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

DEPARTMENT OF

CANCER BIOLOGY

STAFF Jun-Li Luo, Ph.D., MD HaJeung Park, Ph.D. Ann Griffith, Ph.D. Assistant Professor John L. Cleveland, Ph.D. Meredith A. Steeves, Ph.D. Mark A. Hall, Ph.D. Kendall Nettles, Ph.D. Professor and Chairman Weilin Wu, Ph.D. Jun Hyuck Lee, Ph.D. Assistant Professor Howard Petrie, Ph.D. Woonghee Lee, Ph.D. Professor SCIENTIFIC ASSOCIATE RESEARCH ASSOCIATES Hiroshi Nakase, Ph.D. Tina Izard, Ph.D. Associate Professor Chunying Yang, M.D. Antonio Amelio, Ph.D. Robert J. Rounbehler, Ph.D.

Nagi G. Ayad, Ph.D. Mi Ra Chang, Ph.D. Jianjun Shi, Ph.D. Assistant Professor STAFF SCIENTISTS Frank C. Dorsey, Ph.D. Zhen Wu, Ph.D. Philippe R.J. Bois, Ph.D. Joanne R. Doherty, Ph.D. Rangarajan Erumbi, Ph.D. Bhargavakrishna Yekkala, Assistant Professor Ph.D. Ihn Kyung Jang, Ph.D. Irina Getun, Ph.D. Michael Conkright, Ph.D. Sollepura Yogesha, Ph.D. Assistant Professor German Gil, Ph.D. Min Zhao, Ph.D.

Chairman’s Overview battery of state-of-the-art technologies for target discovery and validation, ranging from biochemistry and he Department of Cancer Biology on the Florida cell biology to preclinical models to x-ray crystallogra- campus was established in November 2006. The phy. In addition, unique models have been developed T department has rapidly grown to now include to evaluate the efficacy of new leads in cancer preven- 8 faculty members. The tion and therapeutics. Investigators in the department broad goals of the have interests in understanding the molecular under- research programs of pinnings of all of the major human malignant neoplasms, the department are to including lung, breast, prostate, colon, and brain can- fully define the molecu- cer, and several hematologic malignant neoplasms. lar events that underlie Other interests include pediatric oncology, the inter- human cancer and then play between malignant neoplasms and metabolism, apply this knowledge and the relationships between aging and cancer. to the development of One of the many strengths at the Florida campus novel therapeutic strate- is high-throughput technologies that enable investiga- gies and new agents for tors to rapidly move forward potential leads by using cancer prevention and both genetic and small-molecule screens. Strong col- John L. Cleveland, Ph.D. therapeutics. laborations with the major cancer centers in the State The programs include those that examine the roles of Florida and with cancer researchers at the Califor- of signal transduction pathways, oncogenes, and tumor nia campus of Scripps Research will allow leads that suppressors that are altered in cancer and how these are identified to rapidly advance to translational and alterations control cell division, growth, survival, dif- clinical studies. ferentiation, 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 20 CANCER BIOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’ REPORTS SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1 for destruction by the 26S proteasome. Accordingly, loss Myc-Mediated Pathways in of Cks1 disables the ability of Myc to suppress p27Kip1 and markedly impairs Myc-induced proliferation and Cancer and Development tumorigenesis, whereas loss of p27Kip1 accelerates Myc- induced tumorigenesis. Remarkably, Cks1 overexpression J.L. Cleveland, M.A. Hall, F.C. Dorsey, R. Rounbehler, is a hallmark of all lymphomas with Myc involvement, K. Yekkala, J. Doherty, M. Steeves, C. Yang, T. Bratton, suggesting this pathway is a general route by which Myc S. Prater, W. Li coordinates cell growth and division and that the path- yc oncoproteins function as master regulators way can be targeted by directed therapeutic agents. of transcription and regulate up to 10%–15% Because Myc regulates such a large number of genes M of the genome. Three Myc oncogenes (c-Myc, and is essential for cell growth and division, the adverse N-Myc, L-Myc) are activated in about 70% of human effects of agents that directly target the transcription cancers. Their activation can occur directly via gene functions of Myc might be greater than the agents’ bene- amplification, chromosomal translocations, or somatic ficial effects. We therefore have focused our efforts on missense mutations or indirectly via alterations in signal key transcription targets of Myc that might be suitable transduction pathways or the loss of tumor suppressors therapeutic targets. We found that inhibiting ornithine that normally regulate and/or harness Myc expression. decarboxylase, a direct transcription target of Myc and The pervasive selection for Myc activation in cancer in the rate-limiting enzyme of polyamine biosynthesis, part reflects the essential roles of Myc as a regulator impairs Myc-induced proliferation and tumorigenesis. of cell growth and division, but overexpression of Myc These results were underscored by our findings that also triggers accelerated rates of cell proliferation, tumor heterozygosity in the gene that encodes ornithine decar- angiogenesis, and metastasis. Further, Myc regulates boxylase, a condition that only reduces the enzyme stem cell fate and supercompetition, a scenario in which activity of ornithine decarboxylase and the generation cells that overexpress Myc kill their neighboring, nor- of its product by half, triples the life span of tumor-prone mal cells. mice. Thus, agents that target the polyamine pathway We have used mouse models to dissect the contri- have promise in both the prevention and the treatment bution of key targets downstream of Myc that control of cancer. Currently, we are defining the mechanism by tumorigenesis. In normal cells, Myc triggers apoptosis which targeting ornithine decarboxylase disables the through the Arf-p53 tumor suppressor pathway that is proliferative response of Myc. Our results indicate, quite inactivated in most malignant tumors and by selectively remarkably, that targeting ornithine decarboxylase dis- Kip1 affecting the expression of members of the Bcl-2 fam- ables the ability of Myc to suppress p27 by short- ily of proteins that directly control the intrinsic apoptotic circuiting of the Myc-to-Cks1 pathway. pathway. We have shown that these pathways hold Finally, we recently discovered that additional Myc Myc-induced tumorigenesis in check and that mutations transcription targets that can be exploited in cancer in these apoptotic regulators are a hallmark of most therapy include components of the autophagy pathway, malignant tumors. an ancient survival pathway that directs the digestion Although apoptotic regulators clearly serve as guard- of bulk cytoplasmic material and organelles when cells ians against Myc-induced cancer, we have found that are faced with nutrient- or oxygen-deprived conditions, the ability of Myc to provoke accelerated cell growth is a scenario manifests in the tumor microenvironment. also critical for tumorigenesis. First, Myc coordinately We have shown that agents that disable autophagy have regulates the expression of cytokines that direct cell tremendous potential in cancer prevention and treat- growth and tumor angiogenesis. Second, Myc sup- ment. Currently, we are defining the mechanisms by presses expression of the universal cyclin-dependent which Myc regulates the expression of genes that con- kinase (Cdk) inhibitor p27Kip1 that normally inhibits the trol the autophagy pathway and their potential as tar- activity of cyclin E–Cdk2 and cyclin A–Cdk2 complexes gets for agents to prevent and treat cancer. that are necessary for entry and progression through PUBLICATIONS the DNA synthesis (S) phase of the cell cycle. Notably, Carew, J.S., Nawrocki, S.T., Reddy, V.K., Bush, D., Rehg, J.E., Goodwin, A., we found that Myc suppresses p27Kip1 protein levels 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. Can- by inducing transcription of the Cks1 component of the cer Res. 68:4783, 2008. CANCER BIOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE 21

Garrison, S.P., Jeffers, J.R., Yang, C., Nilsson, J.A., Hall, M.A., Rehg, J.E., Yue, VINCULIN STRUCTURE AND REGULATION 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. Our crystal structures, biochemical studies, and Mol. Cell. Biol. 28:5391, 2008. biological experiments have redefined vinculin structure

Klionsky, D.J., Abeliovich, H., Agostinis, P., et al. Guidelines for the use and inter- and regulation. First, in its resting, inactive conforma- pretation of assays for monitoring autophagy in higher eukaryotes. Autophagy tion, vinculin is held in a closed-clamp conformation 4:151, 2008. through interactions of a 7-helical bundle domain pre- 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 mod- sent in its head domain (Vh1) with a 5-helical bundle els of lymphomagenesis. J. Clin. Invest. 118:79, 2008. in the tail domain (Vt); 3 additional helical bundle

Nawrocki, S.T., Carew, J.S., Douglas, L., Cleveland, J.L., Humphreys, R., Houghton, domains that were identified likely also serve as dock- J.A. Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a ing sites for interactions with partners. Second, contrary p21Cip1-dependent decrease in survivin levels. Cancer Res. 67:6987, 2007. to dogma, we found that, talin itself is a direct activa- 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 forma- tor of vinculin; α-helical vinculin-binding sites (VBSs) tion, the induction of Noxa, and apoptosis in response to the combination of borte- in the central rod domain of talin trigger vinculin activa- zomib and SAHA. Blood 112:2917, 2008. tion by displacing Vt from a distance. More importantly, our structures revealed that this activation of vinculin 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 and displacement of Vt occurred via a heretofore unknown 135:1903, 2008. change in protein structure, by a process we termed Sanjuan, M.A., Dillon, C.P., Tait, S.W., Moshiach, S., Dorsey, F., Connell, S., helical bundle conversion (Fig. 1). Third, our studies 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.

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 C 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 Fig. 1. Vinculin activation by talin through helical bundle con- both cell-cell (cadherin-catenin mediated) and cell-matrix version. Ribbon drawing of the vinculin head (Vh1; cyan) and tail (integrin-talin mediated) junctions, where it provides (Vt; yellow) domains activated by talin’s VBS (red). A, The crystal links to the actin cytoskeleton by binding to talin in structure of the Vh1-Vt complex revealed that vinculin is held in a closed conformation through many hydrophobic interactions between integrin complexes or to α-catenin and α-actinin in the Vh1 and Vt domains. B, The crystal structure of talin-VBS3 cadherin junctions. Previously, little was known about bound to Vh1 shows that talin binds to vinculin at an accessible site the structure and activation of vinculin, although the distal from the Vh1-Vt interface and that talin-VBS3 binding dis- accepted belief was that activation required severing places Vt from a distance. Wholesale structural changes occur upon talin binding, whereby the 4-helical bundle of Vh1 incorporates the intramolecular interactions of the vinculin head and amphipathic VBS helix of talin to form an entirely new 5-helical tail domains. bundle via helical bundle conversion. 22 CANCER BIOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE of the complex composed of α-actinin and vinculin studies showed that IpaA acts as a talin mimic that revealed that α-actinin activates vinculin and alters its disrupts vinculin’s contacts with talin and α-actinin, structure in unique ways. These findings supported our and our results suggest that this mechanism is a gen- model in which the vinculin Vh1 domain functions as eral one that is exploited by other pathogens. Impor- a “molecular switch” that undergoes rapid and unique tantly, our biological studies have shown that this changes in its structure after binding to different acti- interaction is necessary for efficient entry of Shigella vators, which then endow vinculin with the ability to into host cells. bind to unique partners in adherens junctions vs focal Our recent studies have revealed additional layers adhesions. Finally, our studies indicated that adhesion of functional complexity of IpaA. First, we found that signaling involves a chain reaction of structural alter- the second, somewhat lower affinity, VBS of IpaA can ations in which, after their activation, the VBSs of talin bind to a second motif of the vinculin Vh1 domain, a or α-actinin first unravel from their buried locations and situation that would stabilize IpaA-vinculin interactions then bind to and activate vinculin, which then under- (Fig. 3). Second, our biochemical and genetic screens goes wholesale changes in its structure (Fig. 2).

Fig. 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 head- tail interaction. The α-actinin antiparallel homodimer has 2 VBS sites. For clarity, only 1 vinculin molecule is shown bound to the α-actinin homodimer.

TARGETING VINCULIN IN PATHOGEN-HOST INTERACTIONS Fig. 3. A novel second binding site on the vinculin Vh1 domain We have also made significant inroads in under- allows IpaA to bind vinculin in its closed conformation. A, Ribbon standing how vinculin is co-opted by pathogens. Initially, drawing of the vinculin head domain (Vh1; yellow) bound by the 2 we have focused on Shigella flexneri, the principal path- VBSs of S flexneri IpaA. The first IpaA-VBS (blue) binds to Vh1 with ogen of bacillary dysentery. Shigella organisms inject femtomolar affinity by molecular mimicry of the Vh1-talin interaction, via helical bundle conversion. In contrast, the second IpaA-VBS (red) invasin proteins (IpaA-IpaD) that create pores in intestinal binds vinculin by a helix addition mechanism, a scenario that allows epithelial cells and that trigger the formation of filopo- the S flexneri invasin to also recruit pools of inactive vinculin and dial and lamellopodial extensions that surround the bac- that would also facilitate the bridging of 2 molecules of vinculin by teria. IpaA, a protein of approximately 70 kD essential IpaA. B, Surface drawing of full-length human vinculin (yellow, orange, for the pathogenesis of Shigella in vivo, facilitates entry magenta, blue, gray, and cyan) bound to IpaA-VBS (ribbon drawing, of the bacteria into host cells by binding to vinculin. red). The weaker binding IpaA-VBS binds vinculin by a helix addition mechanism, which has no allosteric effects on vinculin, allowing We established that IpaA has 2 high-affinity VBSs that IpaA to bind to vinculin in its closed conformation. Reprinted from bind to the Vh1 domain of vinculin and induce unique Nhieu, G.T., Izard, T. Vinculin binding in its closed conformation by alterations in the domain’s structure. Strikingly, our a helix addition mechanism. EMBO J. 26:4588, 2007. CANCER BIOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE 23 have revealed new cytoskeletal binding partners for IpaA. β-transducin repeat–containing protein and trigger of The physiologic roles of these interactions will be tested, mitotic entry 1 are required for Wee1 destruction and and along with our structural analyses, these studies mitotic entry. These findings indicated that eukaryotic may point to new therapeutic avenues. cells have multiple means of regulating Wee1 degradation, because inactivation of a single critical component PUBLICATIONS Nhieu, G.T., Izard, T. Vinculin binding in its closed conformation by a helix addi- would lead to premature mitosis and disastrous conse- tion mechanism [published correction appears in EMBO J. 27:922, 2008]. EMBO quences for the organism. We are determining the J. 26:4588, 2007. respective roles of these ligases in cancer progression.

