam, the Netherlands
eishmaniasis consists of a group of diseases
caused by parasitic protozoans of the genus
Leishmania. The pathogens are transmitted by
sand flies and can infect skin, mucous membranes,
and certain internal organs. Three major types of leish-
L
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The Scripps Research Institute. All rights reserved.
* Ocean Ridge Biosciences, Jupiter, Florida
epatitis C is caused by a 9.5-kb positive singlestranded RNA virus that encodes 11 proteins
and belongs to the family Flaviviridae. Worldwide, an estimated 170 million persons are carriers of
hepatitis C virus (HCV), including 3 million in the United
States. Liver cirrhosis develops in 20% of those who
are persistently infected, and hepatocellular carcinoma
develops in up to 2.5% of those who have cirrhosis.
No vaccine for HCV is available yet, and the only
treatment is the combination of interferon alfa and ribavirin, which is effective in about 50% of all patients.
Several alternative drugs in development target the
enzymatic activities of the viral protease or the viral
RNA-dependent RNA polymerase. However, even if
H
362 INFECTOLOGY 2005
these drugs are successful initially, ultimately patients
will become resistant to them because of high levels of
mutations in the virus, as has occurred with HIV. The
development of such resistance would be less likely
if drugs acted at the interface of interaction between
2 proteins, especially if one of the proteins was a host
cellular protein.
Viruses recruit a number of cellular proteins in order
to enter cells, integrate cellular organelles (e.g., the
endoplasmic reticulum), go into latency, or start assembly and replication inside a cell. At each step, interactions between host and viral proteins are essential,
but for HCV, most of these interactions have not yet
been elucidated.
Previously, a variety of methods, including use of
the yeast 2-hybrid system and coprecipitation with antibodies to one of the protein partners, were used by our
collaborators to analyze the HCV-HCV and the hepatocyte-HCV interactions. The HCV protein domains involved
in several of these interactions were delineated precisely
by using multiple fragments of cDNAs that encode various HCV proteins. We are continuing these studies, and
we plan to develop compounds that modulate HCVHCV and host-HCV interactions.
HCV-HCV PROTEIN INTERACTIONS
We have selected several HCV interacting domains
for our molecular studies. One of these domains corresponds to the first half of the 191-residue-long capsid
protein. This protein polymerizes to form the main structural element to which other HCV proteins and the viral
RNA are associated to form complete virions. The N-terminal 84-residue-long domain actually forms homodimers without the need for the rest of the protein.
Using either peptide synthesis or expression in
Escherichia coli, we prepared forms of these HCV protein domains labeled with biotin, dinitrophenol, glutathione S-transferase, or a short peptide called FLAG.
We are now studying interactions between the labeled
HCV domains by monitoring energy transfer between
fluorescent antibodies that bind to the different labels.
The homogeneous time-resolved fluorescence assay
that we use for this purpose is sensitive and scalable,
so we should be able to measure inhibition of interaction by a variety of peptides and small chemical compounds. From the National Institutes of Health we have
obtained a set of 428 synthetic peptides, each of which
is 18 residues long, that accounts for the whole HCV
proteome. Each peptide will be evaluated for its potential inhibitory capacity. Other, nonpeptidic compounds
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will be obtained from a variety of sources, including
the extensive small-molecule library being assembled
at Scripps Florida.
Once such HCV inhibitors have been identified and
optimized chemically, we will study their effect on HCV
grown in hepatoma-derived Huh7.5 and other similar
cells. To facilitate titration, we are preparing HCV virions labeled with green flourescent protein or luciferase.
KINASES INVOLVED IN HCV PROTEIN INTERACTIONS
A N D H C V R E P L I C AT I O N
Previous research indicated that a few kinases
thought to play a role in HCV replication act on the
NS5A viral protein. Using a number of potential kinase
inhibitors currently being developed at Scripps Florida
and elsewhere, we will study the effects of the molecules on replication of HCV particles in hepatoma cells.
