Scripps Florida 2008
• Cancer Biology
• Chemistry
• Infectology
• Molecular Therapeutics
• Molecular and Intregrative
Neurosciences
• Translational Research Institute
Cancer Biology
Aberrant cell division in a precancerous cell: Shown is a
differential interference contrast image of an early-passage
p53-null mouse embryo fibroblast. Note that the chromosomes in the cell are being pulled in 3 directions. Daughter
cells that arise are aneuploid and/or polyploid. Work done
by Frank C. Dorsey, Ph.D., research associate, in the laboratory of John L. Cleveland, Ph.D., professor.
Kendall Nettles, Ph.D., Assistant Professor
CANCER BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
19
DEPAR TMENT OF
CANCER BIOLOGY
S TA F F
John L. Cleveland, Ph.D.
Professor and Chairman
Jun-Li Luo, Ph.D., MD
Assistant Professor
Kendall Nettles, Ph.D.
Assistant Professor
HaJeung Park, Ph.D.
Ann Griffith, Ph.D.
Meredith A. Steeves, Ph.D.
Mark A. Hall, Ph.D.
Weilin Wu, Ph.D.
Jun Hyuck Lee, Ph.D.
Howard Petrie, Ph.D.
Professor
Tina Izard, Ph.D.
Associate Professor
Nagi G. Ayad, Ph.D.
Assistant Professor
Philippe R.J. Bois, Ph.D.
Assistant Professor
Michael Conkright, Ph.D.
Assistant Professor
Woonghee Lee, Ph.D.
S C I E N T I F I C A S S O C I AT E
R E S E A R C H A S S O C I AT E S
Hiroshi Nakase, Ph.D.
Chunying Yang, M.D.
Antonio Amelio, Ph.D.
Robert J. Rounbehler, Ph.D.
Mi Ra Chang, Ph.D.
Jianjun Shi, Ph.D.
Frank C. Dorsey, Ph.D.
Zhen Wu, Ph.D.
Joanne R. Doherty, Ph.D.
Rangarajan Erumbi, Ph.D.
Ihn Kyung Jang, Ph.D.
Irina Getun, Ph.D.
Bhargavakrishna Yekkala,
Ph.D.
S TA F F
SCIENTISTS
German Gil, Ph.D.
Sollepura Yogesha, Ph.D.
Min Zhao, Ph.D.
Chairman’s Overview
he Department of Cancer Biology on the Florida
campus was established in November 2006. The
department has rapidly grown to now include
8 faculty members. The
broad goals of the
research programs of
the department are to
fully define the molecular events that underlie
human cancer and then
apply this knowledge
to the development of
novel therapeutic strategies and new agents for
cancer prevention and
John L. Cleveland, Ph.D.
therapeutics.
The programs include those that examine the roles
of signal transduction pathways, oncogenes, and tumor
suppressors that are altered in cancer and how these
alterations control cell division, growth, survival, differentiation, cell migration and metastasis, tumor angiogenesis, transcriptional circuits, and genomic stability
and how they modify the response to therapeutic agents.
In addition, the interplay between tumors and the
immune system in cancer is a new major thrust of
research. Faculty members in the department use a
T
battery of state-of-the-art technologies for target discovery and validation, ranging from biochemistry and
cell biology to preclinical models to x-ray crystallography. In addition, unique models have been developed
to evaluate the efficacy of new leads in cancer prevention and therapeutics. Investigators in the department
have interests in understanding the molecular underpinnings of all of the major human malignant neoplasms,
including lung, breast, prostate, colon, and brain cancer, and several hematologic malignant neoplasms.
Other interests include pediatric oncology, the interplay between malignant neoplasms and metabolism,
and the relationships between aging and cancer.
One of the many strengths at the Florida campus
is high-throughput technologies that enable investigators to rapidly move forward potential leads by using
both genetic and small-molecule screens. Strong collaborations with the major cancer centers in the State
of Florida and with cancer researchers at the California campus of Scripps Research will allow leads that
are identified to rapidly advance to translational and
clinical studies.
20 CANCER BIOLOGY
2008
INVESTIGATORS’ R EPORTS
Myc-Mediated Pathways in
Cancer and Development
J.L. Cleveland, M.A. Hall, F.C. Dorsey, R. Rounbehler,
K. Yekkala, J. Doherty, M. Steeves, C. Yang, T. Bratton,
S. Prater, W. Li
yc oncoproteins function as master regulators
of transcription and regulate up to 10%–15%
of the genome. Three Myc oncogenes (c-Myc,
N-Myc, L-Myc) are activated in about 70% of human
cancers. Their activation can occur directly via gene
amplification, chromosomal translocations, or somatic
missense mutations or indirectly via alterations in signal
transduction pathways or the loss of tumor suppressors
that normally regulate and/or harness Myc expression.
The pervasive selection for Myc activation in cancer in
part reflects the essential roles of Myc as a regulator
of cell growth and division, but overexpression of Myc
also triggers accelerated rates of cell proliferation, tumor
angiogenesis, and metastasis. Further, Myc regulates
stem cell fate and supercompetition, a scenario in which
cells that overexpress Myc kill their neighboring, normal cells.
We have used mouse models to dissect the contribution of key targets downstream of Myc that control
tumorigenesis. In normal cells, Myc triggers apoptosis
through the Arf-p53 tumor suppressor pathway that is
inactivated in most malignant tumors and by selectively
affecting the expression of members of the Bcl-2 family of proteins that directly control the intrinsic apoptotic
pathway. We have shown that these pathways hold
Myc-induced tumorigenesis in check and that mutations
in these apoptotic regulators are a hallmark of most
malignant tumors.
Although apoptotic regulators clearly serve as guardians against Myc-induced cancer, we have found that
the ability of Myc to provoke accelerated cell growth is
also critical for tumorigenesis. First, Myc coordinately
regulates the expression of cytokines that direct cell
growth and tumor angiogenesis. Second, Myc suppresses expression of the universal cyclin-dependent
kinase (Cdk) inhibitor p27Kip1 that normally inhibits the
activity of cyclin E–Cdk2 and cyclin A–Cdk2 complexes
that are necessary for entry and progression through
the DNA synthesis (S) phase of the cell cycle. Notably,
we found that Myc suppresses p27Kip1 protein levels
by inducing transcription of the Cks1 component of the
M
THE SCRIPPS RESEARCH INSTITUTE
SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1
for destruction by the 26S proteasome. Accordingly, loss
of Cks1 disables the ability of Myc to suppress p27Kip1
and markedly impairs Myc-induced proliferation and
tumorigenesis, whereas loss of p27Kip1 accelerates Mycinduced tumorigenesis. Remarkably, Cks1 overexpression
is a hallmark of all lymphomas with Myc involvement,
suggesting this pathway is a general route by which Myc
coordinates cell growth and division and that the pathway can be targeted by directed therapeutic agents.
Because Myc regulates such a large number of genes
and is essential for cell growth and division, the adverse
effects of agents that directly target the transcription
functions of Myc might be greater than the agents’ beneficial effects. We therefore have focused our efforts on
key transcription targets of Myc that might be suitable
therapeutic targets. We found that inhibiting ornithine
decarboxylase, a direct transcription target of Myc and
the rate-limiting enzyme of polyamine biosynthesis,
impairs Myc-induced proliferation and tumorigenesis.
These results were underscored by our findings that
heterozygosity in the gene that encodes ornithine decarboxylase, a condition that only reduces the enzyme
activity of ornithine decarboxylase and the generation
of its product by half, triples the life span of tumor-prone
mice. Thus, agents that target the polyamine pathway
have promise in both the prevention and the treatment
of cancer. Currently, we are defining the mechanism by
which targeting ornithine decarboxylase disables the
proliferative response of Myc. Our results indicate, quite
remarkably, that targeting ornithine decarboxylase disables the ability of Myc to suppress p27Kip1 by shortcircuiting of the Myc-to-Cks1 pathway.
Finally, we recently discovered that additional Myc
transcription targets that can be exploited in cancer
therapy include components of the autophagy pathway,
an ancient survival pathway that directs the digestion
of bulk cytoplasmic material and organelles when cells
are faced with nutrient- or oxygen-deprived conditions,
a scenario manifests in the tumor microenvironment.
We have shown that agents that disable autophagy have
tremendous potential in cancer prevention and treatment. Currently, we are defining the mechanisms by
which Myc regulates the expression of genes that control the autophagy pathway and their potential as targets for agents to prevent and treat cancer.
PUBLICATIONS
Carew, J.S., Nawrocki, S.T., Reddy, V.K., Bush, D., Rehg, J.E., Goodwin, A.,
Houghton, J.A., Casero, R.A., Jr., Marton, L.J., Cleveland, J.L. The novel polyamine analogue CGC-11093 enhances the antimyeloma activity of bortezomib. Cancer Res. 68:4783, 2008.
CANCER BIOLOGY
2008
Garrison, S.P., Jeffers, J.R., Yang, C., Nilsson, J.A., Hall, M.A., Rehg, J.E., Yue,
W., Yu, J., Zhang, L., Onciu, M., Sample, J.T., Cleveland, J.L., Zambetti, G.P.
Selection against PUMA gene expression in Myc-driven B-cell lymphomagenesis.
Mol. Cell. Biol. 28:5391, 2008.
Klionsky, D.J., Abeliovich, H., Agostinis, P., et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy
4:151, 2008.
Maclean, K.H., Dorsey, F.C., Cleveland, J.L., Kastan, M.B. Targeting lysosomal
degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J. Clin. Invest. 118:79, 2008.
Nawrocki, S.T., Carew, J.S., Douglas, L., Cleveland, J.L., Humphreys, R., Houghton,
J.A. Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a
p21Cip1-dependent decrease in survivin levels. Cancer Res. 67:6987, 2007.
Nawrocki, S.T., Carew, J.S., Maclean, K.H., Courage, J.F., Huang, P., Houghton,
J.A., Cleveland, J.L., Giles, F.J., McConkey, D.J. Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA. Blood 112:2917, 2008.
Rodrigues, C.O., Nerlick, S.T.,White, E.L., Cleveland, J.L., King, M.L. A Myc-Slug
(Snail2)/Twist regulatory circuit directs vascular development. Development
135:1903, 2008.
Sanjuan, M.A., Dillon, C.P., Tait, S.W., Moshiach, S., Dorsey, F., Connell, S.,
Komatsu, M., Tanaka, K., Cleveland, J.L., Withoff, S., Green, D.R. Toll-like receptor
signalling in macrophages links the autophagy pathway to phagocytosis. Nature
450:1253, 2007.
THE SCRIPPS RESEARCH INSTITUTE
21
V I N C U L I N S T R U C T U R E A N D R E G U L AT I O N
Our crystal structures, biochemical studies, and
biological experiments have redefined vinculin structure
and regulation. First, in its resting, inactive conformation, vinculin is held in a closed-clamp conformation
through interactions of a 7-helical bundle domain present in its head domain (Vh1) with a 5-helical bundle
in the tail domain (Vt); 3 additional helical bundle
domains that were identified likely also serve as docking sites for interactions with partners. Second, contrary
to dogma, we found that, talin itself is a direct activator of vinculin; α-helical vinculin-binding sites (VBSs)
in the central rod domain of talin trigger vinculin activation by displacing Vt from a distance. More importantly,
our structures revealed that this activation of vinculin
and displacement of Vt occurred via a heretofore unknown
change in protein structure, by a process we termed
helical bundle conversion (Fig. 1). Third, our studies
Schweers, R.L., Zhang, J., Randall, M.S., Loyd, M.R., Li, W., Dorsey, F.C.,
Kundu, M., Opferman, J.T., Cleveland, J.L., Miller, J.L., Ney, P.A. NIX is required
for programmed mitochondrial clearance during reticulocyte maturation. Proc. Natl.
Acad. Sci. U. S. A. 104:19500, 2007.
Structural Dynamics in
Adhesion Complexes
T. Izard, P.R. Bois, J.H. Lee, G.T.V. Nhieu,* H. Park,
E.S. Rangarajan, S.D. Yogesha
* Pasteur Institute, Paris, France
ell migration and morphogenesis are essential
for the development, growth, and survival of
metazoans, and these processes are also involved
in pathophysiologic conditions such as cancer metastasis
and myopathies. Migration and morphogenesis rely on
the ability of a cell to dynamically form and break specific contacts, called adhesion junctions, with neighboring cells (adherens junctions) or the extracellular matrix
(focal adhesions). Vinculin is an essential regulator of
both cell-cell (cadherin-catenin mediated) and cell-matrix
(integrin-talin mediated) junctions, where it provides
links to the actin cytoskeleton by binding to talin in
integrin complexes or to α-catenin and α-actinin in
cadherin junctions. Previously, little was known about
the structure and activation of vinculin, although the
accepted belief was that activation required severing
intramolecular interactions of the vinculin head and
tail domains.
C
F i g . 1 . Vinculin activation by talin through helical bundle con-
version. Ribbon drawing of the vinculin head (Vh1; cyan) and tail
(Vt; yellow) domains activated by talin’s VBS (red). A, The crystal
structure of the Vh1-Vt complex revealed that vinculin is held in a
closed conformation through many hydrophobic interactions between
the Vh1 and Vt domains. B, The crystal structure of talin-VBS3
bound to Vh1 shows that talin binds to vinculin at an accessible site
distal from the Vh1-Vt interface and that talin-VBS3 binding displaces Vt from a distance. Wholesale structural changes occur upon
talin binding, whereby the 4-helical bundle of Vh1 incorporates the
amphipathic VBS helix of talin to form an entirely new 5-helical
bundle via helical bundle conversion.
22 CANCER BIOLOGY
2008
of the complex composed of α-actinin and vinculin
revealed that α-actinin activates vinculin and alters its
structure in unique ways. These findings supported our
model in which the vinculin Vh1 domain functions as
a “molecular switch” that undergoes rapid and unique
changes in its structure after binding to different activators, which then endow vinculin with the ability to
bind to unique partners in adherens junctions vs focal
adhesions. Finally, our studies indicated that adhesion
signaling involves a chain reaction of structural alterations in which, after their activation, the VBSs of talin
or α-actinin first unravel from their buried locations and
then bind to and activate vinculin, which then undergoes wholesale changes in its structure (Fig. 2).
THE SCRIPPS RESEARCH INSTITUTE
studies showed that IpaA acts as a talin mimic that
disrupts vinculin’s contacts with talin and α-actinin,
and our results suggest that this mechanism is a general one that is exploited by other pathogens. Importantly, our biological studies have shown that this
interaction is necessary for efficient entry of Shigella
into host cells.
Our recent studies have revealed additional layers
of functional complexity of IpaA. First, we found that
the second, somewhat lower affinity, VBS of IpaA can
bind to a second motif of the vinculin Vh1 domain, a
situation that would stabilize IpaA-vinculin interactions
(Fig. 3). Second, our biochemical and genetic screens
F i g . 2 . Relays in adherens junctions. Ribbon drawing of α-actinin
(gray and black) and vinculin. Top, In their resting state, both α-actinin
and vinculin are in a closed conformation. The VBS is shown in red.
Bottom, When activated, α-actinin unfurls to expose its VBS, which
then binds to and activates vinculin, resulting in helical bundle conversion of the vinculin Vh1 domain and severing of vinculin’s headtail interaction. The α-actinin antiparallel homodimer has 2 VBS
sites. For clarity, only 1 vinculin molecule is shown bound to the
α-actinin homodimer.
TA R G E T I N G V I N C U L I N I N PAT H O G E N - H O S T
INTERACTIONS
We have also made significant inroads in understanding how vinculin is co-opted by pathogens. Initially,
we have focused on Shigella flexneri, the principal pathogen of bacillary dysentery. Shigella organisms inject
invasin proteins (IpaA-IpaD) that create pores in intestinal
epithelial cells and that trigger the formation of filopodial and lamellopodial extensions that surround the bacteria. IpaA, a protein of approximately 70 kD essential
for the pathogenesis of Shigella in vivo, facilitates entry
of the bacteria into host cells by binding to vinculin.
We established that IpaA has 2 high-affinity VBSs that
bind to the Vh1 domain of vinculin and induce unique
alterations in the domain’s structure. Strikingly, our
F i g . 3 . A novel second binding site on the vinculin Vh1 domain
allows IpaA to bind vinculin in its closed conformation. A, Ribbon
drawing of the vinculin head domain (Vh1; yellow) bound by the 2
VBSs of S flexneri IpaA. The first IpaA-VBS (blue) binds to Vh1 with
femtomolar affinity by molecular mimicry of the Vh1-talin interaction,
via helical bundle conversion. In contrast, the second IpaA-VBS (red)
binds vinculin by a helix addition mechanism, a scenario that allows
the S flexneri invasin to also recruit pools of inactive vinculin and
that would also facilitate the bridging of 2 molecules of vinculin by
IpaA. B, Surface drawing of full-length human vinculin (yellow, orange,
magenta, blue, gray, and cyan) bound to IpaA-VBS (ribbon drawing,
red). The weaker binding IpaA-VBS binds vinculin by a helix addition mechanism, which has no allosteric effects on vinculin, allowing
IpaA to bind to vinculin in its closed conformation. Reprinted from
Nhieu, G.T., Izard, T. Vinculin binding in its closed conformation by
a helix addition mechanism. EMBO J. 26:4588, 2007.
CANCER BIOLOGY
2008
have revealed new cytoskeletal binding partners for IpaA.
The physiologic roles of these interactions will be tested,
and along with our structural analyses, these studies
may point to new therapeutic avenues.
PUBLICATIONS
Nhieu, G.T., Izard, T. Vinculin binding in its closed conformation by a helix addition mechanism [published correction appears in EMBO J. 27:922, 2008]. EMBO
J. 26:4588, 2007.
Regulation of Mitotic Entry and
Exit by Ubiquitin-Mediated
Proteolysis
N. Ayad, S. Simanski, N. Nagarsheth
biquitin-mediated proteolysis is one of the main
ways cells eliminate intracellular proteins. This
elimination is important for cell homeostasis,
development, and growth. Recent studies have also
indicated that one or more components of this system
are overexpressed in cancer cells, making the components attractive targets for pharmacologic inhibition.
Although a fair amount is known about the pathways
leading to ubiquitin-mediated degradation, many essential components have not been identified.
We devised a means of identifying regulators of
the anaphase-promoting complex (APC), an essential
ubiquitin ligase required for the metaphase-to-anaphase
transition and exit from mitosis. We fused cyclin B1, a
known APC substrate, to luciferase and cotransfected
this fusion construct with 14,000 cDNAs. This genomewide screen led to the identification of multiple regulators of the APC, including some that are overexpressed
in breast cancer. Currently, we are identifying the mechanism by which these proteins regulate the APC during
exit from mitosis and are developing high-throughput
screens. Our eventual goal is to eliminate the activity
of the proteins selectively in cancer cells.
APC activity is inhibited before mitosis because its
premature activation would lead to genomic instability.
One way the APC is inhibited is by inhibiting the
cdk1/cyclin B complex required for entry into mitosis.
This complex is kept inactive before mitosis because it
is phosphorylated by the tyrosine kinase Wee1. Wee1
is degraded during the G2 phase and mitosis to tip the
balance to active cdk1 and allow mitotic entry to proceed. We analyzed Wee1 degradation in somatic cells
and found that 2 separate ubiquitin ligases containing
U
THE SCRIPPS RESEARCH INSTITUTE
23
β-transducin repeat–containing protein and trigger of
mitotic entry 1 are required for Wee1 destruction and
mitotic entry. These findings indicated that eukaryotic
cells have multiple means of regulating Wee1 degradation, because inactivation of a single critical component
would lead to premature mitosis and disastrous consequences for the organism. We are determining the
respective roles of these ligases in cancer progression.
PUBLICATIONS
Smith, A., Simanski, S., Fallahi, M., Ayad, N.G. Redundant ubiquitin ligase activities regulate Wee1 degradation and mitotic entry. Cell Cycle 6:2795, 2007.
Anatomy and Regulation of
Mouse Recombination Hot Spots
P.R. Bois, M. Fallahi-Sichani, I. Getun, Z.K. Wu
rossover events are necessary for meiosis progression and for genome reshuffling and diversity
before the generation of gametes. A peculiarity
of meiosis is the programmed nature of double-strand
breaks, which are induced by the conserved endonuclease Spo11. Studies in a variety of model systems
have shown that recombination occurs in distinct regions
termed hot spots. An estimated 5%–10% of the genomes
of higher eukaryotes are recombinogenic; the remainder reside in the “cold.” However, little is known about
the nature and mechanisms that control recombination
hot spots in mammalian genomes.
We have focused on identifying novel hot spots in the
mouse chromosome 19 by directly detecting crossover
events. Characterization of a highly polymorphic hot spot
(HS23.7) revealed quite unexpectedly that repair does
not have to be complete for meiosis to proceed. The persistence of these unrepaired heteroduplex regions at
crossover sites in mature spermatozoa promotes genome
instability that was revealed by the high diversity and
rearrangements observed in laboratory strains and wild
mouse populations. We are identifying and characterizing
more of these recombinogenic regions where chromosomes are reshuffled between generations and provide
the driving mechanism at the origin of diversity.