PUBLICATIONS Regulation of Mitotic Entry and Smith, A., Simanski, S., Fallahi, M., Ayad, N.G. Redundant ubiquitin ligase activities regulate Wee1 degradation and mitotic entry. Cell Cycle 6:2795, 2007. Exit by Ubiquitin-Mediated Proteolysis Anatomy and Regulation of N. Ayad, S. Simanski, N. Nagarsheth Mouse Recombination Hot Spots biquitin-mediated proteolysis is one of the main P.R. Bois, M. Fallahi-Sichani, I. Getun, Z.K. Wu ways cells eliminate intracellular proteins. This U elimination is important for cell homeostasis, rossover events are necessary for meiosis pro- development, and growth. Recent studies have also gression and for genome reshuffling and diversity indicated that one or more components of this system C before the generation of gametes. A peculiarity are overexpressed in cancer cells, making the compo- of meiosis is the programmed nature of double-strand nents attractive targets for pharmacologic inhibition. breaks, which are induced by the conserved endonu- Although a fair amount is known about the pathways clease Spo11. Studies in a variety of model systems leading to ubiquitin-mediated degradation, many essen- have shown that recombination occurs in distinct regions tial components have not been identified. termed hot spots. An estimated 5%–10% of the genomes We devised a means of identifying regulators of of higher eukaryotes are recombinogenic; the remain- the anaphase-promoting complex (APC), an essential der reside in the “cold.” However, little is known about ubiquitin ligase required for the metaphase-to-anaphase the nature and mechanisms that control recombination transition and exit from mitosis. We fused cyclin B1, a hot spots in mammalian genomes. known APC substrate, to luciferase and cotransfected We have focused on identifying novel hot spots in the this fusion construct with 14,000 cDNAs. This genome- mouse chromosome 19 by directly detecting crossover wide screen led to the identification of multiple regula- events. Characterization of a highly polymorphic hot spot tors of the APC, including some that are overexpressed (HS23.7) revealed quite unexpectedly that repair does in breast cancer. Currently, we are identifying the mech- not have to be complete for meiosis to proceed. The per- anism by which these proteins regulate the APC during sistence of these unrepaired heteroduplex regions at exit from mitosis and are developing high-throughput crossover sites in mature spermatozoa promotes genome screens. Our eventual goal is to eliminate the activity instability that was revealed by the high diversity and of the proteins selectively in cancer cells. rearrangements observed in laboratory strains and wild APC activity is inhibited before mitosis because its mouse populations. We are identifying and characterizing premature activation would lead to genomic instability. more of these recombinogenic regions where chromo- One way the APC is inhibited is by inhibiting the somes are reshuffled between generations and provide cdk1/cyclin B complex required for entry into mitosis. the driving mechanism at the origin of diversity. This complex is kept inactive before mitosis because it PUBLICATIONS is phosphorylated by the tyrosine kinase Wee1. Wee1 Bois, P.R. A highly polymorphic meiotic recombination mouse hot spot exhibits is degraded during the G2 phase and mitosis to tip the incomplete repair. Mol. Cell. Biol. 27:7053, 2007. 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 24 CANCER BIOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE Molecular Mechanisms of novel compounds are also being used to explore the biological effects of inhibiting inflammatory programs cAMP-Mediated Transcription in cancer cell proliferation and invasion and the role of estrogen signaling in immune function. M.D. Conkright, A.L. Amelio, N.E. Bruno PUBLICATIONS lucose homeostasis is maintained by coordinat- 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 inde- ing glucose metabolism in skeletal muscle, lipid pendent mechanism. Structure 15:1258, 2007. storage in adipose tissue, and glucose produc- G Nettles, K.W., Bruning, J.B., Gil, G., Nowak, J., Sharma, S.K., Hahm, J.B., Kulp, tion in the liver. Insulin and glucagon are central hor- K., Hochberg, R.B., Zhou, H., Katzenellenbogen, J.A., Katzenellenbogen, B.S., mone regulators of glucose homeostasis. Glucagon Kim, Y., Joachmiak, A., Greene, G.L. NF-κB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses [published correction appears in initiates the gluconeogenic program in hepatocytes by Nat. Chem. Biol. 4:379, 2008]. Nat. Chem. Biol. 4:241, 2008. activating the cAMP signaling pathway, whereas insulin Nettles, K.W., Gil, G., Nowak, J., Métivier, R., Sharma, V.B., Greene, G.L. CBP is inhibits hepatic glucose output. Our recent detection a dosage-dependent regulator of nuclear factor-κB suppression by the estrogen of a new component of the cAMP pathway, transducers receptor. Mol. Endocrinol. 22:263, 2008. 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 CREB- mediated expression of hepatic genes. However, the specific nuclear actions of TORC2 are unknown. Thus, the mechanisms involved in differentiating TORC2- transmitted signals are of biological and clinical interest.

PUBLICATIONS Altarejos, J.Y., Goebel, N., Conkright, M.D., Inoue, H., Xie, J., Arias, C.M., Saw- chenko, 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 identi- fies 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 O 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, reveal- ing the structural features of signaling specificity. The 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 2008 THE SCRIPPS RESEARCH INSTITUTE 79

DEPARTMENT OF Jeffery W. Kelly, Ph.D.* (Andrew) Bin Zhou, Ph.D. Deboshri Banerjee, Ph.D. CHEMISTRY Lita Annenberg Hazen Assistant Professor of Professor of Chemistry Immunochemistry Elizabeth Barrett, Ph.D.****

STAFF Ramanarayanan Roland Barth, Ph.D.***** Krishnamurthy, Ph.D. SENIOR SCIENTIST Clay Bennett, Ph.D. Associate Professor K.C. Nicolaou, Ph.D.* Luis Martinez, Ph.D.***** Moritz Biskup, Ph.D.† Chairman Lucas J. Leman, Ph.D. Universität Karlsruhe Aline W. and L.S. Skaggs Assistant Professor of Karlsruhe, Germany Professor of Chemical Chemistry STAFF SCIENTISTS † Biology Anthony Boitano, Ph.D. Richard A. Lerner, M.D.*** Lisa Eubanks, Ph.D. Darlene Shiley Chair in Genomics Institute of the President, The Scripps Novartis Foundation Chemistry Rajesh Grover, Ph.D. Research Institute San Diego, California Lita Annenberg Hazen Dariush Ajami, Ph.D. Sarah Hanson, Ph.D. Laure Bouchez, Ph.D. Assistant Professor of Professor of Immunochemistry Lubica Supekova, Ph.D. Molecular Assembly Kristopher Boyle, Ph.D. Cecil H. and Ida M. Green Wen Xiong, Ph.D. Phil S. Baran, Ph.D. Chair in Chemistry Christopher Burke, Ph.D Professor Roy Periana, Ph.D.***** Antonio Burtoloso, Ph.D.† Dale L. Boger, Ph.D.* Professor INSTRUMENTATION/ University of Sao Paulo Richard and Alice Cramer SERVICE FACILITIES Sao Paulo, Brazil Professor of Chemistry Evan T. Powers, Ph.D. Associate Professor of Raj K. Chadha, Ph.D. Mark Bushey, Ph.D.† Tobin J. Dickerson, Ph.D. Chemistry Director, X-ray Exxon, Inc. Assistant Professor Crystallography Facility Union City, New Jersey Julius Rebek, Jr., Ph.D.* Albert Eschenmoser, Ph.D.* Professor Dee H. Huang, Ph.D. Darren Bykowski, Ph.D.***** Professor Director, The Skaggs Institute Director, Nuclear Magnetic for Chemical Biology Resonance Facility Petr Capek, Ph.D. Sheng Ding, Ph.D. Associate Professor Edward Roberts, Ph.D. Gary E. Siuzdak, Ph.D. Katerina Capkova, Ph.D. Professor Senior Director, Mass M.G. Finn, Ph.D.* Arani Chanda, Ph.D. Spectrometry Facility Professor Floyd E. Romesberg, Ph.D. Ke Chen, Ph.D. Associate Professor Valery Fokin, Ph.D. Peng Chen, Ph.D. Associate Professor William Roush, Ph.D.***** SENIOR RESEARCH ASSOCIATE Professor Govardhan Cherukupalli, M. Reza Ghadiri, Ph.D.* Ph.D.† Professor Peter G. Schultz, Ph.D.* Suresh Pitram, Ph.D. Epix Pharmaceuticals Professor William A. Greenberg, Ph.D. Lexington, Massachusetts Scripps Family Chair Assistant Professor of RESEARCH ASSOCIATES Jodie Chin, Ph.D. Chemistry K. Barry Sharpless, Ph.D.* W.M. Keck Professor of Srinivas Reddy Chirapu, Ph.D. Inkyu Hwang, Ph.D. Ramzey Abujarour, Ph.D. Chemistry Assistant Professor Rajesh Ambasudhan, Ph.D. Chandramouli Chiruta, Ph.D. Anita D. Wentworth, Ph.D. Kim D. Janda, Ph.D.** Assistant Professor Manuel Amorin Lopez, Ph.D. Dong-Gyu Cho, Ph.D. Professor Ely R. Callaway, Jr., Chair Paul Wentworth, Jr., Ph.D. Mark Ams, Ph.D. So-Hye Cho, Ph.D. in Chemistry Professor Sungwook Choi, Ph.D. Director, Worm Institute of Yoshio Ando, Ph.D. Research and Medicine Chi-Huey Wong, Ph.D.* Deepshikha Angrish, Ph.D. Joyanta Choudhury, Ph.D. Professor of Chemistry Gunnar Kaufmann, Ph.D. Shinji Ashida, Ph.D. Sarwat Chowdhury, Ph.D. Assistant Professor of Jin-Quan Yu, Ph.D. Chemistry Associate Professor Micahel Baksh, Ph.D. Stepan Chuprakov, Ph.D. 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† Jem Efe, Ph.D. Rajesh K. Grover, Ph.D. Michael Jahnz, Ph.D.† Achaogen NOXXON Pharma AG † South San Francisco, Jan Elsner, Ph.D. Jan Grunewald, Ph.D. Berlin, Germany California Celgene Pharmaceuticals Tanja Gulder, Ph.D. San Diego, California Rong Jiang, Ph.D.***** Scott Cockroft, Ph.D.† Richard Guy, Ph.D.**** University of Edinburgh Daniel Ess, Ph.D. Guo Jiantoa, Ph.D. Edinburgh, Scotland Masaki Handa, Ph.D.† Cyrine Ezzili, Ph.D. Hiroyuki Kakei, Ph.D.† Sagami Chemical Research David Colby, Ph.D.† Takeda Pharmaceutical Xingang Fang, Ph.D. Center Purdue University Company Limited Ayase, Kanagawa, Japan West Lafayette, Indiana Simon Ficht, Ph.D.† Osaka, Japan Sanofi-Aventis Deutschland Yuanjun He, Ph.D. Kevin Cole, Ph.D.† Jaroslaw Kalisiak, Ph.D. GmbH Eli Lilly and Company Jason Hein, Ph.D. Indianapolis, Indiana Frankfurt, Germany Seiji Kamioka, Ph.D. Dube Henry, Ph.D. Christine Crane, Ph.D. Joseph Rodolph Fotsing, Moumita Kar, Ph.D. † Ph.D. Marcos Hernandez, Ph.D. Matthew Cremeens, Ph.D.† Senomyx, Inc. Kwang Mi Kim, Ph.D. Gonzaga University San Diego, California Par Holmberg, Ph.D.† F. Scott Kimball, Ph.D. Redmond, Washington Memorial Sloan Kettering Bozena Frackowiak, Ph.D.† Cancer Center Jeremy Kister, Ph.D. Fernando Rodrigo Pinacho Politechnika Opolska New York, New York Crisostomo, Ph.D.† Opole, Poland Keith Korthals, Ph.D. Burnham Institute for Wen-Xu Hong, Ph.D. Graeme Freestone, Ph.D.† Medical Research Larisa Krasnova, Ph.D. Zhangyong Hong, Ph.D. La Jolla, California Metabasis Therapeutics, Inc. San Diego, California Richard J. Hooley, Ph.D.† Arkady Krasovskiy, Ph.D. Jeffrey Culhane, Ph.D. Yu Fu, Ph.D. University of California Luke Lairson, Ph.D. Stephen Dalby, Ph.D. Riverside, California Amelia Fuller, Ph.D.† † Jae Wook Lee, Ph.D. Etzer Darout, Ph.D.† Santa Clara University Tamara Hopkins, Ph.D. Pfizer Inc. Santa Clara, California Boehringer Ingelheim Jinq-Chyi Lee, Ph.D.† Groton, Connecticut Pharmaceuticals, Inc. National Health Research † Jianmin Gao, Ph.D. Ridgefield, Connecticut Institutes † Amy DeBaillie, Ph.D. Boston College Miaoli County, Taiwan Eli Lilly and Company Chestnut Hill, Massachusetts Allen Horhota, Ph.D. Indianapolis, Indiana Jong Seok Lee, Ph.D. Haibo Ge, Ph.D. Tony Horneff, Ph.D. Judith Denery, Ph.D. † Jun-Li Hou, Ph.D. Ki-Bum Lee, Ph.D. Savvas Georgiades, Ph.D.**** Rutgers University Ross Denton, Ph.D.† Claas Hovelmann, Ph.D. Piscataway, New Jersey University of Cambridge Ola Ghoneim, Ph.D.† Cambridge, England Qatar University Fang Hu, Ph.D.† Sejin Lee, Ph.D.† Doha, Qatar Department of Molecular SK Drug Development Center Caroline Desponts, Ph.D. Biology, Scripps Research Daejong, Korea Nathan Gianneschi, Ph.D.† Antonia Di Mola, Ph.D. University of California Xiaoyi Hu, Ph.D. Alexandre Lemire, Ph.D.**** San Diego, California Deguo Du, Ph.D. Zheng-Zheng Huang, Ph.D.† Edward Lemke, Ph.D. Cristina Gil-Lamaignere, Anna Dubrovska, Ph.D. DuPont Central Research Ph.D.† and Development Christophe Letondor, Viktoriya Dubrovskaya, Ph.D. University Hospital Nuestra Wilmington, Delaware Ph.D.**** Señora de la Candelaria † Joshua Dunetz, Ph.D.† Santa Cruz de Tenerife, Spain Ben Hutchins, Ph.D. Chuang-Chuang Li, Ph.D. Pfizer Inc. Peking University † Groton, Connecticut Rodolfo Gonzalez, Ph.D. Der-ren Hwang, Ph.D. Peking, China Academia Sinica Kyle Eastman, Ph.D. Scott Grecian, Ph.D. Taipei, Taiwan Fangzheng Li, Ph.D.***** CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 81