The availability of compounds that disrupt protein
interactions between viral and human host proteins
would help us understand the role of each protein. The
compounds might then be used as the basis for drugs
to complement direct antiviral and immunomodulatory
approaches, thus ultimately providing a broad combination therapy for hepatitis C.
PUBLICATIONS
Coito, C., Diamond, D.L., Neddermann, P., Korth, M.J., Katze, M.G. High-throughput
screening of the yeast kinome: identification of human serine/threonine protein kinases
that phosphorylate the hepatitis C virus NS5A protein. J. Virol. 78:3502, 2004.
Nouet, S., Amzallag, N., Li, J.M., Louis, S., Seitz, I., Cui, T.X., Alleaume, A.M.,
Di Benedetto, M., Boden, C., Masson, M., Strosberg, A.D., Horiuchi, M., Couraud, P.O.,
Nahmias, C. Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2
receptor-interacting protein, ATIP. J. Biol. Chem. 279:28989, 2004.
Translational Research Institute
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The Scripps Research Institute. All rights reserved.
Claes Wahlestedt, Ph.D., Director, CNS Discovery
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The Scripps Research Institute. All rights reserved.
TRANSL ATIONAL RESEARCH INSTITUTE 2005
T R A N S L AT I O N A L
RESEARCH INSTITUTE
Layton H. Smith, Ph.D.
Associate Scientific Director,
Discovery Biology
R E S E A R C H A S S O C I AT E S
I N F O R M AT I C S S TA F F
John B. Bruning, Ph.D.
Mohammad Fallahi-Sichani
Nicholas F. Tsinoremas,
Ph.D.
Senior Director, Informatics
Yen Ting Chen, Ph.D.
Mark M. Gosink, Ph.D.
Julie Conkright, Ph.D.
Christopher C. Mader
Claes Wahlestedt, M.D.,
Ph.D.*
Director, CNS Disorders
Jeffrey E. Habel, Ph.D.
Bruce D. Pascal
Yuanjun He, Ph.D.
Stephan Schuerer, Ph.D.
Rong Jiang, Ph.D.
Mark R. Southern
S TA F F
Patrick R. Griffin, Ph.D.*
Head, Drug Discovery
Jennifer C. Busby, Ph.D.
Associate Scientific Director,
Protein Sciences and
Proteomics
Michael Cameron, Ph.D.
Laboratory Head, Drug
Metabolism and
Pharmacokinetics
SENIOR SCIENTISTS
Magdalena Przydzial, Ph.D.
Thomas D. Bannister, Ph.D.
Derek R. Duckett, Ph.D.
Alessandra Cervino, Ph.D.
Informatics
Assistant Professor
Marcel Koenig, Ph.D.
Louis D. Scampavia, Ph.D.
Yangbo Feng, Ph.D.
Associate Director, Medicinal
Chemistry
S E N I O R S TA F F
Peter S. Hodder, Ph.D.
Associate Director, Lead
Identification
SCIENTISTS
John B. Hogenesch, Ph.D.**
Head, Genome Technologies
Tomas Vojkovsky, Ph.D.
Ted Kamenecka, Ph.D.
Associate Director, Medicinal
Chemistry
Chris Liang, Ph.D.
Director, Medicinal
Chemistry
Phillip LoGrasso, Ph.D.*
Director, Discovery Biology
Kendall W. Nettles, Ph.D.*
Discovery Biology
365
Jeremiah D. Tipton, Ph.D.
* Joint appointment in the
Department of Biochemistry
** Joint appointments in the
Department of Biochemistry and
the Molecular and Integrative
Neurosciences Department
*** Joint appointments in the
Department of Biochemistry and
the Department of Chemistry
Jiu-Xiang Ni, Ph.D.
S TA F F S C I E N T I S T S
Scott A. Busby, Ph.D.
Josephine Harada, Ph.D.
Paul J. Kenny, Ph.D.
Patricia H. McDonald, Ph.D.
Trey Sato, Ph.D.
Thomas Schroeter, Ph.D.