C
PUBLICATIONS
Bois, P.R. A highly polymorphic meiotic recombination mouse hot spot exhibits
incomplete repair. Mol. Cell. Biol. 27:7053, 2007.
24 CANCER BIOLOGY
2008
Molecular Mechanisms of
cAMP-Mediated Transcription
THE SCRIPPS RESEARCH INSTITUTE
novel compounds are also being used to explore the
biological effects of inhibiting inflammatory programs
in cancer cell proliferation and invasion and the role of
estrogen signaling in immune function.
M.D. Conkright, A.L. Amelio, N.E. Bruno
lucose homeostasis is maintained by coordinating glucose metabolism in skeletal muscle, lipid
storage in adipose tissue, and glucose production in the liver. Insulin and glucagon are central hormone regulators of glucose homeostasis. Glucagon
initiates the gluconeogenic program in hepatocytes by
activating the cAMP signaling pathway, whereas insulin
inhibits hepatic glucose output. Our recent detection
of a new component of the cAMP pathway, transducers
of regulated CREB (TORCs), established that cAMP signaling is more sophisticated than previously recognized
and provided new insights into glucose homeostasis.
Collectively, recent studies have indicated that insulin,
glucagon, and energy signals converge on TORC2
phosphorylation to modulate glucose output via CREBmediated expression of hepatic genes. However, the
specific nuclear actions of TORC2 are unknown. Thus,
the mechanisms involved in differentiating TORC2transmitted signals are of biological and clinical interest.
G
PUBLICATIONS
Altarejos, J.Y., Goebel, N., Conkright, M.D., Inoue, H., Xie, J., Arias, C.M., Sawchenko, P.E., Montminy, M. The Creb1 coactivator Crtc1 is required for energy balance and fertility. Nat. Med. 14:1112, 2008.
Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V.,
Orth, A.P., Busby, J., Hogenesch, J.B., Conkright M.D. A coactivator trap identifies NONO (p54nrb) as a new component of the cAMP signaling pathway. Proc.
Natl. Acad. Sci. U. S. A. 104:20314, 2007.
Structural and Molecular
Mechanisms of Nuclear
Receptor Signaling
K.W. Nettles, G. Gil, J. Nowak, M. Zhou
ur overall goal is to understand how small-molecule ligands control specific physiologic outcomes
through the chemical-structural interface of the
ligand with specific nuclear receptors.
We have developed new chemical probes for estrogen receptors that selectively suppress inflammatory
gene expression programs. We characterized a series
of these probes by using x-ray crystallography, revealing the structural features of signaling specificity. The
O
PUBLICATIONS
Bruning, J.B., Chalmers, M.J., Prasad, S., Busby, S.A., Kamenecka, T.M., He, Y.,
Nettles, K.W., Griffin, P.R. Partial agonists activate PPARγ using a helix 12 independent mechanism. Structure 15:1258, 2007.
Nettles, K.W., Bruning, J.B., Gil, G., Nowak, J., Sharma, S.K., Hahm, J.B., Kulp,
K., Hochberg, R.B., Zhou, H., Katzenellenbogen, J.A., Katzenellenbogen, B.S.,
Kim, Y., Joachmiak, A., Greene, G.L. NF-κB selectivity of estrogen receptor ligands
revealed by comparative crystallographic analyses [published correction appears in
Nat. Chem. Biol. 4:379, 2008]. Nat. Chem. Biol. 4:241, 2008.
Nettles, K.W., Gil, G., Nowak, J., Métivier, R., Sharma, V.B., Greene, G.L. CBP is
a dosage-dependent regulator of nuclear factor-κB suppression by the estrogen
receptor. Mol. Endocrinol. 22:263, 2008.
Chemistry
A chiral catalyst for enantioselective carbon-hydrogen activation. Work
done in the laboratory of Jin-Quan Yu, Ph.D., associate professor.
Jin-Quan Yu, Ph.D., Associate Professor
CHEMISTRY
DEPAR TMENT OF
CHEMISTRY
S TA F F
K.C. Nicolaou, Ph.D.*
Chairman
Aline W. and L.S. Skaggs
Professor of Chemical
Biology
Darlene Shiley Chair in
Chemistry
Dariush Ajami, Ph.D.
Assistant Professor of
Molecular Assembly
Phil S. Baran, Ph.D.
Professor
Dale L. Boger, Ph.D.*
Richard and Alice Cramer
Professor of Chemistry
Tobin J. Dickerson, Ph.D.
Assistant Professor
Albert Eschenmoser, Ph.D.*
Professor
Sheng Ding, Ph.D.
Associate Professor
M.G. Finn, Ph.D.*
Professor
Valery Fokin, Ph.D.
Associate Professor
M. Reza Ghadiri, Ph.D.*
Professor
William A. Greenberg, Ph.D.
Assistant Professor of
Chemistry
Inkyu Hwang, Ph.D.
Assistant Professor
Kim D. Janda, Ph.D.**
Professor
Ely R. Callaway, Jr., Chair
in Chemistry
Director, Worm Institute of
Research and Medicine
Gunnar Kaufmann, Ph.D.
Assistant Professor of
Chemistry
2008
Jeffery W. Kelly, Ph.D.*
Lita Annenberg Hazen
Professor of Chemistry
Ramanarayanan
Krishnamurthy, Ph.D.
Associate Professor
Lucas J. Leman, Ph.D.
Assistant Professor of
Chemistry
Richard A. Lerner, M.D.***
President, The Scripps
Research Institute
Lita Annenberg Hazen
Professor of
Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
Roy Periana, Ph.D.*****
Professor
Evan T. Powers, Ph.D.
Associate Professor of
Chemistry
Julius Rebek, Jr., Ph.D.*
Professor
Director, The Skaggs Institute
for Chemical Biology
Edward Roberts, Ph.D.
Professor
THE SCRIPPS RESEARCH INSTITUTE
(Andrew) Bin Zhou, Ph.D.
Assistant Professor of
Immunochemistry
79
Deboshri Banerjee, Ph.D.
Elizabeth Barrett, Ph.D.****
Roland Barth, Ph.D.*****
SENIOR SCIENTIST
Clay Bennett, Ph.D.
Luis Martinez, Ph.D.*****
Moritz Biskup, Ph.D. †
Universität Karlsruhe
Karlsruhe, Germany
S TA F F S C I E N T I S T S
Lisa Eubanks, Ph.D.
Rajesh Grover, Ph.D.
Sarah Hanson, Ph.D.
Lubica Supekova, Ph.D.
Wen Xiong, Ph.D.
Anthony Boitano, Ph.D. †
Genomics Institute of the
Novartis Foundation
San Diego, California
Laure Bouchez, Ph.D.
Kristopher Boyle, Ph.D.
Christopher Burke, Ph.D
SERVICE FACILITIES
Antonio Burtoloso, Ph.D. †
University of Sao Paulo
Sao Paulo, Brazil
Raj K. Chadha, Ph.D.
Director, X-ray
Crystallography Facility
Mark Bushey, Ph.D. †
Exxon, Inc.
Union City, New Jersey
Dee H. Huang, Ph.D.
Director, Nuclear Magnetic
Resonance Facility
Darren Bykowski, Ph.D.*****
Gary E. Siuzdak, Ph.D.
Senior Director, Mass
Spectrometry Facility
Katerina Capkova, Ph.D.
I N S T R U M E N TAT I O N /
Floyd E. Romesberg, Ph.D.
Associate Professor
Petr Capek, Ph.D.
Arani Chanda, Ph.D.
Ke Chen, Ph.D.
Peng Chen, Ph.D.
William Roush, Ph.D.*****
Professor
SENIOR RESEARCH
Peter G. Schultz, Ph.D.*
Professor
Scripps Family Chair
Suresh Pitram, Ph.D.
Govardhan Cherukupalli,
Ph.D. †
Epix Pharmaceuticals
Lexington, Massachusetts
R E S E A R C H A S S O C I AT E S
Jodie Chin, Ph.D.
Ramzey Abujarour, Ph.D.
Srinivas Reddy Chirapu, Ph.D.
Rajesh Ambasudhan, Ph.D.
Chandramouli Chiruta, Ph.D.
Manuel Amorin Lopez, Ph.D.
Dong-Gyu Cho, Ph.D.
Mark Ams, Ph.D.
So-Hye Cho, Ph.D.
Yoshio Ando, Ph.D.
Sungwook Choi, Ph.D.
Deepshikha Angrish, Ph.D.
Joyanta Choudhury, Ph.D.
Shinji Ashida, Ph.D.
Sarwat Chowdhury, Ph.D.
Micahel Baksh, Ph.D.
Stepan Chuprakov, Ph.D.
K. Barry Sharpless, Ph.D.*
W.M. Keck Professor of
Chemistry
Anita D. Wentworth, Ph.D.
Assistant Professor
Paul Wentworth, Jr., Ph.D.
Professor
Chi-Huey Wong, Ph.D.*
Professor of Chemistry
Jin-Quan Yu, Ph.D.
Associate Professor
A S S O C I AT E
80 CHEMISTRY
2008
THE SCRIPPS RESEARCH INSTITUTE
Petr Cigler, Ph.D.
David Edmonds, Ph.D.
Neil Grimster, Ph.D.
Giltae Hwang, Ph.D.
T. Ryan Cirz †
Achaogen
South San Francisco,
California
Jem Efe, Ph.D.
Rajesh K. Grover, Ph.D.
Jan Elsner, Ph.D. †
Celgene Pharmaceuticals
San Diego, California
Jan Grunewald, Ph.D.
Michael Jahnz, Ph.D. †
NOXXON Pharma AG
Berlin, Germany
Daniel Ess, Ph.D.
Richard Guy, Ph.D.****
Ph.D. †
Scott Cockroft,
University of Edinburgh
Edinburgh, Scotland
Ph.D. †
David Colby,
Purdue University
West Lafayette, Indiana
Kevin Cole, Ph.D. †
Eli Lilly and Company
Indianapolis, Indiana
Christine Crane, Ph.D.
Matthew Cremeens, Ph.D. †
Gonzaga University
Redmond, Washington
Fernando Rodrigo Pinacho
Crisostomo, Ph.D. †
Burnham Institute for
Medical Research
La Jolla, California
Cyrine Ezzili, Ph.D.
Xingang Fang, Ph.D.
Simon Ficht, Ph.D. †
Sanofi-Aventis Deutschland
GmbH
Frankfurt, Germany
Joseph Rodolph Fotsing,
Ph.D. †
Senomyx, Inc.
San Diego, California
Bozena Frackowiak, Ph.D. †
Politechnika Opolska
Opole, Poland
Etzer Darout,
Pfizer Inc.
Groton, Connecticut
Ph.D. †
Amy DeBaillie,
Eli Lilly and Company
Indianapolis, Indiana
Graeme Freestone,
Metabasis Therapeutics, Inc.
San Diego, California
Amelia Fuller, Ph.D. †
Santa Clara University
Santa Clara, California
Jianmin Gao, Ph.D. †
Boston College
Chestnut Hill, Massachusetts
Haibo Ge, Ph.D.
Judith Denery, Ph.D.
Ross Denton, Ph.D. †
University of Cambridge
Cambridge, England
Savvas Georgiades, Ph.D.****
Ola Ghoneim, Ph.D. †
Qatar University
Doha, Qatar
Caroline Desponts, Ph.D.
Antonia Di Mola, Ph.D.
Deguo Du, Ph.D.
Anna Dubrovska, Ph.D.
Viktoriya Dubrovskaya, Ph.D.
Joshua Dunetz, Ph.D. †
Pfizer Inc.
Groton, Connecticut
Kyle Eastman, Ph.D.
Yuanjun He, Ph.D.
Jason Hein, Ph.D.
Dube Henry, Ph.D.
Nathan Gianneschi, Ph.D. †
University of California
San Diego, California
Cristina Gil-Lamaignere,
Ph.D. †
University Hospital Nuestra
Señora de la Candelaria
Santa Cruz de Tenerife, Spain
Rodolfo Gonzalez, Ph.D.
Scott Grecian, Ph.D.
Rong Jiang, Ph.D.*****
Guo Jiantoa, Ph.D.
Hiroyuki Kakei, Ph.D. †
Takeda Pharmaceutical
Company Limited
Osaka, Japan
Jaroslaw Kalisiak, Ph.D.
Seiji Kamioka, Ph.D.
Moumita Kar, Ph.D.
Marcos Hernandez, Ph.D.
Kwang Mi Kim, Ph.D.
Par Holmberg, Ph.D. †
Memorial Sloan Kettering
Cancer Center
New York, New York
Wen-Xu Hong, Ph.D.
Yu Fu, Ph.D.
Ph.D. †
Masaki Handa, Ph.D. †
Sagami Chemical Research
Center
Ayase, Kanagawa, Japan
Ph.D. †
Jeffrey Culhane, Ph.D.
Stephen Dalby, Ph.D.
Tanja Gulder, Ph.D.
Zhangyong Hong, Ph.D.
Richard J. Hooley, Ph.D. †
University of California
Riverside, California
Tamara Hopkins, Ph.D. †
Boehringer Ingelheim
Pharmaceuticals, Inc.
Ridgefield, Connecticut
Allen Horhota, Ph.D.
Tony Horneff, Ph.D.
F. Scott Kimball, Ph.D.
Jeremy Kister, Ph.D.
Keith Korthals, Ph.D.
Larisa Krasnova, Ph.D.
Arkady Krasovskiy, Ph.D.
Luke Lairson, Ph.D.
Jae Wook Lee, Ph.D.
Jinq-Chyi Lee, Ph.D. †
National Health Research
Institutes
Miaoli County, Taiwan
Jong Seok Lee, Ph.D.
Claas Hovelmann, Ph.D.
Ki-Bum Lee, Ph.D. †
Rutgers University
Piscataway, New Jersey
Fang Hu, Ph.D. †
Department of Molecular
Biology, Scripps Research
Sejin Lee, Ph.D. †
SK Drug Development Center
Daejong, Korea
Xiaoyi Hu, Ph.D.
Alexandre Lemire, Ph.D.****
Zheng-Zheng Huang, Ph.D. †
DuPont Central Research
and Development
Wilmington, Delaware
Edward Lemke, Ph.D.
Ben Hutchins, Ph.D.
Chuang-Chuang Li, Ph.D. †
Peking University
Peking, China
Jun-Li Hou, Ph.D.
Der-ren Hwang, Ph.D. †
Academia Sinica
Taipei, Taiwan
Christophe Letondor,
Ph.D.****
Fangzheng Li, Ph.D.*****
CHEMISTRY
2008
Hongming Li, Ph.D. †
Schering-Plough
Kenilworth, New Jersey
Joonwoo Nam, Ph.D. †
CytRx Corporation
San Diego, California
Ke Li, Ph.D. †
DuPont Central Research
and Development
Wilmington, Delaware
Tae-Gyu Nam, Ph.D.
THE SCRIPPS RESEARCH INSTITUTE
Troy Ryba, Ph.D. †
Broad Institute of MIT and
Harvard
Cambridge, Massachusetts
Ph.D. †
Sebastian Steiniger, Ph.D.
Antonia Stepan, Ph.D.
James Stover, Ph.D.
Youngha Ryu,
Texas Christian University
Fort Worth, Texas
Bernhard Stump, Ph.D.
Catherine Saccavini, Ph.D.
Hui Kai Sun, Ph.D.*****
Severin Odermatt, Ph.D.****
Nicholas Salzameda, Ph.D.
Shinobu Takizawa, Ph.D.
Yeon-Hee Lim, Ph.D. †
Schering-Plough
Kenilworth, New Jersey
Christian Olsen, Ph.D.
Antonio Sanchez-Ruiz, Ph.D.
Yazmin Osornio, Ph.D.****
Yoshikazu Sasaki, Ph.D.
Tongxiang Lin, Ph.D.
Junguk Park, Ph.D.
Stefan Schiller, Ph.D.
Adam Talbot, Ph.D. †
Institute of Chemical and
Engineering Sciences
Jurong Island, Singapore
Troy Lister, Ph.D. †
Novartis
Cambridge, Massachusetts
Nitin Patil, Ph.D.
Niklas Schone, Ph.D.
Annie Tam, Ph.D.
Yefeng Tang, Ph.D.
Christopher Liu, Ph.D. †
Cubix Pharmaceuticals
Lexington, Massachusetts
Richard Payne, Ph.D. †
University of Sydney
Sydney, Australia
Michael Schramm, Ph.D. †
California State University
Long Beach, California
Wenshe Liu, Ph.D. †
Texas A&M University
College Station, Texas
Xuemei Peng, Ph.D.****
Edward Sessions, Jr., Ph.D.
Murali Peram Surakattula,
Ph.D. †
CytRx Corporation
San Diego, California
Shigeki Seto, Ph.D.
Pi-Hui Liang, Ph.D. †
Academia Sinica
Taipei, Taiwan
Michael Luzung, Ph.D.
Utpal Majumder, Ph.D.
Sreeman Mamidyala, Ph.D.
Andrew Nguyen, M.D., Ph.D.
Romain Noel, Ph.D.
George Nora, Ph.D.*****
Johan Paulsson, Ph.D.
Alexander Mayorov, Ph.D.
Charles Melancon, Ph.D.
Lionel Moisan, Ph.D. †
CEA
Gif-Sur-Yvette, France
Ana Montero, Ph.D.****
Miguel Morales, Ph.D.
Shun Su, Ph.D.
Mariola Tortosa, Ph.D. †
Instituto de Quimica Organica,
CSIC
Madrid, Spain
Craig Turner, Ph.D.****
Roshan Perera, Ph.D. †
University of Texas
Austin, Texas
Takeshi Masuda, Ph.D.
Michael Maue, Ph.D. †
Bayer CropScience AG
Monheim, Germany
Young Jun Seo, Ph.D.
81
Mary Jo Sever, Ph.D.
Alex Shaginian, Ph.D. †
Ardea Biosciences
San Diego, California
David Shaw, Ph.D.
Ramulu Poddutoori, Ph.D.
Weijun Shen, Ph.D.
Jonathan Pokorski, Ph.D.
Xiao Shengxiong, Ph.D.
Agustí Lledó Ponsati, Ph.D.
Bingfeng Shi, Ph.D.
Daniela Radu, Ph.D. †
DuPont Central Research
and Development
Wilmington, Delaware
Yan Shi, Ph.D.
Vincent Trepanier, Ph.D. †
Institute of Chemical and
Engineering Sciences
Jurong Island, Singapore
Jonathan Tripp, Ph.D.
Meng-Lin Tsao, Ph.D. †
University of California
Merced, California
Andrew Udit, Ph.D.
Hiroki Shigehisa, Ph.D.
Hiroyuki Shimamura, Ph.D.
Ronald Rahaim, Ph.D.*****
Siddhartha Shenoy, Ph.D.
Praveen Rao, Ph.D. †
University of Waterloo
Waterloo, Ontario, Canada
Matthew Tremblay, Ph.D.
Ryan Simkovsky, Ph.D.
Chinnappan Sivasankar,
Ph.D.****
Taiki Umezawa, Ph.D. †
Hokkaido University
Sapporo, Japan
Kenji Usui, Ph.D. †
Tokyo Institute of Technology
Tokyo, Japan
Carlos Valdez, Ph.D. †
Rigel Pharmaceuticals, Inc.
South San Francisco,
California
Adam Morgan, Ph.D. †
Concert Pharmaceuticals, Inc.
Lexington, Massachusetts
Per Restorp, Ph.D.
Ting-Wei Mu, Ph.D.
Jin-Kyu Rhee, Ph.D.
Michael Smolinski, Ph.D. †
Kinex Pharmaceuticals
Buffalo, New York
S. Vasudeva Naidu, Ph.D.
Fatima Rivas, Ph.D.
Xinyi Song, Ph.D.
Feng Wang, Ph.D.
Yuya Nakai, Ph.D.
Joshua Roth, Ph.D.*****
Simon Stamm, Ph.D.
Jian Wang, Ph.D.
Kimberly Reynolds, Ph.D.
Punna Venkateshwarlu, Ph.D.
82 CHEMISTRY
Jiangyun Wang, Ph.D. †
Institute of Biophysics
Beijing, China
Lin Wang, Ph.D.
Sheng-Kai Wang, Ph.D.
Weidong Wang, Ph.D.
Xisheng Wang, Ph.D.
Yajuan Wang, Ph.D.
Yuanhua Wang, Ph.D.
Timo Weide, Ph.D.
Albert Willis, Ph.D. †
Pharmagra Labs, Inc.
Brevard, North Carolina
Tao Wu, Ph.D. †
Institute of Chemical and
Engineering Sciences
Jurong Island, Singapore
Heiko Wurdak, Ph.D.
Jian Xie, Ph.D.
Wen Xiong, Ph.D.
Yue Xu, Ph.D.
Junichiro Yamaguchi, Ph.D.
Ryu Yamasaki, Ph.D. †
Tokyo University of Science
Tokyo, Japan
Ura Yasuyuki, Ph.D. †
Nara Women’s University
Nara, Japan
Yan Yin, Ph.D.
2008
Heyue Zhou, Ph.D.