Hongming Li, Ph.D.† Joonwoo Nam, Ph.D.† Troy Ryba, Ph.D.† Sebastian Steiniger, Ph.D. Schering-Plough CytRx Corporation Broad Institute of MIT and Antonia Stepan, Ph.D. Kenilworth, New Jersey San Diego, California Harvard Cambridge, Massachusetts Ke Li, Ph.D.† Tae-Gyu Nam, Ph.D. James Stover, Ph.D. DuPont Central Research † Youngha Ryu, Ph.D. Bernhard Stump, Ph.D. and Development Andrew Nguyen, M.D., Ph.D. Texas Christian University Wilmington, Delaware Fort Worth, Texas Romain Noel, Ph.D. Shun Su, Ph.D. Pi-Hui Liang, Ph.D.† Catherine Saccavini, Ph.D. George Nora, Ph.D.***** Hui Kai Sun, Ph.D.***** Academia Sinica Taipei, Taiwan Severin Odermatt, Ph.D.**** Nicholas Salzameda, Ph.D. Shinobu Takizawa, Ph.D.

† Yeon-Hee Lim, Ph.D.† Christian Olsen, Ph.D. Antonio Sanchez-Ruiz, Ph.D. Adam Talbot, Ph.D. Schering-Plough Institute of Chemical and Yoshikazu Sasaki, Ph.D. Kenilworth, New Jersey Yazmin Osornio, Ph.D.**** Engineering Sciences Stefan Schiller, Ph.D. Jurong Island, Singapore Tongxiang Lin, Ph.D. Junguk Park, Ph.D. Niklas Schone, Ph.D. Annie Tam, Ph.D. Troy Lister, Ph.D.† Nitin Patil, Ph.D. Novartis † Yefeng Tang, Ph.D. Johan Paulsson, Ph.D. Michael Schramm, Ph.D. Cambridge, Massachusetts California State University † † Mariola Tortosa, Ph.D. † Richard Payne, Ph.D. Long Beach, California Christopher Liu, Ph.D. Instituto de Quimica Organica, Cubix Pharmaceuticals University of Sydney Sydney, Australia Young Jun Seo, Ph.D. CSIC Lexington, Massachusetts Madrid, Spain Edward Sessions, Jr., Ph.D. † Xuemei Peng, Ph.D.**** Wenshe Liu, Ph.D. Craig Turner, Ph.D.**** Texas A&M University Murali Peram Surakattula, Shigeki Seto, Ph.D. College Station, Texas Ph.D.† Matthew Tremblay, Ph.D. Mary Jo Sever, Ph.D. CytRx Corporation Michael Luzung, Ph.D. Vincent Trepanier, Ph.D.† San Diego, California † Alex Shaginian, Ph.D. Institute of Chemical and Utpal Majumder, Ph.D. Ardea Biosciences Roshan Perera, Ph.D.† Engineering Sciences San Diego, California Sreeman Mamidyala, Ph.D. University of Texas Jurong Island, Singapore Austin, Texas David Shaw, Ph.D. Takeshi Masuda, Ph.D. Jonathan Tripp, Ph.D. Ramulu Poddutoori, Ph.D. Weijun Shen, Ph.D. Michael Maue, Ph.D.† Meng-Lin Tsao, Ph.D.† Bayer CropScience AG Jonathan Pokorski, Ph.D. Xiao Shengxiong, Ph.D. University of California Monheim, Germany Merced, California Agustí Lledó Ponsati, Ph.D. Bingfeng Shi, Ph.D. Alexander Mayorov, Ph.D. Andrew Udit, Ph.D. † Daniela Radu, Ph.D. Yan Shi, Ph.D. Charles Melancon, Ph.D. DuPont Central Research Taiki Umezawa, Ph.D.† and Development Hiroki Shigehisa, Ph.D. Hokkaido University † Lionel Moisan, Ph.D. Wilmington, Delaware Sapporo, Japan CEA Hiroyuki Shimamura, Ph.D. Gif-Sur-Yvette, France Ronald Rahaim, Ph.D.***** Kenji Usui, Ph.D.† Siddhartha Shenoy, Ph.D. Tokyo Institute of Technology Ana Montero, Ph.D.**** Praveen Rao, Ph.D.† Tokyo, Japan University of Waterloo Ryan Simkovsky, Ph.D. Miguel Morales, Ph.D. Waterloo, Ontario, Canada Chinnappan Sivasankar, Carlos Valdez, Ph.D.† † Rigel Pharmaceuticals, Inc. Adam Morgan, Ph.D. Per Restorp, Ph.D. Ph.D.**** Concert Pharmaceuticals, Inc. South San Francisco, † Lexington, Massachusetts Kimberly Reynolds, Ph.D. Michael Smolinski, Ph.D. California Kinex Pharmaceuticals Ting-Wei Mu, Ph.D. Jin-Kyu Rhee, Ph.D. Buffalo, New York Punna Venkateshwarlu, Ph.D.

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. 82 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE

Jiangyun Wang, Ph.D.† Heyue Zhou, Ph.D. * Joint appointment in The Skaggs Institute for Chemical Institute of Biophysics Hongyan Zhou, Ph.D. Biology Beijing, China ** Joint appointments in The Shoutian Zhu, Ph.D. Skaggs Institute for Chemical Lin Wang, Ph.D. Biology and the Department of Joerg Zimmermann, Ph.D. Immunology and Microbial Sheng-Kai Wang, Ph.D. Science

*** Joint appointments in The Weidong Wang, Ph.D. VISITING INVESTIGATORS Skaggs Institute for Chemical Biology and the Department of Xisheng Wang, Ph.D. Molecular Biology Keisuke Fukuchi, Ph.D. Yajuan Wang, Ph.D. Sankyo Co., Ltd. **** Appointment completed Tokyo, Japan ***** Scripps Florida

Yuanhua Wang, Ph.D. † † Appointment completed; new Christine Hernandez, Ph.D. location shown Timo Weide, Ph.D. University of Philippines Diliman, Philippines Albert Willis, Ph.D.† Pharmagra Labs, Inc. (Edmond) Shie-Liang Hsieh, Brevard, North Carolina Ph.D. National Yang-Ming University † Tao Wu, Ph.D. Taipei, Taiwan Institute of Chemical and Engineering Sciences Masakazu Imamura, Ph.D.† Jurong Island, Singapore Astellas Pharma Inc. Tsukuba, Ibaraki, Japan Heiko Wurdak, Ph.D. Kuniyuki Kishikawa, Ph.D.† Jian Xie, Ph.D. Kyowa Hakko Kogyo Co., Ltd. Sunto-gun, Shizuoka, Japan Wen Xiong, Ph.D. Michael Meijler, Ph.D.† Yue Xu, Ph.D. Ben-Gurion University of the Junichiro Yamaguchi, Ph.D. Negev Be’er Sheva, Israel Ryu Yamasaki, Ph.D.† Tokyo University of Science Takayoshi Suzuki, Ph.D.† Tokyo, Japan Nagoya City University Nagoya, Japan Ura Yasuyuki, Ph.D.† Nara Women’s University Yoshiyuki Yoneda, Ph.D.† Nara, Japan Daiichi Pharmaceutical Co., Ltd. Yan Yin, Ph.D. Tokyo, Japan

Ian Young, Ph.D. SCIENTIFIC ASSOCIATE Zhanqian Yu, Ph.D. Jon Ashley 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 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE 83 Chairman’s Overview role of polyoxygen species. Scientists in Albert Eschen- moser’s group continue to work on the chemical etiol- s the “central science,” chemistry stands between ogy of nucleic acids and the origins of life. biology and medicine and between physics and Barry Sharpless and his group persist in their endeav- A materials science and provides the crucial bridge ors to discover and develop better catalysts for organic for drug discovery and synthesis and to construct, through innovative chemistry development. But chem- and biology, libraries of novel compounds for biological istry has a much more screening. Their click chemistry, which has had a major profound and useful role impact in many areas of the molecular sciences, con- in science and society. It tinues to be an important focus of their research. is the discipline that con- Members of my own group continue to explore chem- tinually creates the myriad ical synthesis and chemical biology, with a focus on the of new materials that we total synthesis of new anticancer agents, antibiotics, all encounter in our every- marine-derived neurotoxins, antimalarial compounds, day lives: pharmaceuti- and other bioactive natural and designed molecules. cals, high-tech materials, Julius Rebek and his group devise biomimetic recep- polymers and plastics, tors, including molecules that bind neurotransmitters and K.C. Nicolaou, Ph.D. insecticides and pesticides, membrane components, for studies in molecular recog- fabrics and cosmetics, fertilizers, and vitamins—basically nition. Larger host receptors can surround 3 or more everything we can touch, feel, and smell. molecular guests and act as chambers in which the Chemistry at Scripps Research focuses on chemical chemical reactions of the guests are accelerated. Scien- synthesis and chemical biology, the areas most relevant tists in the group also synthesize small molecules that act to biomedical research and materials science. The mem- as protein helix mimetics for pharmaceutical applications. bers of our faculty are distinguished teacher-scholars who Peter Schultz and researchers in his laboratory are maintain highly visible and independent research programs expanding the number of genetically encoded amino acids in areas as diverse as biological and chemical catalysis, to include fluorescent, photocaged, metal-binding, chemi- synthesis of natural products, combinatorial chemistry, cally reactive, and posttranslationally modified amino molecular design, supramolecular chemistry, chemical acids. These scientists have also adapted this technol- evolution, materials science, and chemical biology. The ogy to mammalian cells and are applying these tools in chemistry graduate program attracts some of the best- basic and applied problems in cell biology. In addition, qualified candidates from the United States and abroad. members of the group have used cell-based screens to Our major research facilities, under the direction of Dee identify small molecules that selectively differentiate H. Huang (nuclear magnetic resonance), Gary Siuzdak and expand embryonic and adult stem cells and repro- (mass spectrometry), and Raj Chadha (x-ray crystallog- gram lineage-committed cells, as well as novel genes raphy), are second to none and continue to provide cru- and small molecules that affect a number of physiologic cial support to our research programs. In addition, the and disease processes. Mabel and Arnold Beckman Center for the Chemical Chi-Huey Wong and his group further advance the Sciences constantly receives high praise from visitors fields of chemoenzymatic organic synthesis, chemical from around the world for its architectural design and glycobiology, and the development of enzyme inhibitors. operational aspects, both highly conducive to research. A new strategy for the synthesis of glycoproteins based Research in the Department of Chemistry goes on on sugar-assisted glycopeptide ligation has been developed. unabated, establishing international visibility and attract- The programmable 1-pot synthesis of oligosaccharides ing attention, as evidenced by numerous lecture invita- developed by this group has been further used in the tions, visits by outside scholars, and headline news in assembly of glycoarrays for study of saccharides that the media. As of 2007, the Institute for Scientific Infor- bind to proteins. Members of this group also developed mation ranked 2 members of the department as highly new probes to study posttranslational glycosylation and cited researchers (in the top 100 worldwide). identify glycoprotein biomarkers associated with cancer. Richard Lerner and his group continue their research Researchers in Dale Boger’s laboratory continue their on antibodies, chemical synthesis, and the biological work on chemical synthesis; combinatorial chemistry; het- 84 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE erocycle synthesis; anticancer agents, such as vinblastine, Researchers in Floyd Romesberg’s laboratory are using cyclostatin, chlorofusion, and yatakemycin; and antibiotics, diverse techniques ranging from bioorganic and biophysi- such as vancomycin, teicoplanin, and ramoplanin. cal chemistry to bacterial and yeast genetics to under- Scientists in Kim Janda’s laboratory conduct research stand and manipulate the process of evolution. Major grounded on organic chemistry as applied to specific bio- efforts include designing unnatural base pairs and using logical systems. The targeted programs span a wide range directed evolution of DNA polymerases to efficiently syn- of interests, from immunopharmacotherapy to biological thesize unnatural DNA containing the base pairs, using and chemical warfare agents to filarial infections such spectroscopy to understand biological function and how as “river blindness” to quorum sensing in bacteria and it evolves, and understanding how induced and adap- new cancer therapeutic strategies. Recent achievements tive mutations contribute to evolution in eukaryotic and include in vivo detection of botulinum neurotoxin antag- prokaryotic cells. onists, the development of peptides and antibodies as Phil Baran and his group are interested in how the drug delivery modules that home to cancer cells and general challenge of chemoselectivity in organic chemistry active vaccines for nicotine addiction and obesity that can be answered through the auspices of total synthe- are now in preclinical trials. sis. He and his coworkers have developed extremely M. Reza Ghadiri and his group are making important concise chemical solutions to the synthetic challenges contributions in the design and study of a new generation posed by numerous families of natural products. These of antimicrobial agents, based on self-assembling peptide syntheses systematically tackle the issue of chemose- nanotube architecture, to combat multidrug resistant lectivity and are characterized by striking brevity, new infections. In addition, members of the group continue biosynthetic postulates, the invention of new methods, to make novel contributions in several ongoing basic and a minimum use or complete absence of protecting research endeavors, such as designing biosensors, devel- groups and superfluous oxidation state manipulations. oping molecular computation, designing self-reproducing The Frontiers in Chemistry Lecturers (19th Annual systems, understanding the origins of life, and creating Symposium) for the 2007–2008 academic year were emergent chemical systems. M. Christina White, University of Illinois; Ben L. Feringa, M.G. Finn and his group have pioneered the use of University of Groningen, the Netherlands; Ian Paterson, virus particles as chemical reagents and building blocks Cambridge University; and Harry Noller, University of for nanochemical structures. This effort is directed toward California, Santa Cruz. In addition, we enjoyed hosting the development of new diagnostics for disease and cata- the following professors: Samir Zard, Ecole Polytech- lysts for organic reactions. Members of Dr. Finn’s labo- nique, France, as the Bristol-Myers Squibb Lecturer; ratory also develop and investigate new organic and E.J. Corey, Harvard University, as the Pfizer Lecturer; organometallic reactions and use these processes to and Robert Bergman, University of California, Berkeley, synthesize biologically active compounds. as the Novartis Lecturer. 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. 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 O 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 Fig. 1. Structures of recently synthesized natural products. syntheses of 10-hydroxytrilobacin and 3 stereoisomers. We have also developed phosphine-mediated organocat- sites, such as Trypanosoma cruzi, the causative agent alytic reactions, and we recently completed the total of Chagas’ disease, and Plasmodium falciparum, the synthesis of tedanolide. most virulent of the malaria parasites. This research is Our second area of major interest involves problems performed in collaboration with colleagues at the Uni- in bioorganic chemistry and medicinal chemistry. One versity of California, San Francisco. In collaboration with long-term project is the design and synthesis of inhibi- S. Reed, University of California, San Diego, we have tors of cysteine proteases isolated from tropical para- developed a cysteine protease inhibitor with remarkable 86 CHEMISTRY 2008 THE SCRIPPS RESEARCH INSTITUTE