Mathew T. Pletcher, Ph.D.*
Genome Technologies
William R. Roush, Ph.D.***
Executive Director, Medicinal
Chemistry,
Associate Dean, Kellogg
School of Science and
Technology, Florida
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The Scripps Research Institute. All rights reserved.
S E C T I O N C O V E R F O R T H E T R A N S L A T I O N A L R E S E A R C H I N S T I T U T E : Collection
of 3-dimensional chemical structures from top-selling drugs for 2004. The Translational Research
Institute brings together high-throughput screening, discovery biology, medicinal chemistry, informatics,
and drug metabolism with genomic and proteomic technologies to translate basic research into potential
clinical therapeutic agents. Art work by Stephan C. Schuerer, Ph.D., Group Leader, Informatics.
366 TRANSL ATIONAL RESEARCH INSTITUTE 2005
provided by Josephine Harada and Trey Sato, staff scientists, and in genetics, provided by Mat Pletcher, assistant
professor of biochemistry.
Protein Sciences is headed by Jennifer Busby, associate director. Researchers in the Proteomics Laboratory
within Protein Sciences focus on the application of liquid chromatography and mass spectrometry to the identification, quantitation, and characterization of proteins
and posttranslational protein modifications. The laboratory is involved in scientific collaborations within Scripps
Florida and with several external investigators.
Informatics is headed by Nick Tsinoremas, senior
director. The goal of the informatics department is to
provide computational tools and services to the main
research areas of Scripps Florida. Researchers focus on
5 main areas of modern informatics: scientific computing, development of scientific software, computational
biology, drug discovery informatics and chemoinformatics, and data mining and statistical sciences.
Patrick R. Griffin, Ph.D.
Head, Drug Discovery
Chairman’s Overview
he Translational Research Institute combines
the drug discovery efforts at Scripps Florida with
advanced technology platforms: genome technologies, protein sciences, and informatics.
The goal of the drug discovery operation is to discover and develop small-molecule therapeutic agents
for unmet medical needs in inflammation, neurodegeneration, stroke, cancer, and metabolic disorders. The
operation is headed by Pat Griffin, professor of biochemistry. Groups include Lead Identification, headed by
Peter Hodder, associate director; Medicinal Chemistry,
headed by William Roush, executive director and professor of biochemistry; Discovery Biology, headed by
Phil LoGrasso, director and associate professor of biochemistry; Drug Metabolism and Pharmacokinetics,
headed by Mike Cameron, senior scientist; and CNS Disorders, headed by Claes Wahlestedt, director and professor of biochemistry.
Genome Technologies is headed by John Hogenesch,
professor of biochemistry. The goal of this department is
to provide Scripps investigators and external collaborators cutting-edge technologies in cell-based screening,
T
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TRANSL ATIONAL RESEARCH INSTITUTE 2005
367
INVESTIGATORS’ R EPORTS
ties and to determine differential protein binding
affinities on the basis of the site of modification.
Proteomics Core Facility
PUBLICATIONS
Hogan, K.T., Sutton, J.N., Chu, K.U., Busby, J.A.C., Shabanowitz, J., Hunt, D.F.,
Slingluff, C.L., Jr. Use of selected reaction monitoring mass spectrometry for the
detection of specific MHC class I peptide antigens on A3 supertype family members. Cancer Immunol. Immunother. 54:359, 2005.
J.A. Caldwell Busby, J. Tipton, V. Cavett
he Proteomics Core Facility at Scripps Florida has
general high-performance liquid chromatography
and mass spectrometry capabilities for protein
and peptide identification via nanoflow chromatography followed by tandem mass spectrometry and analysis. Current instrumentation includes an ion-trap mass
spectrometer, which is used predominantly for protein
and peptide identification, and a triple quadrupole mass
spectrometer, which is used for relative quantitation
experiments. Each mass spectrometer is interfaced to
nanoflow electrospray ionization sources and capillary
high-performance liquid chromatography columns.
Expertise exists for identifying and mapping posttranslational modifications of proteins and peptides.