Hongyan Zhou, Ph.D.
Shoutian Zhu, Ph.D.
Joerg Zimmermann, Ph.D.
V I S I T I N G I N V E S T I G AT O R S
Keisuke Fukuchi, Ph.D.
Sankyo Co., Ltd.
Tokyo, Japan
Christine Hernandez, Ph.D. †
University of Philippines
Diliman, Philippines
(Edmond) Shie-Liang Hsieh,
Ph.D.
National Yang-Ming University
Taipei, Taiwan
Masakazu Imamura, Ph.D. †
Astellas Pharma Inc.
Tsukuba, Ibaraki, Japan
Kuniyuki Kishikawa, Ph.D. †
Kyowa Hakko Kogyo Co., Ltd.
Sunto-gun, Shizuoka, Japan
Michael Meijler, Ph.D. †
Ben-Gurion University of the
Negev
Be’er Sheva, Israel
Takayoshi Suzuki, Ph.D. †
Nagoya City University
Nagoya, Japan
Yoshiyuki Yoneda, Ph.D. †
Daiichi Pharmaceutical Co.,
Ltd.
Tokyo, Japan
Ian Young, Ph.D.
S C I E N T I F I C A S S O C I AT E
Zhanqian Yu, Ph.D.
Xu Yuan, Ph.D.
Weiqiang Zhan, Ph.D.
Hongjun Zhang, Ph.D.
Xuejun Zhang, Ph.D.
Yanghui Zhang, Ph.D.
Yingchao Zhang, Ph.D. †
Hoffmann-La Roche, Inc.
Nutley, New Jersey
Jon Ashley
THE SCRIPPS RESEARCH INSTITUTE
* Joint appointment in The
Skaggs Institute for Chemical
Biology
** Joint appointments in The
Skaggs Institute for Chemical
Biology and the Department of
Immunology and Microbial
Science
*** Joint appointments in The
Skaggs Institute for Chemical
Biology and the Department of
Molecular Biology
**** Appointment completed
***** Scripps Florida
†
Appointment completed; new
location shown
CHEMISTRY
2008
Chairman’s Overview
s the “central science,” chemistry stands between
biology and medicine and between physics and
materials science and provides the crucial bridge
for drug discovery and
development. But chemistry has a much more
profound and useful role
in science and society. It
is the discipline that continually creates the myriad
of new materials that we
all encounter in our everyday lives: pharmaceuticals, high-tech materials,
polymers and plastics,
K.C. Nicolaou, Ph.D.
insecticides and pesticides,
fabrics and cosmetics, fertilizers, and vitamins—basically
everything we can touch, feel, and smell.
Chemistry at Scripps Research focuses on chemical
synthesis and chemical biology, the areas most relevant
to biomedical research and materials science. The members of our faculty are distinguished teacher-scholars who
maintain highly visible and independent research programs
in areas as diverse as biological and chemical catalysis,
synthesis of natural products, combinatorial chemistry,
molecular design, supramolecular chemistry, chemical
evolution, materials science, and chemical biology. The
chemistry graduate program attracts some of the bestqualified candidates from the United States and abroad.
Our major research facilities, under the direction of Dee
H. Huang (nuclear magnetic resonance), Gary Siuzdak
(mass spectrometry), and Raj Chadha (x-ray crystallography), are second to none and continue to provide crucial support to our research programs. In addition, the
Mabel and Arnold Beckman Center for the Chemical
Sciences constantly receives high praise from visitors
from around the world for its architectural design and
operational aspects, both highly conducive to research.
Research in the Department of Chemistry goes on
unabated, establishing international visibility and attracting attention, as evidenced by numerous lecture invitations, visits by outside scholars, and headline news in
the media. As of 2007, the Institute for Scientific Information ranked 2 members of the department as highly
cited researchers (in the top 100 worldwide).
Richard Lerner and his group continue their research
on antibodies, chemical synthesis, and the biological
A
THE SCRIPPS RESEARCH INSTITUTE
83
role of polyoxygen species. Scientists in Albert Eschenmoser’s group continue to work on the chemical etiology of nucleic acids and the origins of life.
Barry Sharpless and his group persist in their endeavors to discover and develop better catalysts for organic
synthesis and to construct, through innovative chemistry
and biology, libraries of novel compounds for biological
screening. Their click chemistry, which has had a major
impact in many areas of the molecular sciences, continues to be an important focus of their research.
Members of my own group continue to explore chemical synthesis and chemical biology, with a focus on the
total synthesis of new anticancer agents, antibiotics,
marine-derived neurotoxins, antimalarial compounds,
and other bioactive natural and designed molecules.
Julius Rebek and his group devise biomimetic receptors, including molecules that bind neurotransmitters and
membrane components, for studies in molecular recognition. Larger host receptors can surround 3 or more
molecular guests and act as chambers in which the
chemical reactions of the guests are accelerated. Scientists in the group also synthesize small molecules that act
as protein helix mimetics for pharmaceutical applications.
Peter Schultz and researchers in his laboratory are
expanding the number of genetically encoded amino acids
to include fluorescent, photocaged, metal-binding, chemically reactive, and posttranslationally modified amino
acids. These scientists have also adapted this technology to mammalian cells and are applying these tools in
basic and applied problems in cell biology. In addition,
members of the group have used cell-based screens to
identify small molecules that selectively differentiate
and expand embryonic and adult stem cells and reprogram lineage-committed cells, as well as novel genes
and small molecules that affect a number of physiologic
and disease processes.
Chi-Huey Wong and his group further advance the
fields of chemoenzymatic organic synthesis, chemical
glycobiology, and the development of enzyme inhibitors.
A new strategy for the synthesis of glycoproteins based
on sugar-assisted glycopeptide ligation has been developed.
The programmable 1-pot synthesis of oligosaccharides
developed by this group has been further used in the
assembly of glycoarrays for study of saccharides that
bind to proteins. Members of this group also developed
new probes to study posttranslational glycosylation and
identify glycoprotein biomarkers associated with cancer.
Researchers in Dale Boger’s laboratory continue their
work on chemical synthesis; combinatorial chemistry; het-
84 CHEMISTRY
2008
erocycle synthesis; anticancer agents, such as vinblastine,
cyclostatin, chlorofusion, and yatakemycin; and antibiotics,
such as vancomycin, teicoplanin, and ramoplanin.
Scientists in Kim Janda’s laboratory conduct research
grounded on organic chemistry as applied to specific biological systems. The targeted programs span a wide range
of interests, from immunopharmacotherapy to biological
and chemical warfare agents to filarial infections such
as “river blindness” to quorum sensing in bacteria and
new cancer therapeutic strategies. Recent achievements
include in vivo detection of botulinum neurotoxin antagonists, the development of peptides and antibodies as
drug delivery modules that home to cancer cells and
active vaccines for nicotine addiction and obesity that
are now in preclinical trials.
M. Reza Ghadiri and his group are making important
contributions in the design and study of a new generation
of antimicrobial agents, based on self-assembling peptide
nanotube architecture, to combat multidrug resistant
infections. In addition, members of the group continue
to make novel contributions in several ongoing basic
research endeavors, such as designing biosensors, developing molecular computation, designing self-reproducing
systems, understanding the origins of life, and creating
emergent chemical systems.
M.G. Finn and his group have pioneered the use of
virus particles as chemical reagents and building blocks
for nanochemical structures. This effort is directed toward
the development of new diagnostics for disease and catalysts for organic reactions. Members of Dr. Finn’s laboratory also develop and investigate new organic and
organometallic reactions and use these processes to
synthesize biologically active compounds.
Jeff Kelly and his group are exploring the interface
between the chemistry, biology, and pathobiology of
proteome maintenance. The aim of their projects is to
understand the physical and biological basis of protein
folding and the competitive misfolding and aggregation
processes that lead to age-associated neurodegenerative
diseases. Information on proteome maintenance is used
to develop new small-molecule therapeutic strategies for a
variety of diseases, including neurodegenerative diseases.
Anita Wentworth and the researchers in her group are
investigating the chemical basis of complex disease states
and are synthesizing peptide- and small molecule–based
therapeutic agents. These scientists focus on disease
states in which inflammatory and reactive oxygen species are prominent, such as atherosclerosis, Alzheimer’s
disease, and other diseases of ageing.
THE SCRIPPS RESEARCH INSTITUTE
Researchers in Floyd Romesberg’s laboratory are using
diverse techniques ranging from bioorganic and biophysical chemistry to bacterial and yeast genetics to understand and manipulate the process of evolution. Major
efforts include designing unnatural base pairs and using
directed evolution of DNA polymerases to efficiently synthesize unnatural DNA containing the base pairs, using
spectroscopy to understand biological function and how
it evolves, and understanding how induced and adaptive mutations contribute to evolution in eukaryotic and
prokaryotic cells.
Phil Baran and his group are interested in how the
general challenge of chemoselectivity in organic chemistry
can be answered through the auspices of total synthesis. He and his coworkers have developed extremely
concise chemical solutions to the synthetic challenges
posed by numerous families of natural products. These
syntheses systematically tackle the issue of chemoselectivity and are characterized by striking brevity, new
biosynthetic postulates, the invention of new methods,
and a minimum use or complete absence of protecting
groups and superfluous oxidation state manipulations.
The Frontiers in Chemistry Lecturers (19th Annual
Symposium) for the 2007–2008 academic year were
M. Christina White, University of Illinois; Ben L. Feringa,
University of Groningen, the Netherlands; Ian Paterson,
Cambridge University; and Harry Noller, University of
California, Santa Cruz. In addition, we enjoyed hosting
the following professors: Samir Zard, Ecole Polytechnique, France, as the Bristol-Myers Squibb Lecturer;
E.J. Corey, Harvard University, as the Pfizer Lecturer;
and Robert Bergman, University of California, Berkeley,
as the Novartis Lecturer.
CHEMISTRY
2008
THE SCRIPPS RESEARCH INSTITUTE
85
INVESTIGATOR’ S REPORT
Synthesis of Natural Products,
Development of Synthetic
Methods, and Medicinal
Chemistry
W.R. Roush, R. Bates, D. Bykowski, M. Chen, E. Darout,
A. DeBaillie, J. Dunetz, G. Halvorsen, M. Handa, J. Hicks,
T. Hopkins, C.-W. Huh, F. Li, A. Legg, R. Lira, L. Martinez,
C. Nguyen, G. Nora, R. Pragani, R. Rahaim, J. Roth,
H. Sun, M. Tortosa, J. Whitaker, S. Winbush
ur research has 2 major themes. One is the total
synthesis of structurally complex, biologically
active natural products such as those shown
in Figure 1. In each of these syntheses, we emphasize
the discovery, development, and/or illustration of new
reactions and methods for achieving high levels of stereochemical control. These efforts are pursued in parallel
with reaction design, stereochemical studies, and the
development of new synthetic methods. We are particularly interested in stereochemical aspects of intramolecular and transannular Diels-Alder reactions, development
of methods for the diastereoselective and enantioselective reactions of allylmetal compounds with carbonyl
compounds, and nucleophilic phosphine-catalyzed
organic reactions.
Recent research has included stereochemical studies
of transannular Diels-Alder reactions used in total syntheses of spinosyn A and superstolide A and development of new versions of the double allylboration reactions
of aldehydes with γ-boryl-substituted allylboranes for
stereocontrolled synthesis of 1,5-ene-diols, which are
being used in several ongoing syntheses, including those
of tetrafibricin, apoptolidin A, and peloruside. In addition,
we have synthesized highly substituted tetrahydrofurans
via [3+2]-annulation reactions of highly functionalized
allylsilanes; this chemistry was recently applied to total
syntheses of 10-hydroxytrilobacin and 3 stereoisomers.
We have also developed phosphine-mediated organocatalytic reactions, and we recently completed the total
synthesis of tedanolide.
Our second area of major interest involves problems
in bioorganic chemistry and medicinal chemistry. One
long-term project is the design and synthesis of inhibitors of cysteine proteases isolated from tropical para-
O
F i g . 1 . Structures of recently synthesized natural products.
sites, such as Trypanosoma cruzi, the causative 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. In collaboration with
S. Reed, University of California, San Diego, we have
developed a cysteine protease inhibitor with remarkable
86 CHEMISTRY
2008
ability to prevent Entamoeba histolytica from invading
human intestinal tissue. Optimization of this inhibitor
for in vivo applications is in progress. New projects
involve discovery of small molecules that affect cancer
and other disease-related biochemical targets (e.g.,
nuclear hormone receptors), studies of structure-activity
relationships, and optimization of the pharmacologic
profile of certain natural products.
PUBLICATIONS
Chen, Y.-T., Lira, R., Hansell, E., McKerrow, J.H., Roush, W.R. Synthesis of
macrocyclic trypanosomal cysteine protease inhibitors. Bioorg. Med. Chem. Lett.
18:5860, 2008.
Dunetz, J., Roush, W.R. Concerning the synthesis of the tedanolide C(13)-C(23)
fragment via an anti-aldol reaction. Org. Lett. 10:2059, 2008.
Handa, M., Scheidt, K.A., Bossart, M., Zheng, N., Roush, W.R. Studies on the
synthesis of apoptolidin A, I: synthesis of the C(1)-C(11) fragment. J. Org. Chem.
73:1031, 2008.
Handa, M., Smith, W.J. III, Roush, W.R. Studies on the synthesis of apoptolidin
A, II: synthesis of the disaccharide unit. J. Org. Chem. 73:1036, 2008.
Hicks, J.C., Huh, C.W., Legg, A.D., Roush, W.R. Concerning the selective protection of (Z)-1,4-syn-ene-diols and (E)-1,5-anti-ene-diols as allylic triethylsilyl ethers.
Org. Lett. 9:5621, 2007.
Hicks, J.D., Roush, W.R. Synthesis of the C(26)-C(42) and C(43)-C(67) pyrancontaining fragments of amphidinol 3 via a common pyran intermediate. Org. Lett.
10:681, 2008.
Lira, R., Roush, W.R. Enantio- and diastereoselective synthesis of syn-β-hydroxyallylsilanes via a chiral (Z)-γ-silylallylboronate. Org. Lett. 9:4315, 2007.
Methot, J.L., Roush, W.R. Applications of tricoordinated phosphorus compounds in
organic catalysis. In: Organophosphorus Compounds. Trost, B.M. (Ed.). Thieme
Chemistry, New York, in press. Vol. 42 in Science of Synthesis.
Roth, J., Madoux, F., Hodder, P., Roush, W.R. Synthesis of small molecule inhibitors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on isoquinolinone scaffolds. Bioorg. Med. Chem. Lett. 18:2628, 2008.
Roush, W.R. Total synthesis of biologically active natural products. J. Am. Chem.
Soc. 130:6654, 2008.
Tortosa, M., Yakelis, N.A., Roush, W.R. Total synthesis of (+)-superstolide A. J.
Am. Chem. Soc. 130:2722, 2008.
Winbush, S.M., Mergott, D.J., Roush, W.R. Total synthesis of (–)-spinosyn A:
examination of structural features that govern the stereoselectivity of the key
transannular Diels-Alder reaction. J. Org. Chem. 73:1818, 2008.
Scheinost, J.C., Wang, H., Boldt, G.E., Offer, J., Wentworth, P., Jr. Cholesterol secosterol-induced aggregation of methylated amyloid-β peptides, insights into aldehydeinitiated fibrillization of amyloid-β. Angew. Chem. Int. Ed. 47:3919, 2008.
Temperini, C., Cecchi, A., Boyle, N.A., Scozzafava, A., Cabeza, J.E., Wentworth,
P., Jr., Blackburn, G.M., Supuran, C.T. Carbonic anhydrase inhibitors. Interaction
of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methylsulfonamide with 12 mammalian isoforms: kinetic and x-ray crystallographic studies. Bioorg. Med. Chem. Lett.
18:999, 2008.
Wentworth, P., Jr., Witter, D. Antibody-catalyzed water-oxidation pathway. Pure
Appl. Chem. 80:1849, 2008.
THE SCRIPPS RESEARCH INSTITUTE
Infectology
Distinguishing between prion strains 22L and Me7
with the mouse bioassay takes 6 months. The cerebellar
Purkinje cell layer (immunostained for calbindin) remains
intact in Me7-infected, terminally sick mice (top, arrow)
but is obliterated by infection with 22L (bottom, arrow).
With the cell panel assay (insets), the strains can be
distinguished from each other in 2 weeks. 22L prions
efficiently infect the 4 cell lines constituting the panel,
whereas Me7 infects only LD9 and CAD cells. Stained
sections and micrographs prepared by Sukhvir Mahal,
Ph.D., staff scientist, and Alexsandra Sherman, research
assistant; photomontage created by Christopher A. Baker,
Ph.D., research associate. Work done in the laboratory
of Charles Weissmann, Ph.D.
BIOCHEMISTRY
Corinne Lasmézas, D.V.M., Ph.D., Professor, and
Paula Saá Prieto, Ph.D., Research Associate.
INFECTOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
195
DEPAR TMENT OF
INFECTOLOGY
S TA F F
Charles Weissmann, M.D.,
Ph.D.
Professor and Chairman
Joaquin Castilla, Ph.D.
Assistant Professor
S E N I O R S TA F F
R E S E A R C H A S S O C I AT E S
Yervand Karapetyan, M.D.
Ivan Angulo-Herrera, Ph.D.
Minghai Zhou, Ph.D.
Shawn Browning, Ph.D.
SCIENTIST
Corinne Lasmézas, D.V.M.,
Ph.D.
Professor
Sukhvir Mahal, Ph.D.
Natalia Fernández-Borges,
Ph.D.
Maria Herva-Moyana, Ph.D.
SENIOR RESEARCH
Donny Strosberg, Ph.D.
Professor
Tim Tellinghuisen, Ph.D.
Assistant Professor
A S S O C I AT E S
Paula Saá Prieto, Ph.D.
Carlos Coito, Ph.D.
Jiali Li, Ph.D.
Chris Baker, Ph.D.
Anja Oelschlegel, Ph.D.
Chairman’s Overview
he Department of Infectology focuses on prion
diseases and hepatitis. Tim Tellinghuisen and his
colleagues study hepatitis C virus (HCV) RNA
replication and virion
assembly. They identified
the viral protein NS5A as
an essential component
of the viral replicase and
mapped all amino acids
in domains II and III
essential for HCV RNA
replication. They identified regions whose functions are required for
Charles Weissmann, M.D., Ph.D.
generating infectious virus
but not for RNA replication, suggesting that NS5A
regulates the switch between RNA replication and virus
assembly. They are using high-throughput genetic screens
to identify host components required for replicase activity.
Donny Strosberg and his colleagues established for
the first time a cell culture system for HCV genotype 1b.
They identified peptides derived from the HCV core that
inhibit (1) dimerization of core protein, the first step in
viral assembly and (2) release of virions. F.V. Chisari and
his group, Department of Molecular and Experimental
Medicine, independently found that one such peptide
inhibits HCV replication. These findings establish the HCV
core as a target for the development of anti-HCV drugs.
Three groups, those of Corinne Lasmézas and Joaquin
Castilla and my own, study prion biology. Prions consist
T
of a multimeric assembly of PrPSc, a conformer of the
normal host protein PrPC. The seeding hypothesis posits
that prions replicate by recruiting host PrP C into the
PrPSc assembly, a process that entails conformational
rearrangement of the PrPC. Prions occur in the form of
different strains, all associated with the same PrP Sc
sequence but with distinct cell tropisms, both in brain
and in cell culture. The cell-based assay for prions has
been further streamlined by Emery Smith, and the cell
panel assay, which allows rapid distinction between prion
strains, was extended to more strains by Sukhvir Mahal.
It has been proposed that “strain-ness” is encoded either
by distinct conformations of PrP Sc or by the complex
glycans present on the protein. This hypothesis has been
negated by Shawn Browning and Dr. Mahal, who found
that strain specificity is maintained when prions are
propagated under conditions in which complex glycosylation is abrogated.
Dr. Lasmézas and her colleagues determined that
PrPSc in cultured prion-infected cells is undetectable at
the outer cell surface and is therefore likely generated
intracellularly. These investigators characterized a rapid
animal model for prion disease based on a transgenic,
PrP-overexpressing mouse strain and are researching the
mechanism of pathogenesis. Dr. Castilla and his group
have perfected the protein misfolding cyclic amplification
procedure, which allows the cell-free replication of prions,
and have shown that various prion strains can be propagated continuously without losing strain-specific properties.
196 INFECTOLOGY
2008
Investigators’ Reports
Biology of Prion Strains
THE SCRIPPS RESEARCH INSTITUTE
Table 1.
Susceptibility of cell lines to various prion strains.*
Cell line
Prion strain
22L
139A
RML, 79A
Me7
301C
LD9 (EMEM)
+++
+++
+++
+++
–
CAD5*
+++
+++
+++
–
+++
PK1
+++
+++
+++
–
–
PK1/swa
+++
++
–
–
–
++
–
–
–
–
C. Weissmann, C.A. Baker, S. Browning, C. Demczyk,
M. Herva-Moyana, J. Li, S.P. Mahal, A. Oelschlegel,
A. Sherman, E. Smith, I. Suponitsky-Kroyter
rions are thought to consist mainly or entirely
of PrP Sc , an abnormal conformer of a normal
host protein, PrPC, and to propagate via a PrPSccatalyzed conversion of PrPC. Intriguingly, distinct prion
strains, which generate different disease phenotypes,
are associated with the same PrP sequence, suggesting that the phenotypes are encoded posttranslationally. Our major interests are the mechanism of prion
replication, the structural basis of strain specificity,
and the mechanism of strain recognition by cells.