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) pyran- containing 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-hydroxyal- lylsilanes 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 iso- quinolinone 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 seco- sterol-induced aggregation of methylated amyloid-β peptides, insights into aldehyde- initiated 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. 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

DEPARTMENT OF

INFECTOLOGY

STAFF Joaquin Castilla, Ph.D. RESEARCH ASSOCIATES Yervand Karapetyan, M.D. Assistant Professor Charles Weissmann, M.D., Ivan Angulo-Herrera, Ph.D. Minghai Zhou, Ph.D. Ph.D. Professor and Chairman SENIOR STAFF Shawn Browning, Ph.D. SCIENTIST Corinne Lasmézas, D.V.M., Natalia Fernández-Borges, Ph.D. Sukhvir Mahal, Ph.D. Ph.D. Professor Maria Herva-Moyana, Ph.D. SENIOR RESEARCH Donny Strosberg, Ph.D. ASSOCIATES Paula Saá Prieto, Ph.D. Professor Carlos Coito, Ph.D. Jiali Li, Ph.D. Tim Tellinghuisen, Ph.D. Assistant Professor Chris Baker, Ph.D. Anja Oelschlegel, Ph.D.

Chairman’s Overview of a multimeric assembly of PrPSc, a conformer of the normal host protein PrPC. The seeding hypothesis posits he Department of Infectology focuses on prion that prions replicate by recruiting host PrPC into the diseases and hepatitis. Tim Tellinghuisen and his PrPSc assembly, a process that entails conformational T colleagues study hepatitis C virus (HCV) RNA rearrangement of the PrPC. Prions occur in the form of replication and virion different strains, all associated with the same PrPSc assembly. They identified sequence but with distinct cell tropisms, both in brain the viral protein NS5A as and in cell culture. The cell-based assay for prions has an essential component been further streamlined by Emery Smith, and the cell of the viral replicase and panel assay, which allows rapid distinction between prion mapped all amino acids strains, was extended to more strains by Sukhvir Mahal. in domains II and III It has been proposed that “strain-ness” is encoded either essential for HCV RNA by distinct conformations of PrPSc or by the complex replication. They identi- glycans present on the protein. This hypothesis has been fied regions whose func- negated by Shawn Browning and Dr. Mahal, who found that strain specificity is maintained when prions are tions are required for Charles Weissmann, M.D., Ph.D. generating infectious virus propagated under conditions in which complex glycosy- but not for RNA replication, suggesting that NS5A lation is abrogated. regulates the switch between RNA replication and virus Dr. Lasmézas and her colleagues determined that assembly. They are using high-throughput genetic screens PrPSc in cultured prion-infected cells is undetectable at to identify host components required for replicase activity. the outer cell surface and is therefore likely generated Donny Strosberg and his colleagues established for intracellularly. These investigators characterized a rapid the first time a cell culture system for HCV genotype 1b. animal model for prion disease based on a transgenic, They identified peptides derived from the HCV core that PrP-overexpressing mouse strain and are researching the inhibit (1) dimerization of core protein, the first step in mechanism of pathogenesis. Dr. Castilla and his group viral assembly and (2) release of virions. F.V. Chisari and have perfected the protein misfolding cyclic amplification his group, Department of Molecular and Experimental procedure, which allows the cell-free replication of prions, Medicine, independently found that one such peptide and have shown that various prion strains can be propa- inhibits HCV replication. These findings establish the HCV gated continuously without losing strain-specific properties. 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 196 INFECTOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE

Investigators’ Reports T able 1. Susceptibility of cell lines to various prion strains.* Biology of Prion Strains Cell line Prion strain 22L 139A RML, 79A Me7 301C C. Weissmann, C.A. Baker, S. Browning, C. Demczyk, LD9 (EMEM) +++ +++ +++ +++ – M. Herva-Moyana, J. Li, S.P. Mahal, A. Oelschlegel,

A. Sherman, E. Smith, I. Suponitsky-Kroyter CAD5* +++ +++ +++ – +++ rions are thought to consist mainly or entirely PK1 +++ +++ +++ – – of PrPSc, an abnormal conformer of a normal C Sc P host protein, PrP , and to propagate via a PrP - PK1/swa +++ ++ – – – catalyzed conversion of PrPC. Intriguingly, distinct prion strains, which generate different disease phenotypes, R33 ++ – – – – are associated with the same PrP sequence, suggest- * The number of pluses indicates the degree of susceptibility to infection. The ing that the phenotypes are encoded posttranslation- minus sign indicates resistance to infection. ally. Our major interests are the mechanism of prion CPA, differs from that of the prions derived from brain. replication, the structural basis of strain specificity, To determine whether this change reflects a permanent and the mechanism of strain recognition by cells. modification of a strain, we inoculated cell-derived prions Much of our research depends on a cell-based assay into mice; CPA analysis of the infected brains showed for prion infectivity, the standard scrapie cell assay. no differences between the original and the cell-pas- Therefore, considerable effort has gone into automat- saged strains. We are currently considering the possibil- ing the procedure, reducing its cost, and improving its ity that the cell tropism of a prion strain may depend on accuracy. We can now assay about 1000 samples per the host cell in which the strain is generated, reflecting week in sextuplicate, orders of magnitude greater than some host-imparted property (the “cytotype”), for exam- the number that could be processed by using the classi- ple, the glycosylation pattern of the PrP. cal mouse bioassay. The standard scrapie cell assay is It has been proposed that the N-linked complex the basis for the cell panel assay (CPA), which makes glycans attached to PrP might encode the “strain-ness” use of the finding that different cell lines have distinctive of prions. To address this question, we propagated RML, susceptibilities to various prion strains. Using the CPA, 22L, and Me7 prions in CAD5 cells in the presence of we can distinguish between various prion strains within the glycosylation inhibitors deoxymannojirimycin and 2 weeks, as opposed to the year or more required with swainsonine. These inhibitors prevent processing of the the classical mouse bioassay. The panel originally con- precursor of N-linked glycans and result in PrP with sisted of 4 cell lines; however, when exposed to swain- [mannose]9[N-acetylglucosamine]2 as the only glycan, sonine (an α-mannosidase II inhibitor that modifies the rather than a multiplicity of complex sugars. We injected glycans of N-glycosylated proteins), the PK1 cell line cell lysates of the drug-treated, prion-infected cells into (but none of the others) becomes resistant to some prion mice and used the CPA to analyze the infected brains. strains but not to others. Therefore, swainsonine-treated In all instances, the strain-specific properties had been PK1 cells provide an additional criterion for discriminat- retained, proving that complex glycosylation was not ing prion strains. We can currently distinguish 5 groups the strain-determining element and adding weight to of strains (Table 1). the hypothesis that strain-ness is encoded by the con- We determined that the characteristic CPA responses formation of PrPSc. of these strains are the same whether the strains are In another project, we are exploring why certain propagated in wild-type mice or in Tga20 mice, which subclones derived from the same cell line, occurring overexpress PrPC and, conveniently, have a reduced with a frequency of 1 in 5000 to 1 in 10,000, can incubation time. vary by 100 to 1000 times in their susceptibility to We have established a large collection of cell lines prions. We tagged highly susceptible and resistant chronically infected with various prion strains. Interest- subclones with 2 different markers of antibiotic resis- ingly, in many instances, the cell tropism of prions tance and fused the cells. Currently, we are determining derived from such lines, as determined by using the whether susceptibility, as measured in heterokaryons, INFECTOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE 197 is a dominant or a recessive property. In the next step, files in brains of Tga20 mice differed from those in we will generate microcells containing single, tagged C57BL/6 mice for the 3 scrapie strain studied. Each chromosomes from one of the cell lines and fuse the strain had a characteristic profile in Tga20 mice and microcells to cells from the other line to determine which could be readily distinguished by neuropathologic analy- chromosome encodes the critical trait. sis, showing that the level of PrPC expression is not the main determinant of brain tropism. Importantly, despite PUBLICATIONS Julius, C., Hutter, G., Wagner, U., Seeger, H., Kana, V., Kranich, J., Klöhn, P., generating different neuropathologic phenotypes in Weissmann, C., Miele, G., Aguzzi, A. Transcriptional stability of cultured cells C57BL/6 and Tga20 mice, all 3 prion strains retained upon prion infection. J. Mol. Biol. 375:1222, 2008. their intrinsic identity after being replicated in either Mahal, S.P., Baker, C.A., Demczyk, C.A., Smith, E.W. Julius, C., Weissmann, C. mouse line, as determined by the cell panel assay. Prion strain discrimination in cell culture: the cell panel assay. Proc. Natl. Acad. Sci. U. S. A. 104:20908, 2007. This study provides a new reliable method for rapid propagation and characterization of prion strains by Mahal, S.P., Demczyk, C.A., Smith, E.W., Jr., Klöhn, P.-C., Weissmann, C. Assay- ing prions in cell culture: the standard scrapie cell assay (SSCA) and the scrapie using a combination of Tga20 mice and the cell panel cell assay in end point format (SCEPA). Methods Mol. Biol. 459:49, 2008. assay. Fundamentally, our results indicate that despite different biochemical characteristics of PrPC, different expression patterns of PrPC in the 2 murine hosts, and Pathogenesis of Transmissible different genetic background of the 2 mouse strains, prions are propagated faithfully in both types of mice. This Spongiform Encephalopathies finding raises once more the question of the molecular basis of prion-strain properties. C.I. Lasmézas, N. Salès, P. Saá Prieto, M. Zhou, Previously, we found that oligomeric assemblies of Y. Karapetyan, F. Sferrazza, G. Ottenberg recombinant prion protein are toxic to primary cultures rions, the transmissible agents responsible for of cortical neurons and in mice when injected via a prion diseases, are thought to consist mainly of stereotaxic method. We are now devising new interven- P PrPSc, an abnormally folded isoform of the ubiq- tion strategies to block the toxic/infectious PrP species. uitous prion protein PrP. Prions are thought to replicate A first goal is to determine the cellular location of PrP by an autocatalytic process of template-induced con- aggregates, in order to know which cellular compart- formational change. ment to target. The many reports of the presence of In collaboration with C. Weissmann, Department of protease-resistant PrP (PrPres) at the cell membrane Infectology, we are devising a new method for propa- are contradictory. Using a cell-surface biotinylation gating and characterizing different strains of prions. strategy and comprehensive controls to account for Dr. Weissmann and his group have developed a cell- the presence of dead cells and for the intrinsic capac- based infectivity assay, the cell panel assay, in which ity of PrPres to be biotinylated, we found no detectable prion strains are distinguished on the basis of cell tro- PrPres at the cell membrane. This finding has major pism. We have studied the fate of prions in the Tga20 implications for the development of diagnostic mole- mouse model. Compared with wild-type mice, Tga20 cules. We are continuing our efforts to locate the cellu- mice express 8-fold higher levels of PrPC in the brain, lar compartment that must be targeted for therapeutic and clinical disease occurs more quickly after inocula- purposes, and we are setting up new models for PrP- tion of prions. induced toxic effects in cell lines. 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 Inhibitors of Protein-Protein the brains of Tga20 mice has a higher intrinsic resistance to proteolytic digestion by protease than does Interactions in Hepatitis C PrPC from the brains of wild-type mice. We also discov- A.D. Strosberg, C. Coito, R. Henderson, S. Kota, G. Mousseau ered that the levels of PrPC overexpression in different regions of the brain vary and hence could influence the everal small-molecule drugs are in advanced pathologic lesions and PrPSc distribution that occur after clinical development for the treatment of hepati- prion infection. Vacuolation and PrPSc deposition pro- S tis C, a situation that may affect the 170 mil- 198 INFECTOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE lion carriers of hepatitis C virus (HCV) worldwide, includ- tive of SL-175 bound to core106 with a dissociation ing more than 3 million in the United States. Because constant of 1.9 µM and was displaced by the uncoupled of its high mutability, HCV likely will become resistant peptide, with a 50% inhibitory concentration of 18.7 to these potential drugs, which are mostly inhibitors of µM. In a collaborative surface plasmon resonance study a viral protease and the viral polymerase. This past year, with J.-P. Lavergne, Centre National de la Recherche we continued our studies of protein interactions involv- Scientifique, Lyon, France, SL-175 bound core169 with ing HCV proteins to better understand the respective a dissociation constant of 7.2 µM. roles of the proteins and to identify novel target pro- PEPTIDE INHIBITORS OF HCV RELEASE FROM teins that would not induce resistance. The core HCV HEPATOMA CELLS protein, which is highly conserved across all 6 major When added to Huh-7.5 hepatoma cells infected HCV genotypes, is a good candidate for such studies. with the HCV genotype 2a J6/JFH-1 or the 1b CG strain, HCV CORE PROTEIN AS A TARGET peptides SL-173 and SL-175 prevented release of newly The HCV core protein functions primarily as the formed infectious HCV into the medium. Using a dif- structural element of the virus. The core contains several ferent approach, F.V. Chisari and his group, Department residues essential for HCV production. Most of these resi- of Molecular and Experimental Medicine, independently dues are located in the N-terminal two-thirds of the core found that SL-173 inhibits HCV focus formation in vitro protein and mediate core dimerization and most interac- by more than 90% and viral RNA synthesis 11-fold, 72 tions with intracellular proteins. Core dimerization is the hours after infection. The combined results of our 2 first step in nucleocapsid assembly; its inhibition should groups thus establish that the HCV core is a useful novel block formation and release of infectious HCV particles. target for development of anti-HCV drugs. HEPA TOMA CELL CULTURE SYSTEM FOR HCV ASSAYS FOR HCV CORE DIMERIZATION GENOTYPE 1B Using a pair of domains that consist of the first The culture system for HCV used routinely in labo- 106 residues of the HCV core protein (core106) tagged ratories worldwide was derived from strain JFH-1 of with oligonucleotides encoding the octapeptide Flag or an HCV of genotype 2a. In Western countries, however, glutathione-S-transferase (GST), we developed an assay the most prevalent infections are caused by HCV strains based on the use of an antibody to Flag that binds to of genotype 1. To understand differences between HCV Flag-tagged core106 interacting with GST-tagged core106 of different genotypes and further evaluate potential adsorbed on a glutathione-coated microtiter plate. We inhibitors of protein-protein interactions in HCV or designed a 384 well–based sensitive and high-through- between HCV and human host proteins, we developed put time-resolved fluorescence assay with fluorescent and characterized a unique culture system for the HCV antibodies to Flag and GST. Untagged core106 com- genotype 1b CG strain. Two different protocols have been pletely inhibits core106 dimerization. One of our indus- developed: the first one is based on the coculture of trial partners will use this assay to screen 2 million infected, virus-releasing cells with uninfected cells in chemically diversified small molecules to identify novel a 2-chamber system; the second is a direct incubation nonpeptidic inhibitors of HCV production. of uninfected cells with supernatant from cells electro- PEPTIDE INHIBITORS OF CORE DIMERIZATION porated with the RNA from strain CG or from infected We also designed an amplified luminescent prox- cells. Using either system, we have confirmed transfer imity homogeneous assay to monitor core-core interac- of infectivity in several passages. Furthermore, we showed tions on the basis of donor and acceptor beads that that cyclosporine A has comparable inhibitory effects respond to the specific tags on the proteins. Using this on J6/JFH-1 and CG strains of HCV and that antibodies assay, which has a high signal-to-background ratio, we to CD81, a coreceptor for HCV, block infectious particles screened 14 18-residue-long peptides derived from the released into the supernatant of cells infected by the HCV core and identified 2 partially overlapping peptides, 1b CG strain. Initial results also suggest that the 2 SL-173 and SL-174, which caused 68% and 63% inhi- strains are diversely affected by IFN-α; J6/JFH-1 appears bition, respectively, of core106 dimerization. to be sensitive, and CG appears to be resistant. This SL-175, a 3-residue shorter version of SL-173, finding, if confirmed, would reflect the situation in inhibited core106 dimerization by 50%, with a 50% patients; patients infected with genotype 2 HCV gen- inhibitory concentration of 22 µM. Using fluorescence erally respond better to interferon treatment than do polarization, we found that a fluorophore-coupled deriva- patients infected with genotype 1 HCV. INFECTOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE 199