As a core facility, we are responsible for providing
collaborative mass spectrometry services to other faculty
members at Scripps Florida. These collaborative projects
will necessarily determine the development of technology
in the facility. Current efforts in technology development
include on-line sample cleanup and digestion, relative
quantitation of serum proteins, and creation of software for differential analysis of mass spectrometry data.
During its lifetime, a protein can have several locations and duties within a cell. Location, current status,
and 3-dimensional structures of proteins are all influenced by static and dynamic chemical modifications
that occur after translation. These modifications vary,
from small methyl groups, which are a part of the histone codes, to large lipid and glycosylation modifications,
which act as cellular markers and signaling molecules.
Mass spectrometry has the unique advantage of being
able to detect both the small and large changes in
mass that occur because of these modifications.
Phosphorylation is one posttranslational modification that receives attention because of its important role
in signaling pathways. Unfortunately, phosphorylation
is still relatively rare, and enrichment techniques are
needed to detect sites of phosphorylation. In the core
facility, we use immobilized metal affinity chromatography to enrich complex samples for phosphorylated
peptides. This mapping of phosphorylation sites has
been used to identify kinase and phosphatase activi-
T
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Roig, J., Groen, A., Caldwell, J., Avruch, J. Active Nercc1 protein kinase concentrates at centrosomes early in mitosis, and is necessary for proper spindle assembly. Mol. Biol. Cell 16:4827, 2005.
Stover, D.R, Caldwell, J., Marto, J.M., Root, K.R., Mestan, J., Stumm, M., Ornatsky, O.,
Orsi, C., Radosevic, N., Liao, L., Fabbro, D., Moran, M. Differential phosphoprofiles of EGF and GFR kinase inhibitor-treated human tumor cells and mouse xenografts.
Clin. Proteomics J. 1:69, 2004.
Thompson, L.W., Hogan, K.T., Caldwell, J.A., Pierce, R.A., Hendrickson, R.C.,
Deacon, D.H., Settlage, R.E., Brinkerhoff, L.H., Engelhard, V.H., Shabanowitz, J.,
Hunt, D.F., Slingluff, C.L., Jr. Preventing the spontaneous modification of an HLAA2-restricted peptide at an N-terminal glutamine or an internal cysteine residue
enhances peptide antigenicity. J. Immunother. 27:177, 2004.
Drug Discovery
P. Hodder, L. Scampavia, T. Spicer, C. Chung, P. Chase,
N. Kushner, P. Baillargeon
igh-throughput screening requires sophisticated
automation, detection, and assay technologies
to test large (e.g., several hundred thousands)
collections of compounds for biological or biochemical
activity. This method typically is used by large pharmaceutical companies in their efforts to discover new
drugs. With funding from the State of Florida and Palm
Beach County, we are creating a state-of-the-art highthroughput screening operation at Scripps Florida.
Our major goals are to screen large libraries of
compounds and to identify compounds that may result
in new clinical drugs. We intend to accomplish these
goals by using novel high-throughput screening technologies. When these objectives are fully realized, we will
be able to (1) support research at Scripps that requires
high-throughput screening, (2) manage the extensive
collection (>600,000 compounds) of compounds at
Scripps used for drug screening, (3) provide assistance
in the development of assays compatible with highthroughput screening, and (4) explore and implement
novel technologies for the advancement of high-throughput screening. Highlights of our work in 2005 include
the following.
H
CONSTRUCTION OF A HIGH-THROUGHPUT
S C R E E N I N G L A B O R AT O R Y
The Lead Identification Department is building a
state-of-the-art high-throughput screening laboratory
368 TRANSL ATIONAL RESEARCH INSTITUTE 2005
at Scripps Florida. In addition to a fully staffed laboratory devoted to the development of high-throughput
screening assays, the centerpieces of the laboratory are
2 automated robotic systems. The first robotic system
(Fig. 1) is dedicated solely to high-throughput screening. With this system, we can test more than 1 million
samples per day. The system has an on-line storage
capacity of more than 1 million compounds, can be
run 24 hours a day, 7 days a week, and can be used
for both biochemical and cell-based assays. Because
microtiter plates with 1536 wells (assay volumes of
~10 µL per well), are used in the assays, high-throughput screening can be completed rapidly and economically.