Much of our research depends on a cell-based assay
for prion infectivity, the standard scrapie cell assay.
Therefore, considerable effort has gone into automating the procedure, reducing its cost, and improving its
accuracy. We can now assay about 1000 samples per
week in sextuplicate, orders of magnitude greater than
the number that could be processed by using the classical mouse bioassay. The standard scrapie cell assay is
the basis for the cell panel assay (CPA), which makes
use of the finding that different cell lines have distinctive
susceptibilities to various prion strains. Using the CPA,
we can distinguish between various prion strains within
2 weeks, as opposed to the year or more required with
the classical mouse bioassay. The panel originally consisted of 4 cell lines; however, when exposed to swainsonine (an α-mannosidase II inhibitor that modifies the
glycans of N-glycosylated proteins), the PK1 cell line
(but none of the others) becomes resistant to some prion
strains but not to others. Therefore, swainsonine-treated
PK1 cells provide an additional criterion for discriminating prion strains. We can currently distinguish 5 groups
of strains (Table 1).
We determined that the characteristic CPA responses
of these strains are the same whether the strains are
propagated in wild-type mice or in Tga20 mice, which
overexpress PrP C and, conveniently, have a reduced
incubation time.
We have established a large collection of cell lines
chronically infected with various prion strains. Interestingly, in many instances, the cell tropism of prions
derived from such lines, as determined by using the
P
R33
* The number of pluses indicates the degree of susceptibility to infection. The
minus sign indicates resistance to infection.
CPA, differs from that of the prions derived from brain.
To determine whether this change reflects a permanent
modification of a strain, we inoculated cell-derived prions
into mice; CPA analysis of the infected brains showed
no differences between the original and the cell-passaged strains. We are currently considering the possibility that the cell tropism of a prion strain may depend on
the host cell in which the strain is generated, reflecting
some host-imparted property (the “cytotype”), for example, the glycosylation pattern of the PrP.
It has been proposed that the N-linked complex
glycans attached to PrP might encode the “strain-ness”
of prions. To address this question, we propagated RML,
22L, and Me7 prions in CAD5 cells in the presence of
the glycosylation inhibitors deoxymannojirimycin and
swainsonine. These inhibitors prevent processing of the
precursor of N-linked glycans and result in PrP with
[mannose]9[N-acetylglucosamine]2 as the only glycan,
rather than a multiplicity of complex sugars. We injected
cell lysates of the drug-treated, prion-infected cells into
mice and used the CPA to analyze the infected brains.
In all instances, the strain-specific properties had been
retained, proving that complex glycosylation was not
the strain-determining element and adding weight to
the hypothesis that strain-ness is encoded by the conformation of PrPSc.
In another project, we are exploring why certain
subclones derived from the same cell line, occurring
with a frequency of 1 in 5000 to 1 in 10,000, can
vary by 100 to 1000 times in their susceptibility to
prions. We tagged highly susceptible and resistant
subclones with 2 different markers of antibiotic resistance and fused the cells. Currently, we are determining
whether susceptibility, as measured in heterokaryons,
INFECTOLOGY
2008
is a dominant or a recessive property. In the next step,
we will generate microcells containing single, tagged
chromosomes from one of the cell lines and fuse the
microcells to cells from the other line to determine which
chromosome encodes the critical trait.
PUBLICATIONS
Julius, C., Hutter, G., Wagner, U., Seeger, H., Kana, V., Kranich, J., Klöhn, P.,
Weissmann, C., Miele, G., Aguzzi, A. Transcriptional stability of cultured cells
upon prion infection. J. Mol. Biol. 375:1222, 2008.
Mahal, S.P., Baker, C.A., Demczyk, C.A., Smith, E.W. Julius, C., Weissmann, C.
Prion strain discrimination in cell culture: the cell panel assay. Proc. Natl. Acad.
Sci. U. S. A. 104:20908, 2007.
Mahal, S.P., Demczyk, C.A., Smith, E.W., Jr., Klöhn, P.-C., Weissmann, C. Assaying prions in cell culture: the standard scrapie cell assay (SSCA) and the scrapie
cell assay in end point format (SCEPA). Methods Mol. Biol. 459:49, 2008.
Pathogenesis of Transmissible
Spongiform Encephalopathies
C.I. Lasmézas, N. Salès, P. Saá Prieto, M. Zhou,
Y. Karapetyan, F. Sferrazza, G. Ottenberg
rions, the transmissible agents responsible for
prion diseases, are thought to consist mainly of
PrPSc, an abnormally folded isoform of the ubiquitous prion protein PrP. Prions are thought to replicate
by an autocatalytic process of template-induced conformational change.
In collaboration with C. Weissmann, Department of
Infectology, we are devising a new method for propagating and characterizing different strains of prions.
Dr. Weissmann and his group have developed a cellbased infectivity assay, the cell panel assay, in which
prion strains are distinguished on the basis of cell tropism. We have studied the fate of prions in the Tga20
mouse model. Compared with wild-type mice, Tga20
mice express 8-fold higher levels of PrP C in the brain,
and clinical disease occurs more quickly after inoculation of prions.
Many aspects of prion replication in Tga20 mice
were unknown, for instance, the response of these animals to different prion strains. We found that PrPC from
the brains of Tga20 mice has a higher intrinsic resistance to proteolytic digestion by protease than does
PrPC from the brains of wild-type mice. We also discovered that the levels of PrPC overexpression in different
regions of the brain vary and hence could influence the
pathologic lesions and PrPSc distribution that occur after
prion infection. Vacuolation and PrPSc deposition pro-
P
THE SCRIPPS RESEARCH INSTITUTE
197
files in brains of Tga20 mice differed from those in
C57BL/6 mice for the 3 scrapie strain studied. Each
strain had a characteristic profile in Tga20 mice and
could be readily distinguished by neuropathologic analysis, showing that the level of PrPC expression is not the
main determinant of brain tropism. Importantly, despite
generating different neuropathologic phenotypes in
C57BL/6 and Tga20 mice, all 3 prion strains retained
their intrinsic identity after being replicated in either
mouse line, as determined by the cell panel assay.
This study provides a new reliable method for rapid
propagation and characterization of prion strains by
using a combination of Tga20 mice and the cell panel
assay. Fundamentally, our results indicate that despite
different biochemical characteristics of PrPC, different
expression patterns of PrPC in the 2 murine hosts, and
different genetic background of the 2 mouse strains, prions are propagated faithfully in both types of mice. This
finding raises once more the question of the molecular
basis of prion-strain properties.
Previously, we found that oligomeric assemblies of
recombinant prion protein are toxic to primary cultures
of cortical neurons and in mice when injected via a
stereotaxic method. We are now devising new intervention strategies to block the toxic/infectious PrP species.
A first goal is to determine the cellular location of PrP
aggregates, in order to know which cellular compartment to target. The many reports of the presence of
protease-resistant PrP (PrPres) at the cell membrane
are contradictory. Using a cell-surface biotinylation
strategy and comprehensive controls to account for
the presence of dead cells and for the intrinsic capacity of PrPres to be biotinylated, we found no detectable
PrPres at the cell membrane. This finding has major
implications for the development of diagnostic molecules. We are continuing our efforts to locate the cellular compartment that must be targeted for therapeutic
purposes, and we are setting up new models for PrPinduced toxic effects in cell lines.
Inhibitors of Protein-Protein
Interactions in Hepatitis C
A.D. Strosberg, C. Coito, R. Henderson, S. Kota, G. Mousseau
S
everal small-molecule drugs are in advanced
clinical development for the treatment of hepatitis C, a situation that may affect the 170 mil-
198 INFECTOLOGY
2008
lion carriers of hepatitis C virus (HCV) worldwide, including more than 3 million in the United States. Because
of its high mutability, HCV likely will become resistant
to these potential drugs, which are mostly inhibitors of
a viral protease and the viral polymerase. This past year,
we continued our studies of protein interactions involving HCV proteins to better understand the respective
roles of the proteins and to identify novel target proteins that would not induce resistance. The core HCV
protein, which is highly conserved across all 6 major
HCV genotypes, is a good candidate for such studies.
H C V C O R E P R O T E I N A S A TA R G E T
The HCV core protein functions primarily as the
structural element of the virus. The core contains several
residues essential for HCV production. Most of these residues are located in the N-terminal two-thirds of the core
protein and mediate core dimerization and most interactions with intracellular proteins. Core dimerization is the
first step in nucleocapsid assembly; its inhibition should
block formation and release of infectious HCV particles.
THE SCRIPPS RESEARCH INSTITUTE
tive of SL-175 bound to core106 with a dissociation
constant of 1.9 µM and was displaced by the uncoupled
peptide, with a 50% inhibitory concentration of 18.7
µM. In a collaborative surface plasmon resonance study
with J.-P. Lavergne, Centre National de la Recherche
Scientifique, Lyon, France, SL-175 bound core169 with
a dissociation constant of 7.2 µM.
PEPTIDE INHIBITORS OF HCV RELEASE FROM
H E PAT O M A C E L L S
When added to Huh-7.5 hepatoma cells infected
with the HCV genotype 2a J6/JFH-1 or the 1b CG strain,
peptides SL-173 and SL-175 prevented release of newly
formed infectious HCV into the medium. Using a different approach, F.V. Chisari and his group, Department
of Molecular and Experimental Medicine, independently
found that SL-173 inhibits HCV focus formation in vitro
by more than 90% and viral RNA synthesis 11-fold, 72
hours after infection. The combined results of our 2
groups thus establish that the HCV core is a useful novel
target for development of anti-HCV drugs.
H E PAT O M A C E L L C U LT U R E S Y S T E M F O R H C V
A S S AY S F O R H C V C O R E D I M E R I Z AT I O N
Using a pair of domains that consist of the first
106 residues of the HCV core protein (core106) tagged
with oligonucleotides encoding the octapeptide Flag or
glutathione-S-transferase (GST), we developed an assay
based on the use of an antibody to Flag that binds to
Flag-tagged core106 interacting with GST-tagged core106
adsorbed on a glutathione-coated microtiter plate. We
designed a 384 well–based sensitive and high-throughput time-resolved fluorescence assay with fluorescent
antibodies to Flag and GST. Untagged core106 completely inhibits core106 dimerization. One of our industrial partners will use this assay to screen 2 million
chemically diversified small molecules to identify novel
nonpeptidic inhibitors of HCV production.
P E P T I D E I N H I B I T O R S O F C O R E D I M E R I Z AT I O N
We also designed an amplified luminescent proximity homogeneous assay to monitor core-core interactions on the basis of donor and acceptor beads that
respond to the specific tags on the proteins. Using this
assay, which has a high signal-to-background ratio, we
screened 14 18-residue-long peptides derived from the
HCV core and identified 2 partially overlapping peptides,
SL-173 and SL-174, which caused 68% and 63% inhibition, respectively, of core106 dimerization.
SL-175, a 3-residue shorter version of SL-173,
inhibited core106 dimerization by 50%, with a 50%
inhibitory concentration of 22 µM. Using fluorescence
polarization, we found that a fluorophore-coupled deriva-
GENOTYPE 1B
The culture system for HCV used routinely in laboratories worldwide was derived from strain JFH-1 of
an HCV of genotype 2a. In Western countries, however,
the most prevalent infections are caused by HCV strains
of genotype 1. To understand differences between HCV
of different genotypes and further evaluate potential
inhibitors of protein-protein interactions in HCV or
between HCV and human host proteins, we developed
and characterized a unique culture system for the HCV
genotype 1b CG strain. Two different protocols have been
developed: the first one is based on the coculture of
infected, virus-releasing cells with uninfected cells in
a 2-chamber system; the second is a direct incubation
of uninfected cells with supernatant from cells electroporated with the RNA from strain CG or from infected
cells. Using either system, we have confirmed transfer
of infectivity in several passages. Furthermore, we showed
that cyclosporine A has comparable inhibitory effects
on J6/JFH-1 and CG strains of HCV and that antibodies
to CD81, a coreceptor for HCV, block infectious particles
released into the supernatant of cells infected by the
1b CG strain. Initial results also suggest that the 2
strains are diversely affected by IFN-α; J6/JFH-1 appears
to be sensitive, and CG appears to be resistant. This
finding, if confirmed, would reflect the situation in
patients; patients infected with genotype 2 HCV generally respond better to interferon treatment than do
patients infected with genotype 1 HCV.
INFECTOLOGY
2008
PUBLICATIONS
Strosberg, A.D., Nahmias, C. G-protein-coupled receptor signaling through protein
networks. Biochem. Soc. Trans. 35:23, 2007.
Hepatitis C Virus RNA
Replication and Virion Assembly
T.L. Tellinghuisen, J.C. Treadaway, K.L. Foss,
THE SCRIPPS RESEARCH INSTITUTE
199
work. We have also begun applying high-throughput
genetic screens to identify required host components
of the replicase.
Our ultimate goal is to understand, at the molecular level, the assembly, activity, and regulation of the
HCV RNA replication machinery. Greater insight into
the poorly understood replicase components, such as
NS5A, will provide a more complete view of the replicase complex and will fuel the design of new drugs.
I. Angulo-Herrera
epatitis C virus (HCV) is a human pathogen of
global importance; according to some estimates,
nearly 3% of the world’s population are chronically infected. Long-term viral replication in these individuals leads to severe liver disease, including cirrhosis
and, often, hepatocellular carcinoma. The current treatment with agents nonspecific for HCV is poorly tolerated
and is ineffective in about half of the patients, emphasizing the need for effective antiviral drugs specific for
the virus.
The HCV replicase, the multicomponent machine
that replicates the viral RNA, is an ideal drug target.
The core replicase consists of 5 HCV proteins associated with well-characterized polymerase, protease, and
helicase activities. Some HCV replicase proteins, such
as NS5A, are essential for HCV replication; however,
their specific functions remain enigmatic.
We have been characterizing NS5A. Our goal is to
understand the role of this protein in replication and,
more generally, the replicase itself. We have defined
NS5A as an essential, 3-domain metalloprotein component of the replicase. Our crystal structure of domain I
of NS5A has provided a glimpse of the potential interactions of NS5A in the viral replicase.
We recently identified all of the amino acids in the
poorly understood domains II and III that are required
for HCV RNA replication. Additionally, we have discovered an interaction between the membrane anchor of
NS5A and the protein NS4B, another component of the
replicase. This interaction appears to localize NS5A to
the replicase and is essential for RNA replication. We
are identifying regions of NS5A whose functions are
required for the production of infectious virus but not
for RNA replication. Our findings suggest that NS5A
may function as a regulator of the switch between RNA
replication and virus production. We are conducting
biochemical, genetic, and structural experiments to
evaluate the potential interaction surfaces and activities
of NS5A observed in our previous structural and genetic
H
PUBLICATIONS
Lindenbach, B.D., Tellinghuisen, T.L. Insights into hepatitis C virus RNA replication. In: Viral Genome Replication. Götte, M., Cameron, C., Raney, K. (Eds.).
Springer, New York, in press.
Tellinghuisen, T.L., Evans, M.J., Von Hahn, T., You, S., Rice, C.M. Studying hepatitis C virus: making the best of a bad virus. J. Virol. 81:8853, 2007.
Tellinghuisen, T.L., Foss, K.L., Treadaway, J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog. 4:e1000032, 2008.
Tellinghuisen, T.L., Foss, K.L., Treadaway, J.C., Rice, C.M. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus
NS5A protein. J. Virol. 82:1073, 2008.
Tellinghuisen, T.L., Lindenbach, B.D. Reverse transcription PCR based sequence
analysis of hepatitis C virus replicon RNA. In: Hepatitis C: Methods and Protocols,
2nd ed. Tang, J. (Ed.). Humana Press, Totowa, NJ, in press. Vol. 510 in Methods
in Molecular Biology. Walker, J. (Series Ed.).
Tellinghuisen, T.L., Marcotrigiano, J. Preparation of hepatitis C virus NS5A protein
for structural studies. In: Hepatitis C: Methods and Protocols, 2nd ed. Tang, J. (Ed.).
Humana Press, Totowa, NJ, in press. Vol. 510 in Methods in Molecular Biology.
Walker, J. (Series Ed.).
Strain and Species Barrier
Phenomena in a Cell-Free System
J. Castilla, N. Fernández-Borges, J. de Castro
ransmissible spongiform encephalopathies are
fatal neurodegenerative disorders that affect both
humans and animals. The disorders can be classified as genetic, sporadic (putatively spontaneous), or
infectious. The infectious agent associated with these
encephalopathies, the prion, appears to consist of the
single protein PrPSc, an abnormal conformer of the natural host protein PrPC. Prions propagate by converting
host PrPC into PrPSc.
One characteristic of prions is their ability to infect
some species and not others. This phenomenon is known
as the transmission barrier. Interestingly, prions occur
in the form of different strains with distinct biological
and physicochemical properties, even though all the
strains have the same PrP amino acid sequence, albeit
in presumably different conformations. In general, the
T
200 INFECTOLOGY
2008
transmission barrier is manifested as an incomplete
attack rate (i.e., the percentage of animals in a group
in which disease develops after inoculation with prions
is less than 100) and long incubation times (time from
inoculation to the onset of the clinical signs of disease), which become shorter after serial passages of
the prion strain in animals. Compelling evidence indicates that the transmission barriers are closely related
to differences in PrP amino acid sequences between
the donor and recipients of the infectious prions and
the prion strain conformation.
Unfortunately, the molecular basis of the transmission barrier and its relationship to prion-strain conformations are currently unknown, and we cannot predict
the degree of a species barrier simply by comparing
the prion proteins from 2 species. We have conducted
a series of experiments in which we used protein misfolding cyclic amplification, a technique that mimics
in vitro some of the fundamental steps involved in prion
replication in vivo, albeit with accelerated kinetics. The
in vitro generated prions have key prion features: they
are infectious in vivo and maintain their strain specificity.
We have used this technique to efficiently replicate a
variety of prion strains from, among others, mice, hamsters, bank voles, deer, cattle, sheep, and humans. The
correlation between in vivo data and our in vitro results
suggests that protein misfolding cyclic amplification is
a valuable tool for assessing the strength of the transmission barriers between diverse species and for different prion strains. We are using the method to determine
which amino acids in the PrPC sequence contribute to
the strength of the transmission barrier.
These studies are useful in evaluating the potential
risks to humans and animals not only of established
prion strains but also of new (atypical) strains. For
example, although the prion strain that causes classical sheep scrapie cannot cross the human transmission
barrier in vitro, the strain that causes bovine spongiform encephalopathy can cross the human transmission
barrier efficiently after propagation in sheep. In addition, we have generated prions that are infectious to
species hitherto considered resistant to prion diseases.
PUBLICATIONS
Hetz, C., Lee, A.H., González-Romero, D., Thielen, P., Castilla, J., Soto, C., Glimcher, L.H. Unfolded protein response transcription factor XBP-1 does not influence
prion replication or pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 105:757, 2008.
Morales, R., González, D., Soto, C., Castilla, J. Advances in prion detection. In:
Microbial Food Contamination. Wilson, C.L. (Ed.). CRC Press, Boca Raton, FL,
2007, p. 255.
THE SCRIPPS RESEARCH INSTITUTE
Molecular and
Integrative Neurosciences
A, Circadian rhythm profile for
wild-type (WT) littermates and
EP3R–/– mice. Although diurnal
distribution of motor activity follows the light-dark cycle, EP3R–/–
mice have bouts of increased
activity during the light cycle that
are associated with grooming and
eating behavior. Those bouts of
activity are irregular and are better detected during the resting
phase (see carets). B, Continuous recording of core body temperature (CBT) and motor activity (MA) during 5 days at normothermic conditions (room temperature, 30°C) confirms that EP3R–/– and WT mice are
nocturnal and that they follow the low activity–high resting (light cycle) and high activity–low resting (dark
cycle) pattern. C, Averaged data indicate that EP3R–/– mice have an increase in motor activity characterized by bouts of activity that increases the core body temperature (see arrows). The increase in motor
activity is associated with grooming and eating behavior. D, Cumulative data confirm that EP3R–/– mice
are more active during the light period. *P = .03. Work done in the laboratory of Tamas Bartfai, Ph.D.,
professor. Reprinted from Sánchez-Alavez, M., Klein, I., Brownell, S.E., et al. Night eating and obesity in
the EP3R-deficient mouse. Proc. Natl. Acad. Sci. U. S. A. 104:3009, 2007. Copyright 2007 National
Academy of Sciences U.S.A.
Cindy Ehlers, Ph.D., Professor, Gina Stouffer, Research
Assistant, and José Criado, Jr., Ph.D., Staff Scientist
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES
MOLECULAR AND
I N T E G R AT I V E
NEUROSCIENCES
DEPAR TMENT
S TA F F
Tamas Bartfai, Ph.D.