PUBLICATIONS work. We have also begun applying high-throughput Strosberg, A.D., Nahmias, C. G-protein-coupled receptor signaling through protein networks. Biochem. Soc. Trans. 35:23, 2007. genetic screens to identify required host components of the replicase. Our ultimate goal is to understand, at the molecu- Hepatitis C Virus RNA lar level, the assembly, activity, and regulation of the HCV RNA replication machinery. Greater insight into Replication and Virion Assembly the poorly understood replicase components, such as NS5A, will provide a more complete view of the repli- T.L. Tellinghuisen, J.C. Treadaway, K.L. Foss, case complex and will fuel the design of new drugs. I. Angulo-Herrera

PUBLICATIONS epatitis C virus (HCV) is a human pathogen of Lindenbach, B.D., Tellinghuisen, T.L. Insights into hepatitis C virus RNA replica- global importance; according to some estimates, tion. In: Viral Genome Replication. Götte, M., Cameron, C., Raney, K. (Eds.). Springer, New York, in press. H nearly 3% of the world’s population are chronically infected. Long-term viral replication in these indi- 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. viduals leads to severe liver disease, including cirrhosis and, often, hepatocellular carcinoma. The current treat- 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. ment with agents nonspecific for HCV is poorly tolerated and is ineffective in about half of the patients, empha- 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 sizing the need for effective antiviral drugs specific for NS5A protein. J. Virol. 82:1073, 2008. the virus. Tellinghuisen, T.L., Lindenbach, B.D. Reverse transcription PCR based sequence The HCV replicase, the multicomponent machine analysis of hepatitis C virus replicon RNA. In: Hepatitis C: Methods and Protocols, that replicates the viral RNA, is an ideal drug target. 2nd ed. Tang, J. (Ed.). Humana Press, Totowa, NJ, in press. Vol. 510 in Methods in Molecular Biology. Walker, J. (Series Ed.). The core replicase consists of 5 HCV proteins associated with well-characterized polymerase, protease, and 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.). helicase activities. Some HCV replicase proteins, such Humana Press, Totowa, NJ, in press. Vol. 510 in Methods in Molecular Biology. as NS5A, are essential for HCV replication; however, Walker, J. (Series Ed.). their specific functions remain enigmatic. We have been characterizing NS5A. Our goal is to understand the role of this protein in replication and, Strain and Species Barrier more generally, the replicase itself. We have defined NS5A as an essential, 3-domain metalloprotein com- Phenomena in a Cell-Free System ponent of the replicase. Our crystal structure of domain I J. Castilla, N. Fernández-Borges, J. de Castro of NS5A has provided a glimpse of the potential interactions of NS5A in the viral replicase. ransmissible spongiform encephalopathies are We recently identified all of the amino acids in the fatal neurodegenerative disorders that affect both poorly understood domains II and III that are required T humans and animals. The disorders can be clas- for HCV RNA replication. Additionally, we have discov- sified as genetic, sporadic (putatively spontaneous), or ered an interaction between the membrane anchor of infectious. The infectious agent associated with these NS5A and the protein NS4B, another component of the encephalopathies, the prion, appears to consist of the replicase. This interaction appears to localize NS5A to single protein PrPSc, an abnormal conformer of the nat- the replicase and is essential for RNA replication. We ural host protein PrPC. Prions propagate by converting are identifying regions of NS5A whose functions are host PrPC into PrPSc. required for the production of infectious virus but not One characteristic of prions is their ability to infect for RNA replication. Our findings suggest that NS5A some species and not others. This phenomenon is known may function as a regulator of the switch between RNA as the transmission barrier. Interestingly, prions occur replication and virus production. We are conducting in the form of different strains with distinct biological biochemical, genetic, and structural experiments to and physicochemical properties, even though all the evaluate the potential interaction surfaces and activities strains have the same PrP amino acid sequence, albeit of NS5A observed in our previous structural and genetic in presumably different conformations. In general, the 200 INFECTOLOGY 2008 THE SCRIPPS RESEARCH INSTITUTE 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, ham- sters, 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., Glim- cher, 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. Molecular and Integrative Neurosciences

A, Circadian rhythm profile for wild-type (WT) littermates and EP3R–/– mice. Although diurnal distribution of motor activity fol- lows 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 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2008 THE SCRIPPS RESEARCH INSTITUTE 327

MOLECULAR AND Steven J. Henriksen, Ph.D. Amanda Roberts, Ph.D. RESEARCH ASSOCIATES Adjunct Professor Associate Professor INTEGRATIVE Mehrdad Alirezaei, Ph.D. NEUROSCIENCES Paul L. Herrling, Ph.D. Michael G. Rosenfeld, M.D. Michal Bajo, M.D., Ph.D. DEPARTMENT Adjunct Professor Adjunct Professor Hilda Bajova, D.V.M. Tomas Hokfelt, M.D., Ph.D. Pietro P. Sanna, M.D. STAFF Adjunct Professor Associate Professor Fulvia Berton, Ph.D.

Danny Hoyer, Ph.D. George R. Siggins, Ph.D. Vez Repunte Canonigo, Ph.D. Tamas Bartfai, Ph.D. Adjunct Professor Professor Chairman and Professor Zhifeng Chen, Ph.D. Director, Harold L. Dorris Koki Inoue, Ph.D. Iustin Tabarean, Ph.D. Kazuki Hagihara, Ph.D. Neurological Research Adjunct Associate Professor Assistant Professor Institute Izabella Klein, Ph.D. Harvey Karten, M.D. Antoine Tabarin, Ph.D. Adjunct Associate Professor Serge Ahmed, Ph.D. Adjunct Professor Kayo Mitsukawa, Ph.D. Adjunct Assistant Professor Lars Terenius, Ph.D. Henri Korn, M.D., Ph.D. Olivia Osborn, Ph.D. Etienne Baulieu, Ph.D. Adjunct Professor Adjunct Professor Adjunct Professor Covadonga Paneda, Ph.D. Thomas Krucker, Ph.D. Claes Wahlestedt, M.D., Floyd Bloom, M.D. Adjunct Assistant Professor Ph.D.* Gurudutt Pendyala, Ph.D. Professor Emeritus Professor Jerry Pinghwa Pian, Ph.D. Executive Director, Science Stefan Kunz, Ph.D. Tammy Wall, Ph.D. Communication Adjunct Professor Adjunct Associate Professor Jilla Sabeti, Ph.D. Jason Botten, Ph.D. Cary Lai, Ph.D. Friedbert Weiss, Ph.D. Assistant Professor Associate Professor VISITING INVESTIGATORS Professor Karen T. Britton, M.D., Ph.D. Ulo Langel, Ph.D. Hedieh Badie, Ph.D. Adjunct Professor Genomics Institute of the Adjunct Associate Professor STAFF SCIENTISTS Novartis Research Michael Buchmeier, Ph.D. Xiaoying Lu, Ph.D. Foundation Roberto Ciccocioppo, Ph.D. Adjunct Professor Assistant Professor San Diego, California José Criado, Ph.D. Iain L. Campbell, Ph.D. Jan O. Lundstrom, Ph.D. Persephone Borrow, Ph.D. Adjunct Professor Adjunct Professor Walter Francesconi, Ph.D. Edward Jenner Institute for Vaccine Research Zhen Chai, Ph.D. Athina Markou, Ph.D. David Gilder, M.D. Compton, England Adjunct Assistant Professor Adjunct Professor Salvador Huitrón-Reséndiz, Urs Christen, Ph.D. Jerold Chun, M.D., Ph.D. Madis Metsis, Ph.D. Ph.D. La Jolla Institute for Allergy Adjunct Professor Adjunct Associate Professor and Immunology M. Cecilia Marcondes, Ph.D. La Jolla, California Bruno Conti, Ph.D. Benjamin Neuman, Ph.D. Associate Professor Adjunct Assistant Professor Remi Martin-Fardon, Ph.D. Jean E. Gairin, Ph.D. CNRS Tom Nelson, Ph.D. Cindy L. Ehlers, Ph.D. Shirley M. Otis, M.D. Toulouse, France Adjunct Professor Professor Manuel Sánchez-Alavaz, Karine Guillem, Ph.D. M.D., Ph.D. Ralph Feuer, Ph.D. Tommy Phillips, Ph.D. University of Pennsylvania Adjunct Assistant Professor Adjunct Assistant Professor Mitra Rebek, Ph.D. Philadelphia, Pennsylvania John Polich, Ph.D. Howard S. Fox, M.D., Ph.D. Brendan Walker, Ph.D. Katsuro Hagiwara, Ph.D. Associate Professor Associate Professor Rakuno Gakuen University Ebetsu, Japan Hermann H. Gram, Ph.D. Luigi Pulvirenti, M.D. SCIENCE ASSOCIATES Adjunct Associate Professor Adjunct Associate Professor Dirk Homann, M.D., Ph.D. Caroline Lanigan, Ph.D. University of Colorado Donna L. Gruol, Ph.D. Teresa Reyes, Ph.D. Health Sciences Center Associate Professor Adjunct Assistant Professor Sam Madamba Denver, Colorado 328 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2008 THE SCRIPPS RESEARCH INSTITUTE