F i g . 1 . The Scripps Florida high-throughput screening system
will provide fully automated technology to detect compounds that
may lead to the discovery of new drugs.
The second robot stores a copy online of the entire
collection of the compounds used for high-throughput
screening. It has the automation to instantly retrieve
and reformat any member of the collection to a 384or a 1536-well plate. This robot will be used in studies after a high-throughput screening campaign has
been completed, allowing scientists to rapidly select
compounds for further pharmacologic analysis.
The final installation and testing of the robotic systems were scheduled to be completed in November
2005 and should be ready for use by the start of 2006.
ACQUISITION AND MANAGEMENT OF COMPOUNDS
FOR SCREENING
In addition to setting up the screening facility, we
are acquiring a proprietary collection of more than
600,000 druglike compounds to be managed and maintained for high-throughput screening. This collection will
include compounds from commercial sources and novel
compounds discovered by researchers at the Scripps
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facilities. In order to support drug-target profiling and
facilitate grant writing efforts, smaller collections will
be established. Currently, our goal is to have the compounds on hand and ready for high-throughput screening by the first quarter of 2006.
R E S E A R C H C O L L A B O R AT I O N S
The Scripps research facilities have been designated
a member of the Molecular Libraries Screening Center
Network, as part of the National Institutes of Health
Roadmap Initiative (http://nihroadmap.nih.gov/). In
collaboration with H. Rosen, Department of Immunology, Scripps Research, we will support the implementation and execution of high-throughput screening done
to meet the goals of the network. The data acquired via
high-throughput screening will be made publicly available through PubChem, to aid in the discovery of probes
that explore the functions of biological mechanisms in
health and disease.
As part of our commitment to the State of Florida,
we are also setting up processes for Florida academic
researchers to access our expertise in high-throughput
screening. We are creating a specialized collection of
compounds and a screening model tailored to academic research.
PUBLICATIONS
Hale, J.J., Lynch, C.L., Neway, W., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Parent, S.A., Chrebet, G., Bergstrom, J.,
Card, D., Ferrer, M., Hodder, P., Strulovici, B., Rosen, H., Mandala, S. A rational
utilization of high-throughput screening affords selective, orally bioavailable 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonists. J. Med.
Chem. 47:6662, 2004.
Hodder, P., Mull, R., Cassaday, J., Berry, K., Strulovici, B. Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorimetric
imaging plate reader. J. Biomol. Screen. 9:417, 2004.
Zuck, P., O’Donnell, G.T., Cassaday, J., Chase, P., Hodder, P., Strulovici, B., Ferrer, M.
Miniaturization of absorbance assays using the fluorescent properties of white
microplates. Anal. Biochem. 342:254, 2005.
Development of Protein
Kinase Inhibitors
C. Liang, M. Koenig, T. Vojkovsky, Y. He, Y. Feng,
T. Kamenecka, T. Schroeter, A. Weiser
ur goal is to discover protein kinase inhibitors
that can be used as therapeutic agents for the
treatment of human diseases such as cancer
and arthritis. Protein kinases are a class of enzymes
that catalyze the transfer of the γ-phosphate from ATP
to protein substrates. These enzymes play critical roles
O
TRANSL ATIONAL RESEARCH INSTITUTE 2005
in signal transduction for a number of cellular functions.
In particular, they regulate most of the hallmarks of
cancer: cell proliferation, cell survival, cell motility/metastasis, cell cycle/division, and angiogenesis. Protein
kinases are also implicated in inflammatory diseases
such as arthritis and asthma. The approval of the tyrosine kinase inhibitors imatinib mesylate (Gleevec) for
treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors and bevacizumab (Avastin)
for treatment of non–small cell lung cancer validated
protein kinase inhibitors/antagonists as effective, noncytotoxic anticancer agents. These drugs provide new
hope and models in the long fight against cancer. In
addition, later stage clinical data strongly suggest that
p38α MAP kinase inhibitors could be effective antiinflammatory agents. For these reasons, protein kinases
are being investigated as valuable therapeutic targets
by virtually every pharmaceutical company, and according to estimates, about 25% of all current pharmaceutical research is devoted to these targets.