Chairman and Professor
Director, Harold L. Dorris
Neurological Research
Institute
Serge Ahmed, Ph.D.
Adjunct Assistant Professor
Etienne Baulieu, Ph.D.
Adjunct Professor
Floyd Bloom, M.D.
Professor Emeritus
Executive Director, Science
Communication
2008
THE SCRIPPS RESEARCH INSTITUTE
Steven J. Henriksen, Ph.D.
Adjunct Professor
Amanda Roberts, Ph.D.
Associate Professor
Paul L. Herrling, Ph.D.
Adjunct Professor
Michael G. Rosenfeld, M.D.
Adjunct Professor
Tomas Hokfelt, M.D., Ph.D.
Adjunct Professor
Pietro P. Sanna, M.D.
Associate Professor
Danny Hoyer, Ph.D.
Adjunct Professor
George R. Siggins, Ph.D.
Professor
Koki Inoue, Ph.D.
Adjunct Associate Professor
Iustin Tabarean, Ph.D.
Assistant Professor
Harvey Karten, M.D.
Adjunct Professor
Antoine Tabarin, Ph.D.
Adjunct Associate Professor
Henri Korn, M.D., Ph.D.
Adjunct Professor
Lars Terenius, Ph.D.
Adjunct Professor
Thomas Krucker, Ph.D.
Adjunct Assistant Professor
Claes Wahlestedt, M.D.,
Ph.D.*
Professor
Stefan Kunz, Ph.D.
Adjunct Professor
Mehrdad Alirezaei, Ph.D.
Michal Bajo, M.D., Ph.D.
Hilda Bajova, D.V.M.
Fulvia Berton, Ph.D.
Vez Repunte Canonigo, Ph.D.
Kazuki Hagihara, Ph.D.
Izabella Klein, Ph.D.
Kayo Mitsukawa, Ph.D.
Olivia Osborn, Ph.D.
Covadonga Paneda, Ph.D.
Gurudutt Pendyala, Ph.D.
Jerry Pinghwa Pian, Ph.D.
Tammy Wall, Ph.D.
Adjunct Associate Professor
Jilla Sabeti, Ph.D.
Friedbert Weiss, Ph.D.
Professor
V I S I T I N G I N V E S T I G AT O R S
Cary Lai, Ph.D.
Associate Professor
Karen T. Britton, M.D., Ph.D.
Adjunct Associate Professor
Ulo Langel, Ph.D.
Adjunct Professor
Michael Buchmeier, Ph.D.
Adjunct Professor
Xiaoying Lu, Ph.D.
Assistant Professor
Iain L. Campbell, Ph.D.
Adjunct Professor
Jan O. Lundstrom, Ph.D.
Adjunct Professor
Zhen Chai, Ph.D.
Adjunct Assistant Professor
Athina Markou, Ph.D.
Adjunct Professor
Jerold Chun, M.D., Ph.D.
Adjunct Professor
Madis Metsis, Ph.D.
Adjunct Associate Professor
Bruno Conti, Ph.D.
Associate Professor
Benjamin Neuman, Ph.D.
Adjunct Assistant Professor
Cindy L. Ehlers, Ph.D.
Professor
Shirley M. Otis, M.D.
Adjunct Professor
Ralph Feuer, Ph.D.
Adjunct Assistant Professor
Tommy Phillips, Ph.D.
Adjunct Assistant Professor
Howard S. Fox, M.D., Ph.D.
Associate Professor
John Polich, Ph.D.
Associate Professor
Brendan Walker, Ph.D.
Hermann H. Gram, Ph.D.
Adjunct Associate Professor
Luigi Pulvirenti, M.D.
Adjunct Associate Professor
S C I E N C E A S S O C I AT E S
S TA F F S C I E N T I S T S
Roberto Ciccocioppo, Ph.D.
José Criado, Ph.D.
Walter Francesconi, Ph.D.
David Gilder, M.D.
Salvador Huitrón-Reséndiz,
Ph.D.
M. Cecilia Marcondes, Ph.D.
Teresa Reyes, Ph.D.
Adjunct Assistant Professor
R E S E A R C H A S S O C I AT E S
Zhifeng Chen, Ph.D.
Jason Botten, Ph.D.
Assistant Professor
Donna L. Gruol, Ph.D.
Associate Professor
327
Remi Martin-Fardon, Ph.D.
Tom Nelson, Ph.D.
Manuel Sánchez-Alavaz,
M.D., Ph.D.
Mitra Rebek, Ph.D.
Caroline Lanigan, Ph.D.
Sam Madamba
Hedieh Badie, Ph.D.
Genomics Institute of the
Novartis Research
Foundation
San Diego, California
Persephone Borrow, Ph.D.
Edward Jenner Institute for
Vaccine Research
Compton, England
Urs Christen, Ph.D.
La Jolla Institute for Allergy
and Immunology
La Jolla, California
Jean E. Gairin, Ph.D.
CNRS
Toulouse, France
Karine Guillem, Ph.D.
University of Pennsylvania
Philadelphia, Pennsylvania
Katsuro Hagiwara, Ph.D.
Rakuno Gakuen University
Ebetsu, Japan
Dirk Homann, M.D., Ph.D.
University of Colorado
Health Sciences Center
Denver, Colorado
328 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES
Shinchi Iwasaki, M.D., Ph.D.
Osaka City University
Medical School
Osaka, Japan
Rolf Kiessling, Ph.D.
Karolinska Institutet
Stockholm, Sweden
Denise Naniche, Ph.D.,
M.P.H.
Universitat de Barcelona
Barcelona, Spain
Noemi Sevilla, Ph.D.
Universidad Autonoma de
Madrid
Madrid, Spain
Christina Spiropoulou, Ph.D.
Centers for Disease Control
and Prevention
Atlanta, Georgia
Elina Zuniga, Ph.D.
University of California
San Diego, California
* Scripps Florida
2008
THE SCRIPPS RESEARCH INSTITUTE
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES
Chairman’s Overview
I
n the past year, we experienced scientific successes
as well as organizational and policy changes in
the Molecular and Integrative Neurosciences
Department. The scientific work of several faculty members
resulted in high-significance, high-visibility publications
and important new
research grants and
renewals of earlier
grants from the National
Institutes of Health.
Tamas Bartfai, Ph.D.
Particularly noteworthy because of their immediate clinical usefulness
are the findings of George Siggins and his collaborators in the Committee on the Neurobiology of
Addictive Disorders that the widely used antiepileptic compound gabapentin may be useful in
treating alcohol addiction. Pietro Sanna published
important findings on the molecular mechanisms
of alcohol-induced adaptation of nerve cells. Friedbert
Weiss expanded our knowledge of the pharmacologic potential of the subtype-selective antagonists
that can block the endogenous anxiogenic stress
signal corticotropin-releasing factor. Research by
Cindy Ehlers in pharmacogenomics led to new conclusions about the genetic basis of vulnerability of
Native Americans to alcohol addiction, and Donna
Gruol added new data on the effects of the proinflammatory cytokine IL-6 in the brain. John Polich
expanded his noninvasive studies on the human brain
by using attentional tasks.
Bruno Conti made important findings about the
role of the cytokine IL-18 in the regulation of feeding behavior and energy efficiency and through these
mechanisms, the control of body weight. He also
collaborated with Manuel Sánchez-Alavez and Iustin
Tabarean, who uncovered a previously undetected
night-eating phenotype in the commonly studied
strain of mice that lack the gene for prostanoid receptor 3. These mice may be good models of night
bingeing. Xiaoying Lu, Amanda Roberts, and I have
2008
THE SCRIPPS RESEARCH INSTITUTE
329
added to the studies on galanin and galanin receptors
in anxiety and in depressive behaviors.
The scientists of the department have engaged
in many intradepartmental and interdepartmental
collaborations. Numerous high-impact invited lectures and seminars were presented by the faculty
nationally and internationally. For example, I was the
keynote speaker at the largest drug development
meeting (12,000 attendees) in Shanghai in June
2007. Despite a difficult economic climate, scientific
progress in the department was good, and our educational goals for our graduate students and postdoctoral fellows were all successfully met. Several
faculty and students received prestigous stipends.
330 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES
Investigator’s Reports
Neuroscience Discovery and
Pharmacogenomics
C. Wahlestedt, M.A. Faghihi, J. Huang, J. Kocerha,
A.M. Khalil, S. Brothers, F. Modarresi
ur research involves aspects of Alzheimer’s disease, schizophrenia, alcohol addiction, fragile
X syndrome, autism, and aging. In addition to
drug discovery efforts, we focus on basic aspects of
mammalian genomics, genetics, and transcriptomics
(RNA research).
O
2008
THE SCRIPPS RESEARCH INSTITUTE
mals, can be used to elucidate gene functions by rapidly
silencing expression of a target gene. Today, siRNAs
are widely used and have potential for becoming therapeutic agents. We have built up a powerful and versatile portfolio of siRNA technology. Moreover, we have
introduced the use of locked nucleic acids as components of siRNAs (and antisense oligonucleotides)
and have shown a range of beneficial properties of these
modified agents.
G PROTEIN–COUPLED RECEPTORS AS DRUG
TA R G E T S F O R C N S D I S O R D E R S
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
More than half of known drugs bind to G protein–
coupled receptors. We have continued our long-standing work on these receptors. Currently, we are focusing on neuropeptide Y and nociceptin receptors. This
research involves ultra-high-throughput screening.
R E G U L AT O R Y R N A T R A N S C R I P T S
HUMAN GENETICS AND PHARMACOGENOMICS
We are among the few neuroscientists who have
been and continue to be involved in high-throughput
sequencing of transcriptomes (i.e., all the RNA transcripts in a cell) of humans and mice. Such efforts
have provided strong evidence that in contrast to earlier understanding, in mammalian cells, a majority of
the genome is transcribed. Analysis of such data sets
has indicated that most mammalian RNA transcripts
are noncoding.
Thus, conventional protein-coding genes appear to
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 likely corresponds to noncoding RNA. In addition
to small RNAs, many of the noncoding RNAs constitute natural antisense RNA transcripts. We have shown
that many noncoding RNAs identified to date have
substantial conservation across species. We have also
shown that many small noncoding RNAs and antisense
transcripts have differential expression under various
conditions and can affect conventional gene expression.
These novel RNA transcripts also likely are affected by
a range of disease processes in humans. A fruitful study
during the past year has been the investigation of RNA
transcripts in the FMR1 locus, which is related to
fragile X syndrome and to autism spectrum disorders.
We are also involved in several genotyping and
genome-wide association studies related to human CNS
disorders, including schizophrenia. We wish to understand what makes certain individuals susceptible to
disease and how their responses to drug treatment may
differ (pharmacogenomics). One of our goals is to identify
biomarkers associated with human disorders, including Alzheimer’s disease.
RNA INTERFERENCE AND DEVELOPMENT OF
HIGH-THROUGHPUT GENOMICS TECHNOLOGY
RNA interference has become one of the most important gene manipulation technologies. Short interfering
RNA (siRNA), the inducer of RNA interference in mam-
PUBLICATIONS
Dahlgren, C., Zhang, H.Y., Du, Q., Grahn, M., Norstedt, G., Wahlestedt, C.,
Liang, Z. Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Res. 36:e53, 2008.
Faghihi, M.A., Modarresi, F., Khalil, A.M., Wood, D.E., Sahagan, B.E., Morgan,
T.E., Finch, C.E., St-Laurent, G. III, Kenny, P.J., Wahlestedt, C. Expression of a
noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward
regulation of β-secretase. Nat. Med. 14:723, 2008.
Hong, J., Wei, N., Chalk, A., Wang, J., Song, Y., Yi, F., Qiao, R.P., Sonnhammer,
E.L., Wahlestedt, C., Liang, Z., Du, Q. Focusing on RISC assembly in mammalian
cells. Biochem. Biophys. Res. Commun. 368:703, 2008.
Huang, J., Young, B., Pletcher, M.T., Heilig, M., Wahlestedt, C. Association
between the nociceptin receptor gene (OPRL1) single nucleotide polymorphisms
and alcohol dependence. Addict. Biol. 13:88, 2008.
Kemmer, D., Podowski, R.M., Yusuf, D., Brumm, J., Cheung, W., Wahlestedt, C.,
Lenhard, B., Wasserman, W.W. Gene characterization index: assessing the depth
of gene annotation. PLoS ONE 3:e1440, 2008.
Khalil, A.M., Faghihi, M.A., Modarresi, F., Brothers, S.P., Wahlestedt, C. A novel
RNA transcript with antiapoptotic function is silenced in fragile X syndrome. PLoS
ONE 3:e1486, 2008.
Khalil, A.M., Wahlestedt, C. Epigenetic mechanisms of gene regulation during
mammalian spermatogenesis. Epigenetics 3:21, 2008.
Scheele, C., Nielsen, A.R., Walden, T.B., Sewell, D.A., Fischer, C.P., Brogan,
R.J., Petrovic, N., Larsson, O., Tesch, P.A., Wennmalm, K., Hutchinson, D.S.,
Cannon, B., Wahlestedt, C., Pedersen, B.K., Timmons, J.A. Altered regulation of
the PINK1 locus: a link between type 2 diabetes and neurodegeneration? FASEB J.
21:3653, 2007.
St-Laurent, G. III, Wahlestedt, C. Noncoding RNAs: couplers of analog and digital
information in nervous system function? Trends Neurosci. 30:612, 2007.
MOLECULAR THERAPEUTICS
2008
THE SCRIPPS RESEARCH INSTITUTE
Molecular Therapeutics
Identification of posttranslational modifications on peptides
by using high-resolution mass spectrometry and MS3 scanning
for absolute assignment of site of modification. Work done in the
laboratory of Jennifer Caldwell Busby, Ph.D., assistant professor.
347
Jennifer Caldwell Busby, Ph.D., Assistant Professor, and
Kristie Rose, Ph.D., Staff Scientist
MOLECULAR THERAPEUTICS
2008
THE SCRIPPS RESEARCH INSTITUTE
349
DEPAR TMENT OF
MOLECULAR
THERAPEUTICS
S TA F F
Patrick Griffin, Ph.D.
Professor and Chairman
Director, Translational
Research Institute
Jennifer Caldwell-Busby,
Ph.D.*
Assistant Professor
Gregg Fields, Ph.D.
Adjunct Professor
Philip LoGrasso, Ph.D.*
Associate Professor
Mathew T. Pletcher, Ph.D.**
Assistant Professor
S TA F F S C I E N T I S T S
Monica Istrate, Ph.D.
Lisa Cherry, Ph.D.
Brook Miller, Ph.D.
Kristie Rose, Ph.D.
Jun Zhang, Ph.D.
* Joint appointment in the
Translational Research Institute
SENIOR SCIENTISTS
R E S E A R C H A S S O C I AT E S
Scott Busby, Ph.D.
Brian Ember, Ph.D.
Michael Chalmers, Ph.D.
Christie Fowler, Ph.D.
** Joint appointments in the
Department of Biochemistry
and the Translational Research
Institute
Kevin Hayes, Ph.D.
Paul J. Kenny, Ph.D.
Assistant Professor
Chairman’s Overview
he Department of Molecular Therapeutics was
established on the Florida campus of Scripps
Research in 2007. Faculty in the department
use chemical biology
approaches to dissect
signaling pathways and
transcriptional programs.
We rely on state-of-theart multidisciplinary
technology and methods
and a variety of model
systems for target identification, validation,
and preclinical studies.
Currently, the department
Patrick R. Griffin, Ph.D.
has 5 tenure-track faculty members and several
non–tenure track members who oversee key functional
cores on the Florida campus. These investigators have
created strong research programs that take advantage
of the unique high-throughput core facilities at the
Florida campus, including genomics, cell-based screening, and proteomics.
Research activities include discovery and development of therapeutic agents for unmet medical needs in
neurodegeneration, Parkinson’s disease, acute respiratory distress syndrome, spinal cord injury, cardiovascu-
T
Jonathan Hollander, Ph.D.
lar disease, cancer, addiction, and metabolic disorders,
including insulin resistance, obesity, and type 2 diabetes.
Paul Kenny and his group focus on the neuropharmacology of addiction and on establishing the role of several G protein–coupled receptors in addictive behavior.
Phil LoGrasso and members of his laboratory are involved
in the discovery of small-molecule therapeutic agents to
be used as neuroprotective agents in diseases such as
Parkinson’s and are determining the role of rho kinase
in vascular bed modulation and glaucoma. Thomas Burris
and his group are studying the role of orphan nuclear
receptors in circadian rhythms and metabolic disorders
such as obesity. Scientists in Jennifer Caldwel Busby’s
laboratory use state-of-the-art mass spectrometry to identify, quantify, and characterize proteins and protein
modifications to map the signaling pathways related to
diabetes and cancer. Peter Hodder and coworkers focus
on technology and assay development and novel chemical approaches to expand compound libraries. Michael
Cameron and his group are involved in mechanistic
studies of P450s and drug biotransformation mechanisms.
Researchers in my group are dissecting the mechanism
of ligand-dependent activation of orphan nuclear receptors implicated in cancer and metabolic disorders.
350 MOLECULAR THERAPEUTICS
2008
Investigators’ Reports
Probing Protein Dynamics With
Hydrogen-Deuterium Exchange
Mass Spectrometry
P.R. Griffin, S.A. Busby, M.J. Chalmers, S.Y. Dai, J. Zhang,
M. Istrate, R. Garcia-Ordonez, S. Novick, B. Pascal,
J. Conkright, G. Zastrow-Hayes, K. Hayes, T. Schröter,
F. Madoux, D. Minond, P.S. Hodder
e use a wide range of technologies to study
ligand activation of nuclear receptors. During
the past few years, we focused on the ligandbinding domains of the well-characterized nuclear receptors peroxisome proliferator–activated receptor γ (PPARγ)
and the α and β estrogen receptors. Recently, we have
focused on developing hydrogen-deuterium exchange
(HDX) technology for probing the mechanism of activation of several orphan nuclear receptors. In addition, in
collaboration with scientists at Xencor, Monrovia, California, we are studying the dynamics of TNF-α.
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L I G A N D A C T I VAT I O N O F P PA R γ
PPARγ is a multidomain ligand-dependent transcription factor. Ligands regulate PPARγ activation by binding to the receptor’s ligand-binding domain, inducing a
change in the conformational dynamics of the domain
that leads to dissociation of corepressor molecules and
formation of suitable neoepitopes for the binding of coactivator molecules. We used structural, biochemical, and
cell-based techniques to examine the mechanism of
ligand regulation of PPARγ transcriptional activity. We
found that the magnitude of PPARγ agonism is regulated
by coactivator recruitment selectivity of p160 coactivators. In mutagenesis studies, we determined the key
residues on the receptor that facilitate these selective
coactivator interactions.
In other studies, we are using coactivators as chemical tools to generate desired functional responses and to
differentiate pharmacologically beneficial function from
adverse function, a novel unexploited therapeutic avenue
for treating insulin resistance. Our goals are to determine the structure-activity relationships between PPARγ
ligands and their coactivator recruitment selectivity and
to obtain PPARγ ligands with specific coactivator preferences by screening for agonists that favor specifically
the association of a given cofactor. For a large-scale
THE SCRIPPS RESEARCH INSTITUTE
high-throughput screening to identify coactivator-selective
agonists of the receptor, we have developed a validated
time-resolved fluorescence resonance energy transfer assay
for ligand-dependent recruitment of the coactivator to PPARγ.
Scientists at the Scripps Research Institute Molecular Library
Screening Center used these assays to examine the National
Institute of Health small-molecule library. The results
obtained from this research are providing molecular
insight into coactivator recruitment and receptor activation
and will result in chemical tools to dissect the biological role of specific coactivators in modulating PPARγ.
L I G A N D A C T I VAT I O N O F T H E V I TA M I N D R E C E P T O R
In collaboration with scientists at Eli Lilly and Company, Indianapolis, Indiana, we are using HDX to characterize activation of the full-length heterodimer complex
composed of the vitamin D receptor and its coreceptor
retinoid X receptor α. This project is promoting further
development of our HDX platform to facilitate the analysis of large transcriptional complexes. Although this
research is in an early stage, we have data that suggest
HDX is useful for probing dynamics of large transcriptional complexes.
PROBING G PROTEIN–COUPLED RECEPTORS
G protein–coupled receptors are an important family of
transmembrane signaling proteins. Characterization of the
structure and dynamics of these proteins is an analytical
challenge because their transmembrane domains are
hydrophobic. We have begun to expand the application
of HDX to probe the dynamics of these receptors. This
work is being done in collaboration with H. Rosen, Department of Chemical Physiology, and R.C. Stevens, Department of Molecular Biology.
PUBLICATIONS
Bruning, J., Chalmers, M.J., Prasad, S., Busby, S.A., Kamenecka, T., He, Y., Nettles, K.W., Griffin, P.R. Partial agonists activate PPARγ using a helix 12 independent
mechanism. Structure 15:1258, 2007.