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 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2008 THE SCRIPPS RESEARCH INSTITUTE 329

Chairman’s Overview added to the studies on galanin and galanin receptors in anxiety and in depressive behaviors. n the past year, we experienced scientific successes The scientists of the department have engaged as well as organizational and policy changes in in many intradepartmental and interdepartmental I the Molecular and Integrative Neurosciences collaborations. Numerous high-impact invited lec- Department. The sci- tures and seminars were presented by the faculty entific work of sev- nationally and internationally. For example, I was the eral faculty members keynote speaker at the largest drug development resulted in high-sig- meeting (12,000 attendees) in Shanghai in June nificance, high-visi- 2007. Despite a difficult economic climate, scientific bility publications progress in the department was good, and our edu- and important new cational goals for our graduate students and post- research grants and doctoral fellows were all successfully met. Several renewals of earlier faculty and students received prestigous stipends. grants from the National

Institutes of Health. Tamas Bartfai, Ph.D. Particularly notewor- thy 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 antiep- ileptic 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 con- clusions about the genetic basis of vulnerability of Native Americans to alcohol addiction, and Donna Gruol added new data on the effects of the proin- flammatory 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 feed- ing 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 330 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2008 THE SCRIPPS RESEARCH INSTITUTE Investigator’s Reports 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 ther- Neuroscience Discovery and apeutic agents. We have built up a powerful and versatile portfolio of siRNA technology. Moreover, we have Pharmacogenomics introduced the use of locked nucleic acids as components of siRNAs (and antisense oligonucleotides) C. Wahlestedt, M.A. Faghihi, J. Huang, J. Kocerha, and have shown a range of beneficial properties of these A.M. Khalil, S. Brothers, F. Modarresi modified agents. ur research involves aspects of Alzheimer’s dis- G PROTEIN–COUPLED RECEPTORS AS DRUG ease, schizophrenia, alcohol addiction, fragile T ARGETS FOR CNS DISORDERS O X syndrome, autism, and aging. In addition to More than half of known drugs bind to G protein– drug discovery efforts, we focus on basic aspects of coupled receptors. We have continued our long-stand- mammalian genomics, genetics, and transcriptomics ing work on these receptors. Currently, we are focus- (RNA research). ing on neuropeptide Y and nociceptin receptors. This IDENTIFICATION AND FUNCTIONAL ANALYSIS OF research involves ultra-high-throughput screening. REGULATORY RNA TRANSCRIPTS HUMAN GENETICS AND PHARMACOGENOMICS We are among the few neuroscientists who have We are also involved in several genotyping and been and continue to be involved in high-throughput genome-wide association studies related to human CNS sequencing of transcriptomes (i.e., all the RNA tran- disorders, including schizophrenia. We wish to under- scripts in a cell) of humans and mice. Such efforts stand what makes certain individuals susceptible to have provided strong evidence that in contrast to ear- disease and how their responses to drug treatment may lier understanding, in mammalian cells, a majority of differ (pharmacogenomics). One of our goals is to identify the genome is transcribed. Analysis of such data sets biomarkers associated with human disorders, includ- has indicated that most mammalian RNA transcripts ing Alzheimer’s disease. are noncoding. PUBLICATIONS Thus, conventional protein-coding genes appear to Dahlgren, C., Zhang, H.Y., Du, Q., Grahn, M., Norstedt, G., Wahlestedt, C., account for only a minority of human RNA transcripts. Liang, Z. Analysis of siRNA specificity on targets with double-nucleotide mis- matches. Nucleic Acids Res. 36:e53, 2008. A substantial component of the full-length mouse and human cDNA sets that we and others have analyzed 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 does not contain an annotated protein- coding sequence noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward and likely corresponds to noncoding RNA. In addition regulation of β-secretase. Nat. Med. 14:723, 2008. to small RNAs, many of the noncoding RNAs consti- 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 tute natural antisense RNA transcripts. We have shown cells. Biochem. Biophys. Res. Commun. 368:703, 2008. that many noncoding RNAs identified to date have Huang, J., Young, B., Pletcher, M.T., Heilig, M., Wahlestedt, C. Association substantial conservation across species. We have also between the nociceptin receptor gene (OPRL1) single nucleotide polymorphisms shown that many small noncoding RNAs and antisense and alcohol dependence. Addict. Biol. 13:88, 2008. transcripts have differential expression under various Kemmer, D., Podowski, R.M., Yusuf, D., Brumm, J., Cheung, W., Wahlestedt, C., Lenhard, B., Wasserman, W.W. Gene characterization index: assessing the depth conditions and can affect conventional gene expression. of gene annotation. PLoS ONE 3:e1440, 2008. These novel RNA transcripts also likely are affected by Khalil, A.M., Faghihi, M.A., Modarresi, F., Brothers, S.P., Wahlestedt, C. A novel a range of disease processes in humans. A fruitful study RNA transcript with antiapoptotic function is silenced in fragile X syndrome. PLoS ONE 3:e1486, 2008. during the past year has been the investigation of RNA transcripts in the FMR1 locus, which is related to Khalil, A.M., Wahlestedt, C. Epigenetic mechanisms of gene regulation during mammalian spermatogenesis. Epigenetics 3:21, 2008. fragile X syndrome and to autism spectrum disorders. Scheele, C., Nielsen, A.R., Walden, T.B., Sewell, D.A., Fischer, C.P., Brogan, RNA INTERFERENCE AND DEVELOPMENT OF R.J., Petrovic, N., Larsson, O., Tesch, P.A., Wennmalm, K., Hutchinson, D.S., HIGH-THROUGHPUT GENOMICS TECHNOLOGY 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. RNA interference has become one of the most impor- 21:3653, 2007. tant gene manipulation technologies. Short interfering St-Laurent, G. III, Wahlestedt, C. Noncoding RNAs: couplers of analog and digital RNA (siRNA), the inducer of RNA interference in mam- information in nervous system function? Trends Neurosci. 30:612, 2007. MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE 347

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. Jennifer Caldwell Busby, Ph.D., Assistant Professor, and

Kristie Rose, Ph.D., Staff Scientist MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE 349

DEPARTMENT OF

MOLECULAR

THERAPEUTICS

STAFF Philip LoGrasso, Ph.D.* STAFF SCIENTISTS Monica Istrate, Ph.D. Associate Professor Patrick Griffin, Ph.D. Lisa Cherry, Ph.D. Brook Miller, Ph.D. Professor and Chairman Mathew T. Pletcher, Ph.D.** Kristie Rose, Ph.D. Jun Zhang, Ph.D. Director, Translational Assistant Professor Research Institute * Joint appointment in the Translational Research Institute Jennifer Caldwell-Busby, SENIOR SCIENTISTS RESEARCH ASSOCIATES Ph.D.* ** Joint appointments in the Department of Biochemistry Assistant Professor Scott Busby, Ph.D. Brian Ember, Ph.D. and the Translational Research Institute Gregg Fields, Ph.D. Michael Chalmers, Ph.D. Christie Fowler, Ph.D. Adjunct Professor Kevin Hayes, Ph.D. Paul J. Kenny, Ph.D. Assistant Professor Jonathan Hollander, Ph.D.