We synthesized and evaluated more than 500 potential drug candidates during the past year. In 2 projects,
we found compounds that have better in vitro properties than the best known competitors do. Several of
these compounds also have good pharmacokinetic properties and in vivo efficacy. Currently, they are being
evaluated as potential clinical drug candidates. Recently,
we began a collaboration with the NeoRx Corporation,
Seattle, Washington, to discover novel multitargeted
protein kinase inhibitors for the treatment of cancer.
An Integrated Proteomics
Environment and the
Deuterator System
B. Pascal, C. Mader, N.F. Tsinoremas
e are building an integrated proteomics informatics environment to support the proteomics
research activities of the drug discovery group
at Scripps Florida. To date, we have focused on establishing a software infrastructural system to support
acquisition of the primary mass spectral data and on
developing software for the analysis and presentation
of data from hydrogen-deuterium exchange experiments.
Scientists at Scripps Florida are conducting solution-phase amide hydrogen-deuterium exchange exper-
W
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369
iments, which require specific software support. Systems have been put in place for high-throughput data
analysis and visualization. Hydrogen-deuterium exchange
experiments begin with a typical proteomics experiment:
protein is digested with an enzyme, and the resulting
peptides are analyzed by using liquid chromatography
and tandem mass spectrometry. The identity of the
peptides is established by using commercially available
database search tools such as Sequest. Subsequent
solution-phase amide hydrogen-deuterium exchange
experiments are performed for predefined periods
(8–10 time points spanning 1–10,000 seconds). After
incubation with deuterium oxide, the protein is digested,
and the resulting peptides are analyzed by using liquid
chromatography and tandem mass spectrometry. The
increase in peptide mass reveals the extent of hydrogen-deuterium exchange at each time point. Because
the incubation with deuterium oxide precedes the digestion of protein, the measured rate of deuterium incorporation for each peptide reveals the hydrogen-deuterium
exchange rate for the corresponding region of the intact
protein. These hydrogen-deuterium exchange rates can
be determined by using our software, which is titled
the Deuterator. Additional features allow for differential
hydrogen-deuterium exchange analysis between free and
ligand-bound protein.
NEED FOR A UNIFIED SYSTEM
High-throughput analysis for hydrogen-deuterium
data of this type is not generally commercially available
at this time. Our system provides a complete infrastructure for this analysis, from the acquisition of foundational data to analysis and visualization. When possible,
existing components have been used and generic structures built to allow reuse with other projects. Increasing the throughput of hydrogen-deuterium exchange
experiments required constructing an informatics infrastructure to support the increase in the production of
data. We have built a powerful informatics infrastructure that can support the acquisition, management, and
analysis of large data sets (terabytes).
T H E I N T E G R AT E D P R O T E O M I C S E N V I R O N M E N T
The integrated proteomics informatics environment
is used to manage data from the time the data are
created by the mass spectrometer through hydrogendeuterium exchange analysis, and in the future, differential analysis via mass spectrometry. Figure 1 shows
an overall architecture of the environment.
A laboratory information management system (LIMS)
has been put in place to acquire process data and act as
370 TRANSL ATIONAL RESEARCH INSTITUTE 2005
We have developed a system called Morphimi that
converts raw mass spectrometric data to other formats,
usually to mzXML. Morphimi also maintains a repository
of and manages the metadata of the converted mzXML
data. mzXML is an XML-based common file format for
mass spectrometric data, developed at the Institute for
Systems Biology. Having the mass spectrometric data in
mzXML allows access to a number of current and future
tools written for use with the schema (Fig. 1).