Chalmers, M.J., Busby, S.A., Pascal, B.D., Southern, M.R., Griffin, P.R. A twostage differential hydrogen deuterium exchange method for the rapid characterization
of protein/ligand interactions. J. Biomol. Tech. 18:194, 2007.
Dai, S.Y., Chalmers, M.J., Bruning, J., Bramlett, K.S., Osborne, H.E., MontroseRafizadeh, C., Barr, R.J., Wang, M., Burris, T.P., Dodge, J.A., Griffin, P.R. Prediction of the tissue-specificity of selective estrogen receptor modulators using a single
biochemical method. Proc. Natl. Acad. Sci. U. S. A. 105:7171, 2008.
Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright, J.J., Griffin,
P.R., Thacher, S., Hodder, P.S. Potent, selective and cell penetrant inhibitors of SF-1 by
functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008.
MOLECULAR THERAPEUTICS
2008
Mass Spectrometry for
Identification of Proteins
THE SCRIPPS RESEARCH INSTITUTE
351
modifications associated with sites of transcription can
be used as the basis for further experiments to determine the biological roles of the proteins in gene regulation and activation.
J.A. Caldwell Busby, V. Cavett
ur general focus is the use of cutting-edge separation and mass spectrometry techniques to
identify proteins involved in biological events.
The biological applications are determined by the research
needs of a large group of collaborators in various disciplines, with a wide variety of questions to be answered.
We provide these collaborators access to powerful and
novel approaches to examine posttranslational modifications and measure protein levels in multiple samples.
In addition to these collaborative efforts, we are
developing a method to identify and temporally map
chromatin proteins involved in transcriptional regulation. Gene regulation is a fundamental biological process that is studied from a variety of perspectives with
a variety of methods; however, research to date has
been highly gene centric, and only a few reports have
been published on the proteomics of gene regulation.
We target these missing proteomics components, particularly the components of the supermolecular complex of chromatin, including nucleosome substructure
and regulatory and transcription complexes.
Methods for whole-system approaches are difficult
to implement because traditional technologies tend to
focus on the isolation and analysis of individual parts
of the whole—DNA, RNA, or protein. Our techniques
combine advances in molecular biology with the power
of mass spectrometry to identify novel biomolecules
involved in multibiopolymer complexes. In particular,
we are modifying and combining techniques such as
protein-protein and protein-DNA cross-linking, immunoprecipitation methods, chromatin immunoprecipitation,
mass spectrometry, and liquid chromatography to determine the larger regulatory mechanisms involved in the
fate of cells. The keystone of this method is a modified
chromatin immunoprecipitation protocol that maintains
the integrity of the DNA while allowing for the isolation, recovery, and analysis of the protein components of
the nucleosome complex. This advanced method targets
proteins that regulate chromatin function and correlates those proteins with histone modification states
and gene occupancy. Incorporating proteomics into a
traditionally DNA-based experimental protocol provides
a new perspective and a novel approach to genetic
regulation. Newly identified proteins and novel protein
O
PUBLICATIONS
Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V.,
Orth, A.P., Busby, J., Hogenesch, J.B., Conkright, M.D. A coactivator trap identifies NONO (p54nrb) as a component of the cAMP signaling pathway. Proc. Natl.
Acad. Sci. U. S. A. 104:20314, 2007.
Neurobiology of Addiction
P.J. Kenny, P. Bali, C.D. Fowler, J.A. Hollander, H.-I. Im,
P.M. Johnson,* Q. Lu, B.H. Miller
* Kellogg School of Science and Technology, Scripps Research
e focus on understanding the neurobiological
mechanisms of addiction. This knowledge will
be used to develop novel therapeutic agents
for treatment of substance abuse. We use a multidisciplinary approach that includes mouse behavioral genetics, virus-mediated gene expression, RNA and protein
analyses, and in vivo behavioral testing.
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NICOTINE ADDICTION
We seek to identify the subtypes of nicotinic acetylcholine receptors and downstream signaling cascades
through which nicotine promotes tobacco addiction.
Currently, we are assessing the reinforcing effects of
nicotine in mice with null mutations in various subunits of the receptors. In addition, we are testing the
effects on nicotine reinforcement of using lentivirusbased short hairpin RNAs to silence the genes of targeted nicotinic acetylcholine receptor subunits in brain
reward circuitries. Finally, we are using a proteomics
approach to identify the intracellular proteins coupled
to nicotinic acetylcholine receptors in the brains of mice.
Our goal is to identify novel scaffold and signaling proteins involved in transducing the addictive actions of
nicotine. These studies promise to yield significant new
insights into the neurobiological mechanisms of nicotine addiction, with direct relevance for the treatment
of the tobacco habit in humans.
BRAIN SYSTEMS INVOLVED IN ADDICTION
In collaboration with other scientists at Scripps
Research, we found that the neuropeptide orexin
(hypocretin) plays a critical role in drug reward. In
ongoing studies, we are identifying the mechanisms
through which orexin-mediated transmission regulates
drug reward. We are also investigating the roles of
352 MOLECULAR THERAPEUTICS
2008
novel constitutive mechanisms of gene regulation in the
neuroplasticity induced by drugs of abuse that may
promote addiction. Further, we are testing the hypothesis that drug addiction and obesity share common
reward and motivational mechanisms. These studies may
identify novel targets for the development of therapeutics against addiction and obesity.
DEVELOPMENT OF NOVEL ANTIADDICTION
M E D I C AT I O N S
In collaborations with scientists in the Translational
Research Institute, Scripps Florida, we are developing
small-molecule drugs that may be useful as novel therapeutic agents for treatment of substance abuse disorders.
The targets for these drugs are G protein–coupled
receptors that we previously showed play a role in
drug dependence.
PUBLICATIONS
Faghihi, M.A., Modarresi, F., Khalil, A.M., Wood, D.E., Sahagan, B.G., Morgan,
T.E., Finch, C.E., St-Laurent, G. III, Kenny, P.J., Wahlestedt, C. Expression of a
noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward
regulation of β-secretase. Nat. Med. 14:723, 2008.
Johnson, P.M., Hollander, J.A., Kenny, P.J. Decreased brain reward function during
nicotine withdrawal in C57BL6 mice: evidence from intracranial self-stimulation
(ICSS) studies. Pharmacol. Biochem. Behav. 90:409, 2008.
Kenny, P.J., Chartoff, E., Roberto, M., Carlezon, W.A., Jr., Markou, A. NMDA
receptors regulate nicotine-enhanced brain reward function and intravenous nicotine self-administration: role of the ventral tegmental area and central nucleus of
the amygdala. Neuropsychopharmacology 14:723, 2008.
Inhibition of Jun N-Terminal
Kinase 2/3 for the Treatment
of Parkinson’s Disease
P. LoGrasso, M. Cameron, W. Chen, S. Clapp, D. Duckett,
B. Ember, J. Habel, R. Jiang, T. Kamenecka, S. Khan,
L. Ling, Y.-Y. Ling, M. Lopez, A. Pachori, C. Ruiz, Y. Shin,
X. Song, T. Vojkovsky, D. Zadory
poptosis, or programmed cell death, plays a vital
role in the normal development of the nervous
system and is also thought to contribute to the
aberrant neuronal cell death that characterizes many
neurodegenerative diseases. Therefore, blocking neuronal apoptosis could be an approach for treating neurodegenerative diseases. A major pathway implicated
in neuronal cell death and survival is the MAP kinase
pathway, which controls cell proliferation and cell death
in response to many extracellular stimuli. Recent studies have linked Jun N-terminal kinase (JNK) activity with
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THE SCRIPPS RESEARCH INSTITUTE
the cell death associated with Parkinson’s disease and
Alzheimer’s disease.
JNK is linked to many of the hallmark pathophysiologic components of Parkinson’s disease, such as oxidative stress, programmed cell death, and microglial
activation. Many pieces of evidence support JNK as a
target for treatment of the pathologic changes that underlie Parkinson’s disease. One attractive feature of JNK3
as a selective drug target is that this kinase is almost
exclusively expressed in the brain. In contrast, JNK1
and JNK2 are ubiquitously expressed. Despite the ubiquitous expression of JNK2, we are developing a therapy to prevent degeneration of dopaminergic neurons
and halt the progression of Parkinson’s disease by targeting JNK2/3.
Our strategy for inhibiting JNK2/3 is based on the
results of experiments with mice in which the gene for
JNK3 or JNK2 was deleted and mice in which the genes
for both JNK2 and JNK3 or both JNK1 and JNK2 were
deleted. In contrast to mice lacking the gene for JNK1
alone, which had defective T-cell differentiation, mice
lacking the gene for JNK2 alone had normal T- and
B-cell development and normal T-cell proliferation. Moreover, mice lacking the gene for JNK2 alone and mice
lacking the gene for JNK3 alone were protected against
the effects of 1-methyl-4-phenyl-1,2,3,5-tetrahydropyridine (MPTP), a compound used to induce parkinsonian signs in animal models of Parkinson’s disease,
whereas both wild-type mice and mice lacking the
gene for JNK1 were not. In other research, compared
with wild-type mice, mice lacking the genes for both
JNK2 and JNK3 were dramatically protected against
acute MPTP-induced injury of the nigrostriatal pathway.
This protective effect resulted in a 3-fold increase in
the number of neurons positive for tyrosine hydroxylase, an indication of the increase in survival of dopaminergic neurons.
On the basis of these in vitro and in vivo data, we
are synthesizing potent, selective JNK 2/3 inhibitors
that we will test for efficacy in MPTP animal models
of Parkinson’s disease. We have established homogenous time-resolved fluorescence biochemical assays
for JNK3 and counterscreens for JNK1 and p38. We
have generated more than 1000 compounds from 3
different structural classes; many of the compounds
are inhibitory for JNK3 in nanomolar concentrations.
Some of the compounds have a cellular potency of
40–60 nM and in vitro efficacy in promoting primary
survival of dopaminergic neurons. We have tested com-
MOLECULAR THERAPEUTICS
2008
pounds in vivo in rats and mice for drug metabolism
and pharmacokinetic properties. Many of the JNK3
inhibitors have had good oral absorption, good brain
penetration, and good pharmacokinetic properties that
enable efficacy studies.
We have also solved the crystal structure of 10 complexes of JNK3 with inhibitor at approximately 2.2-Å
resolution. This information is being used in structurebased drug design to help guide medicinal chemistry
studies and optimize compounds for potency, selectivity,
brain penetration, oral absorption, half-life, clearance,
and efficacy.
We have also begun investigating the role of JNK
in myocardial infarction. We have set up animal models to test for the ability of JNK inhibitors to decrease
infarct size and preserve cell function in these models.
Finally, we have determined the kinetic mechanism
for JNK3 and have shown that it is a random sequential
mechanism. We are investigating the kinetic mechanism
of JNK1 and are examining differences substrate specificity that may exist between the isoforms. We plan to
investigate the role played by different JNK isoforms and,
more specifically, different splice variants in various
apoptosis scenarios in different cell types. The purposes
of these basic mechanistic studies is to understand
structure-function relationships at the molecular level
and to design specific inhibitors that may be selective
for one isoform or splice variant.
PUBLICATIONS
Ember, B., Kamenecka, T., LoGrasso, P. Kinetic mechanism and inhibitor characterization for c-jun-N-terminal kinase 3α1. Biochemistry 47:3076, 2008.
Jiang, R., Duckett, D., Chen, W., Habel, J., Ling, Y.-Y., LoGrasso, P., Kamenecka,
T.M. 3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors.
Bioorg. Med. Chem. Lett. 17:6378, 2007.
Schröter, T., Minod, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P., Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M., Tsinoremas, N., LoGrasso, P., Hodder, P. Comparison of miniaturized time-resolved
fluorescence energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. Biomol. Screen.
13:17, 2008.
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Translational Research Institute
Neuronal differentiation is essential for the formation
of the mammalian nervous system. Activation of the
Rho Kinase (ROCK) pathway by lysophosphatidic acid
(LPA) causes neurite retraction. In order to examine the
effect of ROCK inhibitors on the prevention of LPAinduced neurite retraction, PC12 cells were allowed
to differentiate for 4 days in the presence of nerve
growth factor before treatment with ROCK inhibitors
and then stimulation with LPA. Cells were fixed and
stained for α-tubulin, and nuclei were visualized by
using Hoechst 33342 dye. Cells were imaged in a 96well format with the IN Cell 1000 platform. Images
were analyzed for neurite length (red) and cell count
(green) by using the Developer Toolbox. Work done in
the laboratory of Thomas Schröter, Ph.D., senior scientist.
Thomas Schröter, Ph.D., Senior Scientist
TRANSL ATIONAL RESEARCH INSTITUTE
T R A N S L AT I O N A L
RESEARCH INSTITUTE
S TA F F
Patrick Griffin, Ph.D.*
Director
2008
William Roush, Ph.D.**
Executive Director, Medicinal
Chemistry
Associate Dean, Kellogg
School of Science and
Technology
THE SCRIPPS RESEARCH INSTITUTE
Romain Noel, Ph.D.
Sanjay Saldanha, Ph.D.
E. Hamp Sessions, Ph.D.
Anthony Smith, Ph.D.
Xinyi Song, Ph.D.
Thomas D. Bannister, Ph.D.
Associate Scientific Director,
Medicinal Chemistry
SENIOR SCIENTISTS
Prem Subramaniam, Ph.D.
Yenting Chen, Ph.D.
Dusica Vidovic, Ph.D.
Jennifer Caldwell Busby,
Ph.D.*
Associate Scientific Director,
Proteomics
Michael Cameron, Ph.D.*
Associate Scientific Director,
Drug Metabolism and
Pharmacokinetics
Derek R. Duckett, Ph.D.
Associate Scientific Director,
Discovery Biology
Rong Jiang, Ph.D.
Kristen Clarke Ware, Ph.D.
Marcel Koenig, Ph.D.
Yan Yin, Ph.D.
Jiuxiang Ni, Ph.D.
Alok Pachori, Ph.D.
Louis Scampavia, Ph.D.
Thomas Schröter, Ph.D.
Peter Hodder, Ph.D.
Scientific Director, Lead
Identification
Ted Kamenecka, Ph.D.
Associate Scientific Director,
Medicinal Chemistry
Dmitriy Minond
Timothy Spicer
Youseung Shin, Ph.D.
Tomas Vojkovsky, Ph.D.
Yangbo Feng, Ph.D.
Associate Scientific Director,
Medicinal Chemistry
S C I E N T I F I C A S S O C I AT E S
HTS ROBOTICS
ENGINEERS
S TA F F S C I E N T I S T S
Pierre Baillargeon
Lisa Cherry, Ph.D.
Peter Chase
Juliana Conkright, Ph.D.
Lina Deluca
Dympna Harmey, Ph.D.
Sahba Tabrizifard, Ph.D.
I N F O R M AT I C S S TA F F
Congxin Liang, Ph.D.
Scientific Director, Medicinal
Chemistry
SENIOR RESEARCH
A S S O C I AT E
Philip LoGrasso, Ph.D.*
Senior Director, Discovery
Biology
Franck Madoux, Ph.D.
Caty Chung
Yasel Cruz
Kashif Hoda
Bruce Pascal
Patricia McDonald, Ph.D.
Associate Scientific Director,
Discovery Biology
R E S E A R C H A S S O C I AT E S
Becky Mercer, Ph.D.
Associate Scientific Project
Manager, Lead
Identification
Melissa Crisp, Ph.D.
Mathew T. Pletcher, Ph.D.**
Assistant Professor, RNA
Core
Stephan Schuerer
Sarwat Chowdhury, Ph.D.
Mark Southern
Brian Ember, Ph.D.
Xingang Fang, Ph.D.
Yuanjun He, Ph.D.
Xiaohai Li, Ph.D.
* Joint appointment in the
Department of Molecular
Therapeutics
** Joint appointment in the
Department of Chemistry
367
368 TRANSL ATIONAL RESEARCH INSTITUTE
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Director’s Overview
he Translational Research Institute merges drug
discovery efforts at the Scripps Research Florida
campus with advanced technology platforms to
rapidly identify and validate biological pathways
that can be targeted for
therapeutic intervention.
The goal of the drug discovery operation is to
discover and develop
small-molecule therapeutic
agents for unmet medical
needs in neurodegeneration, Parkinson’s disease,
acute respiratory distress
syndrome, glaucoma,
Patrick R. Griffin, Ph.D.
spinal cord injury, cancer,
and metabolic disorders, including insulin resistance,
type 2 diabetes, and obesity, by targeting G protein–
coupled receptors, proteases, ion channels, and kinases.
The drug discovery component of the Translational
Research Institute is fully integrated with the following
groups: Lead Identification and High-Throughput Screening, headed by Peter Hodder, Department of Molecular
Therapeutics; Medicinal Chemistry, headed by William
Roush, Department of Chemistry; Discovery Biology,
headed by Phil LoGrasso, Department of Molecular
Therapeutics; Drug Metabolism and Pharmacokinetics,
headed by Mike Cameron, Department of Molecular
Therapeutics; and Informatics, headed by Mark Southern.
The Lead Identification team enables drug-target lead
identification via ultra-high-throughput screening technology. Using state-of-the-art automation and instrumentation,
members in this group are responsible for developing
and executing biochemical or cell-based high-throughput
screening assays in a miniaturized 1536-well microtiter
plate format. In addition to its support of internal Scripps
Research objectives, the group participates in the National
Institutes of Health Molecular Libraries Probe Production
Centers Network (MLPCN), in which qualified assays
are screened against the network’s high-throughput
screening compound library. Several internal and external investigators have accessed the group’s expertise
via collaborative or core-charge mechanisms.
The genomics core is headed by Brandon Young.
Scientists in this core oversee genotyping and gene
expression profiling. The services provided by the core
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THE SCRIPPS RESEARCH INSTITUTE
allow Scripps Research investigators to examine the
genome at both the genetic and the transcriptional level
for the genes that underlie common diseases. In collaboration with colleagues on the Florida campus, members
of the core have been involved in projects to identify the
genes responsible for pathologic conditions, such as
addiction and alcoholism, systemic lupus erythematosus, autism, obsessive-compulsive disorder, diabetes,
obesity, and prion diseases.
The cell-based screening platform is headed by
Julie Conkright, Department of Molecular Therapeutics.
The faculty advisor to the core is Michael Conkright,
Department of Cancer Biology. In this group, high-throughput technologies are used to provide a systematic description of the function of genes encoded by the human
genome and a more comprehensive understanding of the
genetic basis for human disease. Members of the group
provide investigators access to genome-wide collections
of cDNAs and short interfering RNAs that can be used
to examine cellular models of signal transduction pathways and phenotypes. In addition, the cell-based screening platform participates in one of the center-based
initiatives of the Scripps Research MLPCN center.
The proteomics platform is headed by Jennifer
Caldwell Busby, Department of Molecular Therapeutics.
The focus of this core is using liquid chromatography
and state-of-the-art mass spectrometry technology to
identify, quantify, and characterize proteins and protein
modifications. Researchers in the core are involved in
scientific collaborations in which novel technologies are
used to identify biologically important proteins and protein modifications. Large-scale differential analysis is
being used to map the pathways related to insulin sensitization and adipogenesis. In other projects, chromatographic enrichment techniques are used to identify sites
of phosphorylation and other posttranslational modifications. Researchers in the proteomics core collaborate
with other scientists to create experiments that will
provide meaningful mass spectrometric results.
TRANSL ATIONAL RESEARCH INSTITUTE
2008
Investigators’ Reports
Drug Discovery: Medicinal
Chemistry Efforts
T.D. Bannister, Y. Feng, T.M. Kamenecka, C. Liang,
W.R. Roush, Y. Chen, S. Chowdhury, X. Fang, Y. He,
R. Jiang, M. Koenig, R. Noel, E.H. Sessions, Y. Shin,
X. Song, T. Vojkovsky, Y. Yin
e seek to discover new compounds to treat
diseases for which current therapies are inadequate. In our major programs during the past
year, we targeted glaucoma, Parkinson’s disease, and
breast cancer. In each program, we have attempted to
block the action of a specific protein kinase that is overactive or overabundant in affected patients and that
hastens the progression of disease. In 2 of the programs,
we began by identifying chemical leads from a highthroughput biochemical screen of the Scripps collection of more than 700,000 compounds. The structural
information from these screens, in combination with
computational and biological analysis of compounds
made in other laboratories targeting the same enzymes,
provides insights for modifying the structures to obtain
unique and patentable leads with the required druglike
biochemical, physical, and pharmacologic properties. All
of these are evaluated internally by Scripps scientists in
the biology, pharmacology, and drug metabolism and
pharmacokinetics groups of the Translation Research
Institute, who work closely with the medicinal chemists
on fully integrated interdisciplinary project teams.
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GLAUCOMA
We are designing inhibitors of the serine-threonine
kinase ROCK, or Rho kinase, which regulates intraocular
pressure by controlling the outflow of aqueous humor.