Chairman’s Overview lar disease, cancer, addiction, and metabolic disorders, including insulin resistance, obesity, and type 2 diabetes. he Department of Molecular Therapeutics was Paul Kenny and his group focus on the neuropharma- established on the Florida campus of Scripps cology of addiction and on establishing the role of sev- T Research in 2007. Faculty in the department eral G protein–coupled receptors in addictive behavior. use chemical biology Phil LoGrasso and members of his laboratory are involved approaches to dissect in the discovery of small-molecule therapeutic agents to signaling pathways and be used as neuroprotective agents in diseases such as transcriptional programs. Parkinson’s and are determining the role of rho kinase We rely on state-of-the- in vascular bed modulation and glaucoma. Thomas Burris art multidisciplinary and his group are studying the role of orphan nuclear technology and methods receptors in circadian rhythms and metabolic disorders and a variety of model such as obesity. Scientists in Jennifer Caldwel Busby’s systems for target iden- laboratory use state-of-the-art mass spectrometry to identification, validation, tify, quantify, and characterize proteins and protein and preclinical studies. modifications to map the signaling pathways related to Currently, the department diabetes and cancer. Peter Hodder and coworkers focus Patrick R. Griffin, Ph.D. has 5 tenure-track facul- on technology and assay development and novel chemi- ty members and several cal approaches to expand compound libraries. Michael non–tenure track members who oversee key functional Cameron and his group are involved in mechanistic cores on the Florida campus. These investigators have studies of P450s and drug biotransformation mechanisms. created strong research programs that take advantage Researchers in my group are dissecting the mechanism of the unique high-throughput core facilities at the of ligand-dependent activation of orphan nuclear recep- Florida campus, including genomics, cell-based screen- tors implicated in cancer and metabolic disorders. ing, 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- 350 MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE Investigators’ Reports high-throughput screening to identify coactivator-selective agonists of the receptor, we have developed a validated time-resolved fluorescence resonance energy transfer assay Probing Protein Dynamics With for ligand-dependent recruitment of the coactivator to PPARγ. Scientists at the Scripps Research Institute Molecular Library Hydrogen-Deuterium Exchange Screening Center used these assays to examine the National Mass Spectrometry Institute of Health small-molecule library. The results obtained from this research are providing molecular P.R. Griffin, S.A. Busby, M.J. Chalmers, S.Y. Dai, J. Zhang, insight into coactivator recruitment and receptor activation M. Istrate, R. Garcia-Ordonez, S. Novick, B. Pascal, and will result in chemical tools to dissect the biologi- J. Conkright, G. Zastrow-Hayes, K. Hayes, T. Schröter, cal role of specific coactivators in modulating PPARγ. F. Madoux, D. Minond, P.S. Hodder LIGAND ACTIVATION OF THE VITAMIN D RECEPTOR In collaboration with scientists at Eli Lilly and Com- e use a wide range of technologies to study pany, Indianapolis, Indiana, we are using HDX to charac- ligand activation of nuclear receptors. During terize activation of the full-length heterodimer complex the past few years, we focused on the ligand- W composed of the vitamin D receptor and its coreceptor binding domains of the well-characterized nuclear recep- retinoid X receptor α. This project is promoting further tors peroxisome proliferator–activated receptor γ (PPARγ) development of our HDX platform to facilitate the analy- and the α and β estrogen receptors. Recently, we have sis of large transcriptional complexes. Although this focused on developing hydrogen-deuterium exchange research is in an early stage, we have data that suggest (HDX) technology for probing the mechanism of activa- HDX is useful for probing dynamics of large transcription of several orphan nuclear receptors. In addition, in tional complexes. collaboration with scientists at Xencor, Monrovia, Cali- PROBING G PROTEIN–COUPLED RECEPTORS fornia, we are studying the dynamics of TNF-α. G protein–coupled receptors are an important family of LIGAND ACTIVA TION OF PPARγ transmembrane signaling proteins. Characterization of the PPARγ is a multidomain ligand-dependent transcrip- structure and dynamics of these proteins is an analytical tion factor. Ligands regulate PPARγ activation by bind- challenge because their transmembrane domains are ing to the receptor’s ligand-binding domain, inducing a hydrophobic. We have begun to expand the application change in the conformational dynamics of the domain of HDX to probe the dynamics of these receptors. This that leads to dissociation of corepressor molecules and work is being done in collaboration with H. Rosen, Depart- formation of suitable neoepitopes for the binding of coac- ment of Chemical Physiology, and R.C. Stevens, Depart- tivator molecules. We used structural, biochemical, and ment of Molecular Biology. cell-based techniques to examine the mechanism of ligand regulation of PPARγ transcriptional activity. We PUBLICATIONS Bruning, J., Chalmers, M.J., Prasad, S., Busby, S.A., Kamenecka, T., He, Y., Net- found that the magnitude of PPARγ agonism is regulated tles, K.W., Griffin, P.R. Partial agonists activate PPARγ using a helix 12 independent by coactivator recruitment selectivity of p160 coactiva- mechanism. Structure 15:1258, 2007. tors. In mutagenesis studies, we determined the key Chalmers, M.J., Busby, S.A., Pascal, B.D., Southern, M.R., Griffin, P.R. A two- residues on the receptor that facilitate these selective stage differential hydrogen deuterium exchange method for the rapid characterization of protein/ligand interactions. J. Biomol. Tech. 18:194, 2007. coactivator interactions. Dai, S.Y., Chalmers, M.J., Bruning, J., Bramlett, K.S., Osborne, H.E., Montrose- In other studies, we are using coactivators as chemi- Rafizadeh, C., Barr, R.J., Wang, M., Burris, T.P., Dodge, J.A., Griffin, P.R. Predic- cal tools to generate desired functional responses and to tion of the tissue-specificity of selective estrogen receptor modulators using a single biochemical method. Proc. Natl. Acad. Sci. U. S. A. 105:7171, 2008. differentiate pharmacologically beneficial function from adverse function, a novel unexploited therapeutic avenue 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 for treating insulin resistance. Our goals are to deter- functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008. mine the structure-activity relationships between PPARγ ligands and their coactivator recruitment selectivity and to obtain PPARγ ligands with specific coactivator pref- erences by screening for agonists that favor specifically the association of a given cofactor. For a large-scale MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE 351 Mass Spectrometry for modifications associated with sites of transcription can be used as the basis for further experiments to deter- Identification of Proteins mine the biological roles of the proteins in gene regulation and activation. J.A. Caldwell Busby, V. Cavett PUBLICATIONS ur general focus is the use of cutting-edge sep- Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V., aration and mass spectrometry techniques to Orth, A.P., Busby, J., Hogenesch, J.B., Conkright, M.D. A coactivator trap identi- fies NONO (p54nrb) as a component of the cAMP signaling pathway. Proc. Natl. O identify proteins involved in biological events. Acad. Sci. U. S. A. 104:20314, 2007. 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. Neurobiology of Addiction We provide these collaborators access to powerful and novel approaches to examine posttranslational modifi- P.J. Kenny, P. Bali, C.D. Fowler, J.A. Hollander, H.-I. Im, cations and measure protein levels in multiple samples. P.M. Johnson,* Q. Lu, B.H. Miller In addition to these collaborative efforts, we are * Kellogg School of Science and Technology, Scripps Research developing a method to identify and temporally map chromatin proteins involved in transcriptional regula- e focus on understanding the neurobiological tion. Gene regulation is a fundamental biological pro- mechanisms of addiction. This knowledge will cess that is studied from a variety of perspectives with W be used to develop novel therapeutic agents a variety of methods; however, research to date has for treatment of substance abuse. We use a multidisci- been highly gene centric, and only a few reports have plinary approach that includes mouse behavioral genet- been published on the proteomics of gene regulation. ics, virus-mediated gene expression, RNA and protein We target these missing proteomics components, par- analyses, and in vivo behavioral testing. ticularly the components of the supermolecular com- NICOTINE ADDICTION plex of chromatin, including nucleosome substructure We seek to identify the subtypes of nicotinic acetyl- and regulatory and transcription complexes. choline receptors and downstream signaling cascades Methods for whole-system approaches are difficult through which nicotine promotes tobacco addiction. to implement because traditional technologies tend to Currently, we are assessing the reinforcing effects of focus on the isolation and analysis of individual parts nicotine in mice with null mutations in various sub- of the whole—DNA, RNA, or protein. Our techniques units of the receptors. In addition, we are testing the combine advances in molecular biology with the power effects on nicotine reinforcement of using lentivirus- of mass spectrometry to identify novel biomolecules based short hairpin RNAs to silence the genes of tar- involved in multibiopolymer complexes. In particular, geted nicotinic acetylcholine receptor subunits in brain we are modifying and combining techniques such as reward circuitries. Finally, we are using a proteomics protein-protein and protein-DNA cross-linking, immuno- approach to identify the intracellular proteins coupled precipitation methods, chromatin immunoprecipitation, to nicotinic acetylcholine receptors in the brains of mice. mass spectrometry, and liquid chromatography to deter- Our goal is to identify novel scaffold and signaling pro- mine the larger regulatory mechanisms involved in the teins involved in transducing the addictive actions of fate of cells. The keystone of this method is a modified nicotine. These studies promise to yield significant new chromatin immunoprecipitation protocol that maintains insights into the neurobiological mechanisms of nico- the integrity of the DNA while allowing for the isola- tine addiction, with direct relevance for the treatment tion, recovery, and analysis of the protein components of of the tobacco habit in humans. the nucleosome complex. This advanced method targets BRAIN SYSTEMS INVOLVED IN ADDICTION proteins that regulate chromatin function and corre- In collaboration with other scientists at Scripps lates those proteins with histone modification states Research, we found that the neuropeptide orexin and gene occupancy. Incorporating proteomics into a (hypocretin) plays a critical role in drug reward. In traditionally DNA-based experimental protocol provides ongoing studies, we are identifying the mechanisms a new perspective and a novel approach to genetic through which orexin-mediated transmission regulates regulation. Newly identified proteins and novel protein drug reward. We are also investigating the roles of 352 MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE novel constitutive mechanisms of gene regulation in the the cell death associated with Parkinson’s disease and neuroplasticity induced by drugs of abuse that may Alzheimer’s disease. promote addiction. Further, we are testing the hypoth- JNK is linked to many of the hallmark pathophysi- esis that drug addiction and obesity share common ologic components of Parkinson’s disease, such as oxi- reward and motivational mechanisms. These studies may dative stress, programmed cell death, and microglial identify novel targets for the development of therapeu- activation. Many pieces of evidence support JNK as a tics against addiction and obesity. target for treatment of the pathologic changes that under- DEVELOPMENT OF NOVEL ANTIADDICTION lie Parkinson’s disease. One attractive feature of JNK3

MEDICATIONS as a selective drug target is that this kinase is almost In collaborations with scientists in the Translational exclusively expressed in the brain. In contrast, JNK1 Research Institute, Scripps Florida, we are developing and JNK2 are ubiquitously expressed. Despite the ubiq- small-molecule drugs that may be useful as novel thera- uitous expression of JNK2, we are developing a ther- peutic agents for treatment of substance abuse disorders. apy to prevent degeneration of dopaminergic neurons The targets for these drugs are G protein–coupled and halt the progression of Parkinson’s disease by tar- receptors that we previously showed play a role in geting JNK2/3. drug dependence. Our strategy for inhibiting JNK2/3 is based on the results of experiments with mice in which the gene for PUBLICATIONS JNK3 or JNK2 was deleted and mice in which the genes 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 for both JNK2 and JNK3 or both JNK1 and JNK2 were noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward deleted. In contrast to mice lacking the gene for JNK1 regulation of β-secretase. Nat. Med. 14:723, 2008. alone, which had defective T-cell differentiation, mice Johnson, P.M., Hollander, J.A., Kenny, P.J. Decreased brain reward function during nicotine withdrawal in C57BL6 mice: evidence from intracranial self-stimulation lacking the gene for JNK2 alone had normal T- and (ICSS) studies. Pharmacol. Biochem. Behav. 90:409, 2008. B-cell development and normal T-cell proliferation. More-

Kenny, P.J., Chartoff, E., Roberto, M., Carlezon, W.A., Jr., Markou, A. NMDA over, mice lacking the gene for JNK2 alone and mice receptors regulate nicotine-enhanced brain reward function and intravenous nico- lacking the gene for JNK3 alone were protected against tine self-administration: role of the ventral tegmental area and central nucleus of the amygdala. Neuropsychopharmacology 14:723, 2008. the effects of 1-methyl-4-phenyl-1,2,3,5-tetrahydro- pyridine (MPTP), a compound used to induce parkinsonian signs in animal models of Parkinson’s disease, Inhibition of Jun N-Terminal whereas both wild-type mice and mice lacking the gene for JNK1 were not. In other research, compared Kinase 2/3 for the Treatment with wild-type mice, mice lacking the genes for both JNK2 and JNK3 were dramatically protected against of Parkinson’s Disease acute MPTP-induced injury of the nigrostriatal pathway. This protective effect resulted in a 3-fold increase in P. LoGrasso, M. Cameron, W. Chen, S. Clapp, D. Duckett, the number of neurons positive for tyrosine hydroxy- B. Ember, J. Habel, R. Jiang, T. Kamenecka, S. Khan, lase, an indication of the increase in survival of dopa- L. Ling, Y.-Y. Ling, M. Lopez, A. Pachori, C. Ruiz, Y. Shin, minergic neurons. X. Song, T. Vojkovsky, D. Zadory On the basis of these in vitro and in vivo data, we poptosis, or programmed cell death, plays a vital are synthesizing potent, selective JNK 2/3 inhibitors role in the normal development of the nervous that we will test for efficacy in MPTP animal models A system and is also thought to contribute to the of Parkinson’s disease. We have established homoge- aberrant neuronal cell death that characterizes many nous time-resolved fluorescence biochemical assays neurodegenerative diseases. Therefore, blocking neu- for JNK3 and counterscreens for JNK1 and p38. We ronal apoptosis could be an approach for treating neu- have generated more than 1000 compounds from 3 rodegenerative diseases. A major pathway implicated different structural classes; many of the compounds in neuronal cell death and survival is the MAP kinase are inhibitory for JNK3 in nanomolar concentrations. pathway, which controls cell proliferation and cell death Some of the compounds have a cellular potency of in response to many extracellular stimuli. Recent stud- 40–60 nM and in vitro efficacy in promoting primary ies have linked Jun N-terminal kinase (JNK) activity with survival of dopaminergic neurons. We have tested com- MOLECULAR THERAPEUTICS 2008 THE SCRIPPS RESEARCH INSTITUTE 353

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 structure- based 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., Bail- largeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M., Tsi- noremas, 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. 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 LPA- induced 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 96- well 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 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 367

TRANSLATIONAL William Roush, Ph.D.** Romain Noel, Ph.D. RESEARCH INSTITUTE Executive Director, Medicinal Chemistry Sanjay Saldanha, Ph.D.

STAFF Associate Dean, Kellogg School of Science and E. Hamp Sessions, Ph.D. Technology Patrick Griffin, Ph.D.* Anthony Smith, Ph.D. Director Xinyi Song, Ph.D. Thomas D. Bannister, Ph.D. SENIOR SCIENTISTS Associate Scientific Director, Prem Subramaniam, Ph.D. Medicinal Chemistry Yenting Chen, Ph.D. Dusica Vidovic, Ph.D. Jennifer Caldwell Busby, Rong Jiang, Ph.D. Ph.D.* Kristen Clarke Ware, Ph.D. Associate Scientific Director, Marcel Koenig, Ph.D. Proteomics Yan Yin, Ph.D. Jiuxiang Ni, Ph.D. Michael Cameron, Ph.D.* Alok Pachori, Ph.D. Associate Scientific Director, SCIENTIFIC ASSOCIATES Drug Metabolism and Louis Scampavia, Ph.D. Pharmacokinetics Dmitriy Minond Thomas Schröter, Ph.D. Derek R. Duckett, Ph.D. Timothy Spicer Associate Scientific Director, Youseung Shin, Ph.D. Discovery Biology Tomas Vojkovsky, Ph.D. HTS ROBOTICS Yangbo Feng, Ph.D. ENGINEERS Associate Scientific Director, Medicinal Chemistry STAFF SCIENTISTS Pierre Baillargeon

Peter Hodder, Ph.D. Lisa Cherry, Ph.D. Peter Chase Scientific Director, Lead Identification Juliana Conkright, Ph.D. Lina Deluca