The mass spectrometer produces binary RAW files,
which are then submitted to the LIMS. Morphimi then
retrieves these files and converts them to mzXML and
places them in the Morphimi archive. Sequest sequence
searches are performed from the RAW files, and the
results are submitted into the LIMS. The Deuterator then
retrieves the search result file from the LIMS and the
mzXML file from the Morphimi archive and proceeds
with peak picking and centroid calculations. The resulting data set is made available via a Web interface, and
the reviewed results are sent to the statistical analysis
and visualization tools, which are also available through
the Web interface (Fig. 2).
F i g . 1 . Overview of the integrated proteomics environment.
1. Mass spectrometer (MS) produces raw spectral data. 2. The
LIMS system captures and organizes project information and maintains an archive of the raw MS data. 3. Morphimi MBean is used
to manage conversion of raw MS data to mzXML. 4. Morphimi maintains an index and archive of mzXML data. 5. Peak Picking toolkit
filters and prepares mzXML data for analysis. 6. The Deuterator performs hydrogen-deuterium exchange analysis. 7. The Diff MS application suite will provide tools for differential analysis of MS data.
8. Various open source libraries are used to analyze the data.
9. Various open source libraries are used to visualize the data.
10. Protein database search tools (Sequest and MASCOT are currently supported) are used to identify the peptides.
a repository for the raw data produced by the mass spectrometers. This solution has been provided by Genologics,
Victoria, British Columbia, and is called ProteusLIMS.
This system is being used to set up projects and upload
raw and associated data files, which immediately become
available to the downstream analysis software.
The Institute for Systems Biology, Seattle, Washington, provides a variety of open source software tools for
analysis and organization of data obtained in proteomics
research. The goal of the Sashimi (http://sashimi
.sourceforge.net) project begun at the institute is to provide the scientific community free open source software
tools for the analysis of mass spectrometric data. We
have incorporated a number of components from the
institute within our system.
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F i g . 2 . Deuterator work flow diagram. H/D indicates hydrogen-
deuterium.
The peptide set obtained from the database search
is initially grouped according to sequence and charge
(multiple occurrences of the same peptide ion often occur
within the raw data set). Redundant data are removed,
and the retention time for each peptide ion becomes the
mean of all measured values. A retention time window
is then created for each peptide within the set. This
range is used by the software to go into the raw data
and calculate the average of all spectral points for all
scans located within this region. The monoisotopic mass
of each peptide is then calculated. Accounting for the
TRANSL ATIONAL RESEARCH INSTITUTE 2005
371
charge state of the given peptide, the 0% and the 100%
deuterium incorporation mass-to-charge ratio limits for
the given peptide are calculated. The intensity-weighted
mean mass-to-charge ratio between these values is
calculated, defined as the centroid mass-to-charge ratio,
and the values for each peptide set are presented in the
main interface page.
On the basis of queries to the LIMS, the user interface enables drilling down from the project level to the
sample level and finally to the individual time point
(Fig. 3). All processed time points are immediately
available through this interface.
F i g . 5 . Spectral viewer.
file format. The extracted ion graph displays the ion
intensities for a given mass across all scans. This graph
is used to zone in to the areas with the best data for a
given peptide’s centroid mass (Fig. 5).
FUTURE DEVELOPMENT
F i g . 3 . Project, sample, and time point selection.
The main analysis page has 3 sections. The grid
presents all of the calculations and allows users to select
a peptide for viewing (Fig. 4). By using the tab browser
functionality in the Firefox browser, many time points
can be seen simultaneously.
F i g . 4 . Selection of a peptide for viewing.
The spectral viewer initially displays the raw spectral data for the calculated range of the selected peptide (Fig. 5), and navigation controls can be used to
further refine the range. The centroid is automatically
recalculated for each refinement. When the user is satisfied, the results can be saved into the database. At any
time, the entire grid data can be exported to a common
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Rendering components will be built that provide
extended graphing capabilities, 2- and 3-dimensional
color gradient maps, and data grids that show changes
in deuterium incorporation between different experiments. Analysis tools will be built that will calculate
the theoretical isotopic envelope and fit it onto the
spectral output, as well as deconvolute overlapping
peptides. These components will be tightly integrated
into the system.