Excess ROCK activity is associated with high intraocular pressure, which is a primary risk factor for glaucoma,
and with retinal damage. Application of a ROCK inhibitor increases outflow, lowers intraocular pressure, and
preserves retinal neurons. Current antiglaucoma drugs
have limited efficacy or cause side effects, including
discomfort, hyperemia (red eye), and/or undesired
changes in cardiovascular function. No glaucoma drugs
on the market act by directly altering the Rho kinase
pathway, but ROCK inhibitors have strong pressurelowering and neuroprotective effects and thus could
be a valuable new treatment. An ideal ROCK inhibi-
THE SCRIPPS RESEARCH INSTITUTE
369
tor applied topically to the eye must simultaneously
have many properties, including high ROCK affinity,
aqueous solubility, excellent corneal permeability, high
cellular penetration, and low ocular clearance, to provide
a long-lasting effect. Most importantly, the inhibitor must
be selective for ROCK over other enzymes and receptors
so that no serious side effects occur.
We have synthesized thousands of new ROCK inhibitors in multiple chemical classes; many have low nanomolar potency in both biochemical and cell-based assays,
high selectivity, and a profile of properties appropriate for
preclinical development. For example, SR-3677 was
tested in an animal model for glaucoma by our collaborator, V. Rao, Duke University, Durham, North Carolina.
The inhibitor lowered intraocular pressure more than
30% within 1 hour, an efficacy comparable to that of
antiglaucoma drugs in current use. The reduction in pressure waned after 2 hours, however, so we are designing
other compounds intended to have a similarly powerful
yet more sustained effect. As expected, the reduction
in pressure was due to an increased rate of fluid outflow.
We have also made compounds that distinguish
between the enzyme isoforms ROCK-I and ROCK-II to
test their precise roles. An inhibitor selective for ROCK-II,
for example, would lack any unwanted side effects due
to ROCK-I inhibition. Such effects are unclear, because
no other isoform-selective ROCK inhibitors targeting
glaucoma are known.
PARKINSON’S DISEASE
In collaboration with the National Institute of Neurological Disorders and Stroke, we are developing a therapy to interrupt the loss of dopamine-containing neurons
in the midbrain that is a hallmark of Parkinson’s disease.
Activation of the transcription factor c-Jun by c-Jun
N-terminal kinase (JNK) promotes neurodegeneration.
Inhibitors of JNK, which exist in 3 isoforms, JNK1,
JNK2, and JNK3, are neuroprotective in animal models
of Parkinson’s disease. Our approach, using inhibitors
selective for JNK2 and JNK3, would be a quantum leap
in the clinical treatment of Parkinson’s disease for several reasons. All current therapies merely treat the symptoms of the disease rather than address the underlying
pathologic changes, they tend to lose therapeutic efficacy over time, and they typically elicit undesired side
effects. Our challenge is to develop a compound that
is a potent, selective, and cell-permeable JNK2/3 inhibitor; has the pharmacokinetic properties for oral dosing
(ideally once a day); has good brain penetration; and
has a benign toxicology profile.
370 TRANSL ATIONAL RESEARCH INSTITUTE
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We have synthesized thousands of new JNK inhibitors in multiple chemical scaffolds and are evaluating
compounds with the best combination of properties in
several preclinical animal models for Parkinson’s disease. For example, in a pilot study, the Scripps JNK
inhibitor SR-3306 delivered systemically to rodents via
osmotic minipump at 10 mg/kg reduced CNS-mediated
behaviors that occur after a chemically induced brain
lesion used to mimic the parkinsonian condition. Newer
generation compounds, including SR-3562, will soon
be evaluated in animal models and are particularly
promising because of improved properties, including
high oral bioavailability (45%), high cell-based potency
(0.06 µM), and excellent distribution to the brain of
rodents after oral dosing.
CANCER
In collaboration with Poniard Pharmaceuticals,
South San Francisco, California, we have synthesized
many potent and selective novel inhibitors of focal adhesion kinase (FAK). FAK inhibitors could be an important new means of treating solid tumors, including breast
cancer. FAK has been implicated in promoting detachment of tumor cells and metastasis, characteristics of
almost all advanced-stage solid tumors that are responsible for most of the suffering and death related to cancer. By blocking FAK and thus stopping the first step
of metastasis, the detachment of cancer cells from their
primary site, we hope to halt this process and thereby
interrupt progression of the disease. We have recently
completed our FAK chemistry efforts after identifying
highly potent and selective FAK inhibitors, including SR2516. This lead compound is effective in in vitro tumor
metastasis models, is efficacious in animal models of
tumor progression, has desirable pharmaceutical properties suitable for convenient once-a-day oral dosing,
and is being licensed for further development.
FUTURE DIRECTIONS
We are continuing our research on glaucoma and
Parkinson’s disease and have smaller or exploratory
efforts in other areas, including methods for treating
diabetes, for curbing drug addiction, and for targeting
cancer progression by other mechanisms. We hope to
expand these efforts. Many of the compounds identified in the ROCK and JNK inhibitor programs are also
likely to be useful in the treatment of other diseases.
For example, animal data suggest that ROCK inhibitors
might be an effective treatment for multiple sclerosis.
Strong preclinical evidence shows that JNK inhibitors,
in addition to treating Parkinson’s disease, also may
THE SCRIPPS RESEARCH INSTITUTE
prevent neuronal damage in a host of other disorders
including stroke, Alzheimer’s disease, and amyotrophic
lateral sclerosis.
We anticipate that in each of our research programs
we can continue to synthesize novel compounds with the
right combination of properties that would permit development of the compounds as safe and effective agents
for stopping the progression of important diseases.
PUBLICATIONS
Chen, Y.T., Bannister, T.D., Weiser, A., Griffin, E., Lin, L., Ruiz, C., Cameron,
M.D., Schürer, S., Duckett, D., Schröter, T., Lograsso, P., Feng, Y. Chroman-3amides as potent Rho kinase inhibitors. Bioorg. Med. Chem. Lett., in press.
Feng, Y., Cameron, M.D., Frackowiak, B., Griffin, E., Lin, L., Ruiz, C., Schröter,
T., LoGrasso, P. Structure-activity relationships and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of
ROCK-II. Bioorg. Med. Chem. Lett. 17:2355, 2007.
Feng, Y., Yin, Y., Weiser, A., Griffin, E., Cameron, M.D., Lin, L., Ruiz, C., Schürer,
S.C., Inoue, T., Rao, P.E., Schröter, T., LoGrasso, P. Discovery of substituted 4(pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as potent and highly selective
Rho kinase (ROCK-II) inhibitors. J. Med. Chem. 51:6642, 2008.
Jiang, R., Duckett, D., Chen, W., Habel, J., Ling, Y.Y., LoGrasso, P., Kamenecka,
T.M. 3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors.
Bioorg. Med. Chem. Lett. 17:6378, 2007.
LoGrasso, P., Kamenecka, T. Inhibitors of c-jun-N-Terminal Kinase (JNK). Mini Rev.
Med. Chem. 8:755, 2008.
Sessions, E.H., Yan, Y., Bannister, T.D., Pocas, J., Cameron, M.D., Ruiz, C.,
Schürer, S.C., Schröter, T., LoGrasso, P., Feng, Y. Benzimidazole- and benzoxazole-based inhibitors of Rho kinase. Bioorg. Med. Chem. Lett., in press.
Proteomics Laboratory
J.A. Caldwell Busby, V. Cavett
he Proteomics Laboratory at Scripps Florida provides proteomics services and expertise to scientific collaborators at Scripps Research facilities
in both Florida and California, universities within the
state of Florida, and other educational institutions. We
use cutting-edge mass spectrometry technology to identify
proteins, map modifications that occur after translation, and do relative quantitation experiments with a
variety of sample types.
In its lifetime, a protein can have several locations
and functions within a cell. Location, function, 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 and acetyl groups, which are part of the histone
codes, to large lipid and glycosylation modifications,
which act as cellular markers and signaling molecules.
With mass spectrometry, we can detect both the small
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2008
and the large changes in mass that occur in proteins
because of these modifications, and we can identify
the specific amino acids modified.
Relative changes in protein levels or level of posttranslational modification between multiple samples provide biologically relevant information about cellular
pathways and proteins of interest. Large-scale studies of
this type require rigorous sample preparation and highly
tuned algorithms for comparing different mass spectrometric analyses. We are currently validating methods for
both sample fractionation and data analysis for these
types of large-scale differential protein experiments.
Mass spectrometers at the facility include an ion trap
spectrometer, which is used mostly to identify proteins
and peptides, and a triple quadrupole mass spectrometer,
which is used for relative quantitation experiments. A
new addition is a mass spectrometer that can be used to
perform accurate mass and high-resolution experiments.
Each mass spectrometer is interfaced to nano-flow electrospray ionization sources and capillary high-performance liquid chromatography columns.
Data analysis is performed primarily via automated
workflow on a cluster maintained by the bioinformatics
group. Automation of the front-end processing allows
a more thorough review of the data and more time
for development of innovative software in collaboration
with information technology groups at Scripps Research
and beyond.
Drug Metabolism and
Pharmacokinetics Laboratory
M.D. Cameron, L. Lin, C. Ruiz, S. Khan, Z. Li
he Drug Metabolism and Pharmacokinetics Laboratory at Scripps Florida provides in vitro and
in vivo evaluation of the pharmacokinetic and
pharmacodynamic properties of new chemical entities.
We work on project teams within the drug discovery
group of the Department of Molecular Therapeutics and
support chemistry efforts within the Scripps Research
Institute Molecular Screening Center. We help bridge
medicinal chemistry and pharmacology by evaluating
the metabolic fate and identifying the liabilities of
compounds. Pharmacokinetic studies provide basic
parameters, including peak plasma concentration, bioavailability, exposure, half-life, clearance, volume of
distribution, and tissue distribution. Research interests
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THE SCRIPPS RESEARCH INSTITUTE
371
include P450 structure-function relationships and the
formation of reactive intermediates during metabolism.
The laboratory is equipped with a liquid chromatography–tandem mass spectrometry system and a Q-trap
hybrid triple quadrupole/linear ion-trap mass spectrometer.
PUBLICATIONS
Cameron, M.D., Wen, B., Roberts, A.G., Atkins, W.M., Campbell, A.P., Nelson,
S.D. Cooperative binding of acetaminophen and caffeine within the P450 3A4
active site. Chem. Res. Toxicol. 20:1434, 2007.
Cameron, M.D., Wright, J., Black, C.B., Ye, N. In vitro prediction and in vivo verification of enantioselective human tofisopam metabolite profiles. Drug Metab. Dispos. 35:1894, 2007.
Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright. J.J., Griffin,
P.R., Thacher, S., Hodder, P. Potent, selective and cell penetrant inhibitors of SF-1
by functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008.
Miller, B.H., Schultz, L.E., Gulati, A., Cameron, M.D., Pletcher, M.T. Genetic regulation of behavioral and neuronal responses to fluoxetine. Neuropsychopharmacology 33:1312, 2008.
Cell-Based Screening Core
J.J. Conkright, G. Zastrow, J. Cartzendafner, M. Morris
he Cell-Based Screening Core provides highthroughput screening of functional genomic
platforms and consults with researchers from
Scripps, both in California and Florida; universities
in Florida; and other outside academic institutions to
perform these screens. We curate 2 large libraries: the
Mammalian Genome Collection cDNA library and the
Qiagen Druggable siRNA library. Screening these libraries allows investigators to determine if overexpression
of a single gene (Mammalian Genome Collection cDNA
library) or reduction in expression levels of a single gene
(Qiagen siRNA library) positively or negatively influences
their particular biological readout. These libraries provide investigators a unique tool to identify novel factors and pathways involved in biological systems. The
findings can lead to new areas of research and novel
targets for drug development.
In addition to our large libraries, we have 2 small
libraries that we built: a transcription factor library and
a nuclear receptor library. These libraries are important
new tools for investigators who study the effects of proteins and signaling pathways on gene expression. These
libraries are also a mechanism for studies of the specificity of new potential drugs and chemical probes that
modulate gene expression.
A third area of expertise we provide is the generation of mutagenesis screens. Determining the regions
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372 TRANSL ATIONAL RESEARCH INSTITUTE
2008
or residues in a protein that are important for its biological function can be a key component in dissecting
how the protein interacts with other factors. Chemical
mutagenesis of a gene permits an unbiased approach
to identifying these biologically critical residues of
the protein. We perform the mutagenesis and provide
screening sets for investigators to examine the effect
the mutation has on a chosen biological end point.
Last, we counsel researchers on how to validate
their screens and counterscreen the rank-order hits.
These tasks are extremely important to prove the statistical significance of a finding and to ascertain the
specificity of the finding for that precise biological
function or pathway.
PUBLICATIONS
Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V.,
Orth, A.P., Busby, J., Hogenesch, J.B., Conkright, M.D. A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway. Proc. Natl.
Acad. Sci. U. S. A. 104:20314, 2007.
Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright, J.J., Griffin,
P.R., Thacher, S., Hodder, P. Potent, selective and cell penetrant inhibitors of SF-1
by functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008.
Discovery Biology: Kinases
THE SCRIPPS RESEARCH INSTITUTE
United States. The prognosis for these patients is poor,
and treatment options are limited. We will focus on
defining the role Jun N-terminal kinase signaling plays
in tumor maintenance and cell dispersal and whether
inhibition of this kinase has therapeutic potential in this
devastating disease.
In addition, we are involved in the Scripps-Pfizer
collaboration that was started in 2007. Since then, several assays have been designed for high-throughput
screening of targets of therapeutic interest to Pfizer.
PUBLICATIONS
Jiang, R., Duckett, D., Chen, W., Habel, J., Ling, Y.Y., LoGrasso, P., Kamenecka,
T.M. 3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors.
Bioorg. Med. Chem. Lett. 17:6378, 2007.
Lansing, T.J., McConnell, R.T., Duckett, D.R., Sephar, G.R., Knick, V.B., Hassler,
D.F., Noro, N., Furuta, M., Emmitte, K.A., Gilmer, T.M., Mook, R.A., Jr., Cheung, M.
In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1.
Mol. Cancer Ther. 6:450, 2007.
Rech, J.C., Yato, M., Duckett, D., Ember, B., LoGrasso, P.V., Bergman, R.G., Ellman, J.R. Synthesis of potent bicyclic bisarylimidazole c-jun N-terminal kinase
inhibitors by cyclic C-H bond activation. J. Am. Chem. Soc. 129:490, 2007.
Rhodes, N., Heerding, D.A., Duckett, D.R., Eberwein, D., Knick, V.B., Lansing,
T.J., McConnell, R.J., Gilmer, T.M., Zhang, S.Y., Robell, K., Kahana, J., Geske,
R.S., Kleymenova, E.V., Choudhry, A.E., Lai, Z., Leber, J.D., Minthorn, E.A.,
Strum, S.L., Wood, E.R., Huang, P.S., Copeland, R.A., Kumar, R. Characterization
of an Akt kinase inhibitor with potent pharmacodynamic and antitumor activity.
Cancer Res. 68:2366, 2008.
D.R. Duckett, J. Anderson, W. Chen, D. Harmey, Y.Y. Ling
e are investigating the use of small-molecule
kinase inhibitors of biologic interest and therapeutic potential. Protein kinases are important components of signal transduction pathways, and
deregulation of kinase activity in humans can lead to
disease. Kinases have become one of the most important target classes for drug development. We are optimizing a novel class of kinase inhibitors for treatment
of Parkinson’s disease. Although the cause of Parkinson’s
disease is unknown, a strong correlation exists between
loss of primary dopaminergic neurons within the substantia nigra and progression to the diseased state.
Our current goal is to develop an inhibitor of the
Jun N-terminal family of kinases; our aim is to protect
the primary dopaminergic neurons from cell death, thus
slowing or halting the progression of the disease. Working closely with scientists in other disciplines necessary for lead optimization (chemistry, pharmacology,
and drug metabolism), we were successful in securing
funding from the National Institute of Neurological Disorders and Stroke for this research.
We are also investigating the role of MAP kinases
in primary brain cancers. In 2008, brain tumors will
be diagnosed in approximately 20,000 patients in the
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Probe and Drug Discovery: The
Lead Identification Department
P. Hodder, A. Abovich, P. Baillargeon, P. Chase, M. Crisp,
L. DeLuca, R. Einsteder, K. Emery, F. Madoux, B. Mercer,
D. Minond, M. Petrillo, A. Porto, S. Saldanha, L. Scampavia,
M. Spaargaren, T. Spicer, V. Fernandez-Vega
he Lead Identification Department is responsible
for developing and executing high-throughput
screening (HTS) assays and for supporting downstream medicinal chemistry and related “hit-to-lead”
efforts (Fig. 1). The anchors of the department are 2
fully automated robotic platforms. One supports screening of 384- and 1536-well microtiter plates in a variety of biochemical and cell-based assay formats. The
other is used to manage and distribute the more than
600,000 compounds used for drug discovery at Scripps
Research and 300,000 compounds for the Molecular
Libraries Probe Production Centers Network. The facility
also contains an assay development laboratory, equipped
with bacterial culture, protein purification, compound
characterization, and tissue culture laboratories as well
as semi-automated equipment for liquid handling and
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TRANSL ATIONAL RESEARCH INSTITUTE
2008
THE SCRIPPS RESEARCH INSTITUTE
373
F i g . 1 . The uHTS laboratory of the
Lead Identification Department houses equipment and instrumentation
necessary to develop and support a
uHTS campaign and medicinal chemistry follow-up efforts. The anchor
of the department is a fully automated uHTS platform (center right),
which is used to screen libraries of
compounds for biological activity in a
variety of pharmacologically relevant
assays, including cell-based, protein, RNA, and DNA targets. Flanking the uHTS platform are an assay development laboratory (left and center) containing equipment and instrumentation necessary to develop an HTS assay and a mammalian tissue culture suite (upper right). Behind
the uHTS platform (not shown) is a fully automated compound management platform capable of storing, retrieving, and aliquoting desirable compounds from the screening file and a liquid chromatography–mass spectrometry platform (bottom right) used to perform routine compound quality
assurance/quality control. Not shown are fully equipped protein expression/purification and microbiology laboratories.
detection. Supporting this operation is an integrated
laboratory information management system, which tracks
HTS assay data and compound usage and quality. Additionally, we are involved in developing metallo-β-lactamase class B1 chemical probes.
related collaborations (Table 1) and have contributed to
the discovery more than 10 novel leads (chemical
probes) of G protein–coupled receptors, metalloproteinases, nuclear receptors/transcription factors, and
kinases (http://molscreen.florida.scripps.edu/).
THE SCRIPPS RESEARCH INSTITUTE MOLECULAR
DISCOVERY AND DEVELOPMENT OF CLASS B
SCREENING CENTER
M E TA L L O - β- L A C TA M A S E I N H I B I T O R S
Established in July 2005, the Scripps Research
Institute Molecular Screening Center is a national
resource for small-molecule screening and the development of chemical probes. It is 1 of 9 members in
the Molecular Libraries Probe Production Centers Network, a translational research initiative sponsored by
National Institutes of Health (NIH) and part of the NIH
Roadmap. The mission of the Scripps center is to screen
the NIH library of more than 300,000 individual compounds against peer-reviewed targets; the goal is to discover proof-of-concept probes. The results are available
to the scientific community through the PubChem Web
site of the National Center for Biotechnology Information: http://pubchem.ncbi.nlm.nih.gov. Currently, the
Lead Identification department serves as the HTS core
within the Scripps screening center; our responsibilities
are to develop biological and biochemical assays, perform HTS campaigns, manage the resulting data, act
as steward of the NIH screening library, and provide
assay support for the development of probes.
The diversity of bacterial β-lactamases continues to
outpace the development of useful β-lactam–based antibiotics. Although the development of class B β-lactamase
inhibitors has been an active area of past research, an
array of potent, class-specific small-molecule inhibitors
has yet to be fully characterized in the clinically relevant
VIM-2 metallo-β-lactamase system. Additionally, VIM2 inhibitors that are effective inhibitors of other class B
β-lactamases will be of great interest. Such compounds
will be useful as tools for characterizing gram-negative
pathogens or as adjuvant in antibiotic therapy.
One of our goals is to develop HTS-ready assays suitable for rapid identification of compounds that modulate
the activity of Ambler molecular class B (Bush-JakobyMedeiros group 3) metallo-β-lactamases, specifically
VIM-2 and IMP-1 enzymes. In preliminary research
efforts, we have developed HTS-ready fluorescence- and
absorbance-based VIM-2 and IMP-1 inhibition assays. In
collaboration with K.B. Sharpless, Department of Chemistry, we have screened a diverse click-chemistry library
or compounds designed specifically to inhibit metallo-βlactamases. Currently we are developing several novel
scaffolds that appear to be specific inhibitors.
OTHER SCREENING ACTIVITIES
Since the inauguration of the ultra-HTS (uHTS) operation in November of 2005, we have also been actively
screening the Scripps collection of compounds against
drug discovery targets not only from the MLPCN but also
from scientists at Scripps Research and from outside
partners. So far, members of the department have initiated and successfully completed more that 50 uHTS-
PUBLICATIONS
Chung, C.C., Ohwaki, K., Schneeweis, J.E., Stec, E., Varnerin, J.P., Goudreau,
P.N., Chang, A., Cassaday, J., Yang, L., Yamakawa, T., Kornienko, O., Hodder, P.,
Inglese, J., Ferrer, M., Strulovici, B., Kusunoki, J., Tota, M.R., Takagi, T. A fluorescence-based thiol quantification assay for ultra-high-throughput screening for
inhibitors of coenzyme a production. Assay Drug Dev. Technol. 6:361, 2008.
374 TRANSL ATIONAL RESEARCH INSTITUTE
2008
THE SCRIPPS RESEARCH INSTITUTE
T a b l e 1 . Summary of collaborations in the development of HTS and HTS assays.
Target class
Antibacterial
ATPase
Target name (Abbreviation)
β-Lactamase
VIM-2
Pseudomonas aeruginosa
p97
IMP-1
5HT1a
5HT1e
GalR2
GPR7
S1P1
S1P2
G protein–coupled receptor
S1P3
GLP-1
GPR119
m Opioid heterodimers
NPY- Y1
NPY- Y2
RBBP9
b-gluc
Aquaporins (AQP)
Ion channel
TRPML3
TRPN1
JAK2
JNK3
Kinase
P. Hodder, Scripps Research, Jupiter, Florida
M. Teitler, Albany Medical College, Albany, New York
S. Brown, Scripps Research, La Jolla, California
O. Civelli, University of California, Irvine, California
H. Rosen, Scripps Research, La Jolla, California
S1P4
AGTRL-1 (APJ)
Hydrolase
Collaborator, affiliation
R. Miller, Pfizer, Groton, Connecticut
R. Deshaies, California Institute of Technology, Pasadena, California
ROCK2
PKA
FAK
L. Smith, Burnham Institute for Medical Research, Orlando, Florida
P. LoGrasso, Scripps Research, Jupiter, Florida
P. McDonald. Scripps Research, Jupiter, Florida
L. Devi, Mt. Sinai School of Medicine, New York, New York
C. Wahlestedt, Scripps Research, Jupiter, Florida
B. Cravatt, Scripps Research, La Jolla, California
J. Kelly, Scripps Research, La Jolla, California
M. Yeager, Scripps Research, La Jolla, California
S. Heller, Stanford University, Stanford, California
R. Levine, G. Gilliland, Sloan Kettering, New York, New York
P. LoGrasso, Scripps Research, Jupiter, Florida
T. Schröter, Scripps Research, Jupiter, Florida
P. Hodder, Scripps Research, Jupiter, Florida
ADAMTS4
MMP13
Metalloproteinase
Falciparum M18 metalloprotease
IDE
NADPH oxidase
Nox-1
SHP-1
Estrogen receptor
Nuclear receptor
RAR
SF1 (NR5A1)
RORa (NR1F1)
Phosphotransferase
Proliferation/viability
TPT1
Jurkat E6.1 cells
EphB4-ephrinB2
Protein/protein
HCV core homodimer
NS5B/CYPB
Hsp70
Protein misfolding
AL-09
PERK
Protein/RNA
Reductase
Stem cell proliferation
G. Fields, Florida Atlantic University, Boca Raton, Florida
MMP8
HIV Rev-RRE RNA
msrA
Notch
D. Gardiner, Queensland Institute of Medical Research, Queensland, Australia
M. Leissring, Mayo Clinic, Jacksonville, Florida
G. Bokoch, Scripps Research, La Jolla, California
P. Griffin, Scripps Research, Jupiter, Florida
K. Nettles, Scripps Research, Jupiter, Florida
P. Griffin, Scripps Research, Jupiter, Florida
X. Li, Orphagen Pharmaceuticals, San Diego, California
H. Harding, New York University, New York, New York
P. Hodder, Scripps Research, Jupiter, Florida
P. Kuhn, Scripps Research, La Jolla, California
D. Strosberg, Scripps Research, Jupiter, Florida
R. Morimoto, Northwestern University, Chicago, Illinois
M. Ramirez-Alvarado, Mayo Clinic, Rochester, Minnesota
D. Ron, New York University, New York, New York
J. Williamson, Scripps Research, La Jolla, California
H. Weissbach, Florida Atlantic University, Boca Raton, Florida
H. Petrie, Scripps Research, Jupiter, Florida
PPARg/Src1
PPARg/Src2
P. Griffin, Scripps Research, Jupiter, Florida
PPARg/Src3
Transcription factor
NF-kB
STAT1
STAT3
KLF5
AHR
Ubiquitin proteolysis
WEE1
J. Reed, Burnham Institute for Medical Research, La Jolla, California
D. Frank, Dana-Farber Cancer Institute, Boston, Massachusetts
V. Yang, Emory University, Atlanta, Georgia
M. Denison, University of California, Davis, California
N. Ayad, Scripps Research, Jupiter, Florida
TRANSL ATIONAL RESEARCH INSTITUTE
2008
Lauer-Fields, J.L., Minond, D., Chase, P.S., Baillargeon, P.E., Saldanha, S.A.,
Stawikowska R., Hodder, P., Fields, G.B. High throughput screening of potentially
selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate. Bioorg.
Med. Chem. Lett., in press.
Lauer-Fields, J.L., Spicer, T.P., Chase, P.S., Cudic, M., Burstein, G.D., Nagase,
H., Hodder, P., Fields, G.B. Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate. Anal. Biochem. 373:43, 2008.
Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright, J.J., Griffin,
P.R., Thacher, S., Hodder, P. Potent, selective and cell penetrant inhibitors of SF-1
by functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008.
Roth, J., Madoux, F., Hodder, P., Roush, W.R. Synthesis of small molecule inhibitors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on isoquinolinone scaffolds. Bioorg. Med. Chem. Lett. 18:2628, 2008.
Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P.,
Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M.,
Tsinoremas, N., LoGrasso, P., Hodder, P. Comparison of miniaturized time-resolved
fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. Biomol.
Screen. 13:17, 2008.
Schürer, S.C., Brown, S.J., Gonzales-Cabrera P.J., Schaeffer, M.T., Chapman, J.,
Jo, E., Chase, P., Spicer, T., Hodder, P., Rosen, H. Ligand-binding pocket shape
differences between sphingosine 1-phosphate (S1P) receptors S1P1 and S1P3
determine efficiency of chemical probe identification by ultrahigh-throughput
screening. ACS Chem. Biol. 3:486, 2008.
Discovery Biology:
G Protein–Coupled Receptors
P. McDonald, D. Obradovich, A. Smith, E. Sturchler,
S. Tabrizifard
protein–coupled receptors (GPCRs) are the largest
and most versatile family of cell-surface receptors. The ubiquitous cell-surface distribution and
involvement of these proteins in almost all biological
processes explain why the largest percentage of currently marketed therapeutic drugs target these receptors.
We focus on developing biochemical and cell-based
functional assays to monitor GPCR activity that involve
high-throughput and high-content technologies. Using
a multidisciplinary approach that involves collaborations with disciplines such as lead identification, chemistry, drug metabolism and pharmacokinetics, and in
vivo pharmacology, we aim to identify and develop
small-molecule modulators of GPCRs for the treatment
of metabolic diseases such as type 2 diabetes mellitus
and obesity.
We have developed a series of novel cell-based
assays for the glucagon-like peptide 1 receptor (GLP-1R)
to promote a drug discovery program for this clinically
validated target of type 2 diabetes. In parallel with the
GLP-1R assays, we have developed similar assays for
2 other closely related receptors, GLP-2R and glucagon
receptor, which serve as counterscreens for selectivity
against GLP-1R. We are also working on an orphan
G
THE SCRIPPS RESEARCH INSTITUTE
375
GPCR that has been implicated in type 2 diabetes and
obesity and that signals and functions in an analogous
manner to GLP-1R. In collaboration with P. Kenny,
Molecular Therapeutics, we are also developing smallmolecule inhibitors of a GPCR previously shown to be
involved in drug dependence that may lead to a novel
therapy for substance abuse.
As part of the collaboration between Scripps and
Pfizer, Inc., that was initiated in 2007, we are designing
and developing 3–4 assays per year for GPCR targets of
therapeutic interest to Pfizer.
In Vivo Pharmacology
A.S. Pachori, M. Ganno, S. Khan, S. Clapp, D. Hansen
he In Vivo Pharmacology group at Scripps Florida
is an integrated group of investigators involved
in preclinical studies in support of drug discovery
efforts at Scripps Research in both Florida and California. We develop appropriate animal models of diseases
for ongoing projects such as studies in hypertension,
glaucoma, Parkinson’s disease, diabetes, and heart failure. These models are then used to test the efficacy of
new compounds for a particular therapeutic area. An
important aspect of establishing the efficacy of a compound is to determine if the compound is altering its
intended target. We initially evaluate the role of a particular target in primary cell culture and then evaluate
the target in vivo. The cell culture experiments are also
used to screen novel compounds for the effects of the
compounds on targets.
In addition, we evaluate the pharmacodynamic
properties of new chemical entities in vivo by doing
dose-response and time-course studies to determine
the effects of the compounds on the intended target.
For these evaluations, we use standard techniques such
as Western blotting, enzyme-linked immunosorbent
assays and immunohistochemistry. We also collaborate with the drug metabolism and pharmacokinetics
laboratory to monitor plasma and tissue concentrations of chemical entities; the results help us further
refine and develop the disease models. Finally, we evaluate the compounds for toxicity.
In the past year, we have successfully developed
animal models of hypertension to test the efficacy of
Rho kinase inhibitors as novel antihypertensive agents.
We have screened several novel Rho kinase inhibitors
for their efficacy and toxicity and established the need
for isoform-selective inhibitors to avoid toxicity issues.
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376 TRANSL ATIONAL RESEARCH INSTITUTE
2008
We have also established the use of primary dopaminergic neurons in an in vitro target modulation assay
to screen compounds for treatment of Parkinson’s disease. In addition, we have adopted a strategy to explore
and seek alternative therapeutic uses for novel compounds currently under development. For example, we
have expanded the use for novel Jun N-terminal kinase
inhibitors for Parkinson’s disease to include their use
as cytoprotective agents against heart failure induced
by ischemia-reperfusion injury. Our preliminary data indicate that in rodents, these inhibitors can successfully
reduce tissue damage in a dose-dependent manner.
We are also exploring the use of Rho kinase inhibitors,
which were originally developed as antihypertensive
agents, in the treatment of glaucoma. This strategy
will help us not only expand our portfolio but also discover new approaches for some of the biggest unmet
needs of patients.
Omics Informatics
B.D. Pascal
he Omics Informatics group at Scripps Florida
addresses support and software needs of various
laboratories. Our goals are to identify and integrate
existing solutions where possible and to build new solutions only when necessary. A primary specialization is
the analysis and management of mass spectrometric data
and development of proteomics software research tools
that enable proteomics researchers to validate, visualize, and share their data.
T
HD DESKTOP
Scientists at Scripps Florida are using hydrogendeuterium exchange mass spectrometry to characterize
protein dynamics and protein-protein or protein-ligand
interactions. The Deuterator software, released last year,
addresses some of the data analysis bottlenecks by
providing a platform to automate and visualize centroid
calculations. Despite these efforts, the task of assembling and visualizing the resulting data is still a manual operation left to the end user. The current software,
HD Desktop, leverages the existing code base and stores
all data in a relational database. Novel rendering and
analysis tools have been presented in an integrated user
interface (Fig. 1).
M A S S S P E C T R O M E T R Y C A L I B R AT I O N S O F T WA R E
Although every mass spectrometry laboratory will
vary in instrument manufacturer and experimental
designs, most proteomics laboratories have a common
THE SCRIPPS RESEARCH INSTITUTE
F i g . 1 . HD Desktop experiment view.
need to measure and validate the calibration of the instruments. An automated method to process and display the
mass calibration shift at various time points allows the
maximization of instrument run time and contributes to
the standardization and validation of proteomics data.
We have developed an automated method for determining calibrated mass drift on high-resolution instruments. On a daily basis, the quality of the data collection
is monitored by routine analysis of a tryptic digest of a
β-casein standard. The resulting binary files are collected,
moved into the laboratory information management system, and then processed through an automated workflow, which conducts file conversions and peak quality
assessments and sends the files to a compute cluster for
peptide identification search. The results are then parsed,
and the difference between the observed and calculated
mass is stored in a database; the data are made available through a Web-based interface (Fig. 2, page 377).
PUBLICATIONS
Chalmers, M.J., Busby, S.A., Pascal, B.D., Southern, M.R., Griffin, P.R. A twostage differential hydrogen deuterium exchange method for the rapid characterization of protein/ligand interactions. J. Biomol. Tech. 18:194, 2007.
Pascal, B.D., Chalmers, M.J., Busby, S.A., Mader, C.C., Southern, M.R., Tsinoremas,
N.F., Griffin, P.R. The Deuterator: software for the determination of backbone amide
deuterium levels from H/D exchange MS data. BMC Bioinformatics 8:156, 2007.
Drug Discovery Biology:
Cell Biology
T. Schröter, A.M.W. Handy, E. Griffin, J.R. Pocas, K. Clarke,
C. Hahmann
W
ith more than 500 members, protein kinases
are important drug discovery targets for a
wide variety of therapeutic indications. These
TRANSL ATIONAL RESEARCH INSTITUTE
2008
THE SCRIPPS RESEARCH INSTITUTE
377
For the cancer program, in 2007, we successfully
finished a collaboration with scientists at Poniard Pharmaceuticals, Inc., South San Francisco, California, to
discover novel inhibitors of focal adhesion kinase. This
kinase has been implicated in tumor cell detachment
and metastasis. We supported the program by developing biochemical and cell-based assays to monitor
the effect of newly discovered small molecules on biochemical inhibition of focal adhesion kinase and on
cellular growth, migration, and invasion. We are also
collaborating with researchers at Pfizer, Inc., in developing biochemical and cell-based high-throughput
screening assays for a diverse set of novel disease
targets, including protease inhibitors, hydrolases, and
membrane transporters.
PUBLICATIONS
Feng, Y., Cameron, M.D., Frackowiak, B., Griffin, E., Lin, L., Ruiz, C., Schröter,
T., LoGrasso, P. Structure-activity relationships and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of
ROCK-II. Bioorg. Med. Chem. Lett. 17:2355, 2007.
F i g . 2 . Data flow of mass spectrometry calibration software.
kinases control signal transduction pathways, and in
humans, deregulation of their activity can lead to
diseases such as glaucoma and cancer. The serine-threonine kinase Rho kinase (ROCK) regulates intraocular
pressure by controlling the outflow of aqueous humor. In
glaucoma, increased intraocular pressure leads to loss of
retinal ganglion cells and, ultimately, loss of vision.
Inhibition of ROCK activity increases outflow, lowers
intraocular pressure, and preserves retinal neurons. We
concentrate on developing biochemical and cell-based
functional assays to monitor ROCK activity via both highcontent and high-throughput screening technologies.
Working closely with researchers in high-throughput screening, medicinal chemistry, pharmacology, and
drug metabolism and pharmacokinetics, we identified
small-molecule lead compounds from a high-throughput screening of the Scripps collection of more than
700,000 compounds. During lead optimization, we
screened thousands of new ROCK inhibitors for the
biochemical activity against the enzyme and the close
family members protein kinase A, Akt1, and MRCKα.
Hundreds of these compounds were chosen, and their
cell-based activity against ROCK was tested by using
target modulation and functional assays. Changes in
myosin light-chain phosphorylation were measured by
using a 96-well immunocytochemical assay and infrared
imaging, and changes in the formation of stress fibers
and neurite protection were evaluated by using a highcontent imaging system.
Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P.,
Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M.,
Tsinoremas, N., Lograsso, P., Hodder, P. Comparison of miniaturized time-resolved
fluorescence resonance energy transfer and enzyme-coupled luciferase highthroughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J.
Biomol. Screen. 13:17, 2008.
Chemical Informatics Program
S.C. Schürer, D. Vidović, C. Chung
e collaborate with scientists in the Scripps
Research Institute Molecular Screening Center,
and we also received a grant from the Columbia University Molecular Library Screening Center. Both
of these centers are part of the national Molecular Libraries Screening Centers Network. We are also involved in
drug discovery efforts within the Translational Research
Institute. We have developed a platform of industry-standard software tools for analysis, visualization, hypothesis
building, and modeling of large and focused experimental
screening data sets. Our platform enables us to generate
and evaluate a large variety of structural, pharmacophore,
and physicochemical 2- and 3-dimensional descriptors.
The platform includes computational chemistry tools for
3-dimensional pharmacophore-alignment ligand-based
quantitative structure-activity relationships, ligand-protein
docking (in a variety of approaches and scoring functions), homology modeling, molecular modeling and
dynamics, and statistical tools.
We have also developed various interactive reporting
and visualization protocols that are used in collaborative
research. Our platform provides broad chemical informat-
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378 TRANSL ATIONAL RESEARCH INSTITUTE
2008
ics and computational chemistry capabilities. Examples
of research projects in which these technologies play a
key role include analysis of data on toxic effects in cells
and animals; development of small-molecule modulators
of a broad variety of targets, including metalloproteases,
phosphatases, kinases, nuclear receptors, and sphingosine
lipid receptors (for which we also modeled the receptor
structures); and image-based high-content assays, including probing the inflammatory pathway at the stage of
NF-κB translocation and expression of E-selectin or vascular adhesion molecule 1 and targeting the aggregation
of the protein huntingtin.
In collaboration with the screening informatics team,
we play a key role in implementing work flow, procedures, and business rules and in integrating discovery
informatics with the operational informatics infrastructure to facilitate discovery processes. Examples include
the Scripps Research compound registration system;
integration of the Molecular Libraries Screening Centers
Network chemoinformatics server, which is hosted at
Scripps Research, with PubChem; integration of images
from image-based screening with the operational infrastructure; and publication of screening data to PubChem.
To date, more than 5.5 million data points and more
than 120 assays obtained by using these protocols have
been published.
Other chemoinformatics research efforts are focused
on structure-based comprehensive analyses of target-similarity relationships in the phosphatase and kinase gene
families, ligand-based and target “fishing,” and the development of integrative applied chemoinformatics methods.
PUBLICATIONS
Guha, R., Schürer, S.C. Utilizing high throughput screening data for predictive toxicology models: protocols and application to MLSCN assays. J. Comput. Aided Mol.
Des. 22:367, 2008.
Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P.,
Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M.,
Tsinoremas, N., Lograsso, P., Hodder, P. Comparison of miniaturized time-resolved
fluorescence resonance energy transfer and enzyme-coupled luciferase highthroughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J.
Biomol. Screen. 13:17, 2008.
Xie, Y., Deng, S., Thomas, C.J., Liu, Y., Zhang, Y.Q., Rinderspacher, A., Huang,
W., Gong, G., Wyler, M., Cayanis, E., Aulner, N., Többen, U., Chung, C., Pampou, S., Southall, N., Vidović, D., Schürer, S., Branden, L., Davis, R.E., Staudt,
L.M., Inglese, J., Austin, C.P., Landry, D.W., Smith, D.H., Auld, D.S. Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating
NFκB activity within two separate high-throughput screens of NFκB activation.
Bioorg. Med. Chem. Lett. 18:329, 2008.
Xie, Y., Liu, Y., Gong, G., Rinderspacher, A., Deng, S.X., Smith, D.H., Többen, U.,
Tzilianos, E., Branden, L., Vidović, D., Chung, C., Schürer, S., Tautz, L., Landry,
D.W. Discovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine
phosphatase. Bioorg. Med. Chem. Lett. 18:2840, 2008.
THE SCRIPPS RESEARCH INSTITUTE
Screening Informatics Program
M.R. Southern, K. Hoda, Y. Cruz
cientists in the Screening Informatics Program
at Scripps Florida collaborate broadly with
researchers in the Scripps Research Institute
Molecular Screening Center, part of the national Molecular Libraries Screening Centers Network, and in drug
discovery efforts within the Translational Research
Institute. Our responsibilities include high- and lowthroughput screening assays and downstream drug
metabolism and pharmacology, medicinal chemistry,
and probe development. We have an operational environment for data management and quality assurance
and a knowledge environment that facilitates efficient
optimization of probes. These activities take place at
both the Florida and the California sites.
The software systems have been built primarily
by using the MDL Discovery Experiment Management
Framework from Symyx Technologies, Inc., Santa Clara,
California, and support specific work flows involving
tasks such as chemical compound registration, plate
and sample registration, assay development, and entire
screening campaigns. On top of these systems, we have
developed in-house software that is tightly coupled to
provide additional functionality and to improve our efficiency. Examples include robotic automation, plate mapping operations, and structure search. A key component
is our Assay Exploration Data Warehouse, which along
with its Web-based front end is known to end users
as ChemInfo.
ChemInfo is assay metric and structure centric,
enabling exploration of assay data by compound, target,
or assays. It integrates chemical descriptors, physical
properties, and data on drug metabolism and pharmacokinetics to facilitate probe optimization. Complicated
Venn-like queries are possible. ChemInfo contains individual and aggregated assay data from our internal
assays as well as from PubChem. The database has
up to 30 users within Scripps Research.
S
PUBLICATIONS
Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P.,
Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M.,
Tsinoremas, N., Lograsso, P., Hodder, P. Comparison of miniaturized time-resolved
fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput
screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. Biomol.
Screen. 13:17, 2008.