Ted Kamenecka, Ph.D. Dympna Harmey, Ph.D. Associate Scientific Director, Medicinal Chemistry Sahba Tabrizifard, Ph.D. INFORMATICS STAFF Congxin Liang, Ph.D. Scientific Director, Medicinal SENIOR RESEARCH Caty Chung Chemistry ASSOCIATE Yasel Cruz Philip LoGrasso, Ph.D.* Franck Madoux, Ph.D. Senior Director, Discovery Kashif Hoda Biology Bruce Pascal Patricia McDonald, Ph.D. RESEARCH ASSOCIATES Associate Scientific Director, Stephan Schuerer Discovery Biology Sarwat Chowdhury, Ph.D. Mark Southern Becky Mercer, Ph.D. Melissa Crisp, Ph.D. Associate Scientific Project Brian Ember, Ph.D. Manager, Lead * Joint appointment in the Identification Xingang Fang, Ph.D. Department of Molecular Therapeutics Mathew T. Pletcher, Ph.D.** Yuanjun He, Ph.D. ** Joint appointment in the Assistant Professor, RNA Department of Chemistry Core Xiaohai Li, Ph.D. 368 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE Director’s Overview allow Scripps Research investigators to examine the genome at both the genetic and the transcriptional level he Translational Research Institute merges drug for the genes that underlie common diseases. In collab- discovery efforts at the Scripps Research Florida oration with colleagues on the Florida campus, members T campus with advanced technology platforms to of the core have been involved in projects to identify the rapidly identify and vali- genes responsible for pathologic conditions, such as date biological pathways addiction and alcoholism, systemic lupus erythemato- that can be targeted for sus, autism, obsessive-compulsive disorder, diabetes, therapeutic intervention. obesity, and prion diseases. The goal of the drug dis- The cell-based screening platform is headed by covery operation is to Julie Conkright, Department of Molecular Therapeutics. discover and develop The faculty advisor to the core is Michael Conkright, small-molecule therapeutic Department of Cancer Biology. In this group, high-through- agents for unmet medical put technologies are used to provide a systematic descrip- needs in neurodegeneration of the function of genes encoded by the human tion, Parkinson’s disease, genome and a more comprehensive understanding of the acute respiratory distress genetic basis for human disease. Members of the group syndrome, glaucoma, provide investigators access to genome-wide collections Patrick R. Griffin, Ph.D. spinal cord injury, cancer, of cDNAs and short interfering RNAs that can be used and metabolic disorders, including insulin resistance, to examine cellular models of signal transduction path- type 2 diabetes, and obesity, by targeting G protein– ways and phenotypes. In addition, the cell-based screen- coupled receptors, proteases, ion channels, and kinases. ing platform participates in one of the center-based The drug discovery component of the Translational initiatives of the Scripps Research MLPCN center. Research Institute is fully integrated with the following The proteomics platform is headed by Jennifer groups: Lead Identification and High-Throughput Screen- Caldwell Busby, Department of Molecular Therapeutics. ing, headed by Peter Hodder, Department of Molecular The focus of this core is using liquid chromatography Therapeutics; Medicinal Chemistry, headed by William and state-of-the-art mass spectrometry technology to Roush, Department of Chemistry; Discovery Biology, identify, quantify, and characterize proteins and protein headed by Phil LoGrasso, Department of Molecular modifications. Researchers in the core are involved in Therapeutics; Drug Metabolism and Pharmacokinetics, scientific collaborations in which novel technologies are headed by Mike Cameron, Department of Molecular used to identify biologically important proteins and pro- Therapeutics; and Informatics, headed by Mark Southern. tein modifications. Large-scale differential analysis is The Lead Identification team enables drug-target lead being used to map the pathways related to insulin sen- identification via ultra-high-throughput screening technol- sitization and adipogenesis. In other projects, chromato- ogy. Using state-of-the-art automation and instrumentation, graphic enrichment techniques are used to identify sites members in this group are responsible for developing of phosphorylation and other posttranslational modifi- and executing biochemical or cell-based high-throughput cations. Researchers in the proteomics core collaborate screening assays in a miniaturized 1536-well microtiter with other scientists to create experiments that will plate format. In addition to its support of internal Scripps provide meaningful mass spectrometric results. 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 exter- nal 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 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 369 Investigators’ Reports tor applied topically to the eye must simultaneously have many properties, including high ROCK affinity, aqueous solubility, excellent corneal permeability, high Drug Discovery: Medicinal cellular penetration, and low ocular clearance, to provide a long-lasting effect. Most importantly, the inhibitor must Chemistry Efforts be selective for ROCK over other enzymes and receptors so that no serious side effects occur. T.D. Bannister, Y. Feng, T.M. Kamenecka, C. Liang, We have synthesized thousands of new ROCK inhibi- W.R. Roush, Y. Chen, S. Chowdhury, X. Fang, Y. He, tors in multiple chemical classes; many have low nanomo- R. Jiang, M. Koenig, R. Noel, E.H. Sessions, Y. Shin, lar potency in both biochemical and cell-based assays, X. Song, T. Vojkovsky, Y. Yin high selectivity, and a profile of properties appropriate for e seek to discover new compounds to treat preclinical development. For example, SR-3677 was diseases for which current therapies are inad- tested in an animal model for glaucoma by our collabora- W equate. In our major programs during the past tor, V. Rao, Duke University, Durham, North Carolina. year, we targeted glaucoma, Parkinson’s disease, and The inhibitor lowered intraocular pressure more than breast cancer. In each program, we have attempted to 30% within 1 hour, an efficacy comparable to that of block the action of a specific protein kinase that is over- antiglaucoma drugs in current use. The reduction in pres- active or overabundant in affected patients and that sure waned after 2 hours, however, so we are designing hastens the progression of disease. In 2 of the programs, other compounds intended to have a similarly powerful we began by identifying chemical leads from a high- yet more sustained effect. As expected, the reduction throughput biochemical screen of the Scripps collec- in pressure was due to an increased rate of fluid outflow. tion of more than 700,000 compounds. The structural We have also made compounds that distinguish information from these screens, in combination with between the enzyme isoforms ROCK-I and ROCK-II to computational and biological analysis of compounds test their precise roles. An inhibitor selective for ROCK-II, made in other laboratories targeting the same enzymes, for example, would lack any unwanted side effects due provides insights for modifying the structures to obtain to ROCK-I inhibition. Such effects are unclear, because unique and patentable leads with the required druglike no other isoform-selective ROCK inhibitors targeting biochemical, physical, and pharmacologic properties. All glaucoma are known. of these are evaluated internally by Scripps scientists in PARKINSON’S DISEASE the biology, pharmacology, and drug metabolism and In collaboration with the National Institute of Neuro- pharmacokinetics groups of the Translation Research logical Disorders and Stroke, we are developing a ther- Institute, who work closely with the medicinal chemists apy to interrupt the loss of dopamine-containing neurons on fully integrated interdisciplinary project teams. in the midbrain that is a hallmark of Parkinson’s disease. GLAUCOMA Activation of the transcription factor c-Jun by c-Jun We are designing inhibitors of the serine-threonine N-terminal kinase (JNK) promotes neurodegeneration. kinase ROCK, or Rho kinase, which regulates intraocular Inhibitors of JNK, which exist in 3 isoforms, JNK1, pressure by controlling the outflow of aqueous humor. JNK2, and JNK3, are neuroprotective in animal models Excess ROCK activity is associated with high intraocu- of Parkinson’s disease. Our approach, using inhibitors lar pressure, which is a primary risk factor for glaucoma, selective for JNK2 and JNK3, would be a quantum leap and with retinal damage. Application of a ROCK inhib- in the clinical treatment of Parkinson’s disease for sev- itor increases outflow, lowers intraocular pressure, and eral reasons. All current therapies merely treat the symp- preserves retinal neurons. Current antiglaucoma drugs toms of the disease rather than address the underlying have limited efficacy or cause side effects, including pathologic changes, they tend to lose therapeutic effi- discomfort, hyperemia (red eye), and/or undesired cacy over time, and they typically elicit undesired side changes in cardiovascular function. No glaucoma drugs effects. Our challenge is to develop a compound that on the market act by directly altering the Rho kinase is a potent, selective, and cell-permeable JNK2/3 inhibi- pathway, but ROCK inhibitors have strong pressure- tor; has the pharmacokinetic properties for oral dosing lowering and neuroprotective effects and thus could (ideally once a day); has good brain penetration; and be a valuable new treatment. An ideal ROCK inhibi- has a benign toxicology profile. 370 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE

We have synthesized thousands of new JNK inhib- prevent neuronal damage in a host of other disorders itors in multiple chemical scaffolds and are evaluating including stroke, Alzheimer’s disease, and amyotrophic compounds with the best combination of properties in lateral sclerosis. several preclinical animal models for Parkinson’s dis- We anticipate that in each of our research programs ease. For example, in a pilot study, the Scripps JNK we can continue to synthesize novel compounds with the inhibitor SR-3306 delivered systemically to rodents via right combination of properties that would permit devel- osmotic minipump at 10 mg/kg reduced CNS-mediated opment of the compounds as safe and effective agents behaviors that occur after a chemically induced brain for stopping the progression of important diseases. lesion used to mimic the parkinsonian condition. Newer generation compounds, including SR-3562, will soon PUBLICATIONS Chen, Y.T., Bannister, T.D., Weiser, A., Griffin, E., Lin, L., Ruiz, C., Cameron, be evaluated in animal models and are particularly M.D., Schürer, S., Duckett, D., Schröter, T., Lograsso, P., Feng, Y. Chroman-3- promising because of improved properties, including amides as potent Rho kinase inhibitors. Bioorg. Med. Chem. Lett., in press. high oral bioavailability (45%), high cell-based potency Feng, Y., Cameron, M.D., Frackowiak, B., Griffin, E., Lin, L., Ruiz, C., Schröter, (0.06 µM), and excellent distribution to the brain of T., LoGrasso, P. Structure-activity relationships and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of rodents after oral dosing. ROCK-II. Bioorg. Med. Chem. Lett. 17:2355, 2007. CANCER Feng, Y., Yin, Y., Weiser, A., Griffin, E., Cameron, M.D., Lin, L., Ruiz, C., Schürer, In collaboration with Poniard Pharmaceuticals, 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 South San Francisco, California, we have synthesized Rho kinase (ROCK-II) inhibitors. J. Med. Chem. 51:6642, 2008. many potent and selective novel inhibitors of focal adhe- Jiang, R., Duckett, D., Chen, W., Habel, J., Ling, Y.Y., LoGrasso, P., Kamenecka, sion kinase (FAK). FAK inhibitors could be an impor- T.M. 3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors. tant new means of treating solid tumors, including breast Bioorg. Med. Chem. Lett. 17:6378, 2007. cancer. FAK has been implicated in promoting detach- LoGrasso, P., Kamenecka, T. Inhibitors of c-jun-N-Terminal Kinase (JNK). Mini Rev. ment of tumor cells and metastasis, characteristics of Med. Chem. 8:755, 2008. almost all advanced-stage solid tumors that are respon- Sessions, E.H., Yan, Y., Bannister, T.D., Pocas, J., Cameron, M.D., Ruiz, C., sible for most of the suffering and death related to can- Schürer, S.C., Schröter, T., LoGrasso, P., Feng, Y. Benzimidazole- and benzoxa- zole-based inhibitors of Rho kinase. Bioorg. Med. Chem. Lett., in press. cer. 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 Proteomics Laboratory completed our FAK chemistry efforts after identifying J.A. Caldwell Busby, V. Cavett highly potent and selective FAK inhibitors, including SR- 2516. This lead compound is effective in in vitro tumor he Proteomics Laboratory at Scripps Florida pro- metastasis models, is efficacious in animal models of vides proteomics services and expertise to scien- tumor progression, has desirable pharmaceutical prop- T tific collaborators at Scripps Research facilities erties suitable for convenient once-a-day oral dosing, in both Florida and California, universities within the and is being licensed for further development. state of Florida, and other educational institutions. We FUTURE DIRECTIONS use cutting-edge mass spectrometry technology to identify We are continuing our research on glaucoma and proteins, map modifications that occur after transla- Parkinson’s disease and have smaller or exploratory tion, and do relative quantitation experiments with a efforts in other areas, including methods for treating variety of sample types. diabetes, for curbing drug addiction, and for targeting In its lifetime, a protein can have several locations cancer progression by other mechanisms. We hope to and functions within a cell. Location, function, and expand these efforts. Many of the compounds identi- 3-dimensional structures of proteins are all influenced fied in the ROCK and JNK inhibitor programs are also by static and dynamic chemical modifications that occur likely to be useful in the treatment of other diseases. after translation. These modifications vary from small For example, animal data suggest that ROCK inhibitors methyl and acetyl groups, which are part of the histone might be an effective treatment for multiple sclerosis. codes, to large lipid and glycosylation modifications, Strong preclinical evidence shows that JNK inhibitors, which act as cellular markers and signaling molecules. in addition to treating Parkinson’s disease, also may With mass spectrometry, we can detect both the small TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 371 and the large changes in mass that occur in proteins include P450 structure-function relationships and the because of these modifications, and we can identify formation of reactive intermediates during metabolism. the specific amino acids modified. The laboratory is equipped with a liquid chromatogra- Relative changes in protein levels or level of post- phy–tandem mass spectrometry system and a Q-trap translational modification between multiple samples pro- hybrid triple quadrupole/linear ion-trap mass spectrometer. vide biologically relevant information about cellular pathways and proteins of interest. Large-scale studies of PUBLICATIONS Cameron, M.D., Wen, B., Roberts, A.G., Atkins, W.M., Campbell, A.P., Nelson, this type require rigorous sample preparation and highly S.D. Cooperative binding of acetaminophen and caffeine within the P450 3A4 tuned algorithms for comparing different mass spectro- active site. Chem. Res. Toxicol. 20:1434, 2007. metric analyses. We are currently validating methods for Cameron, M.D., Wright, J., Black, C.B., Ye, N. In vitro prediction and in vivo veri- fication of enantioselective human tofisopam metabolite profiles. Drug Metab. Dis- both sample fractionation and data analysis for these pos. 35:1894, 2007. types of large-scale differential protein experiments. Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright. J.J., Griffin, Mass spectrometers at the facility include an ion trap P.R., Thacher, S., Hodder, P. Potent, selective and cell penetrant inhibitors of SF-1 spectrometer, which is used mostly to identify proteins by functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008. and peptides, and a triple quadrupole mass spectrometer, Miller, B.H., Schultz, L.E., Gulati, A., Cameron, M.D., Pletcher, M.T. Genetic reg- which is used for relative quantitation experiments. A ulation of behavioral and neuronal responses to fluoxetine. Neuropsychopharmacol- ogy 33:1312, 2008. 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 elec- trospray ionization sources and capillary high-perfor- Cell-Based Screening Core mance liquid chromatography columns. J.J. Conkright, G. Zastrow, J. Cartzendafner, M. Morris Data analysis is performed primarily via automated workflow on a cluster maintained by the bioinformatics he Cell-Based Screening Core provides high- group. Automation of the front-end processing allows throughput screening of functional genomic a more thorough review of the data and more time T platforms and consults with researchers from for development of innovative software in collaboration Scripps, both in California and Florida; universities with information technology groups at Scripps Research in Florida; and other outside academic institutions to and beyond. perform these screens. We curate 2 large libraries: the Mammalian Genome Collection cDNA library and the Qiagen Druggable siRNA library. Screening these librar- Drug Metabolism and ies allows investigators to determine if overexpression of a single gene (Mammalian Genome Collection cDNA Pharmacokinetics Laboratory library) or reduction in expression levels of a single gene (Qiagen siRNA library) positively or negatively influences M.D. Cameron, L. Lin, C. Ruiz, S. Khan, Z. Li their particular biological readout. These libraries pro- he Drug Metabolism and Pharmacokinetics Lab- vide investigators a unique tool to identify novel fac- oratory at Scripps Florida provides in vitro and tors and pathways involved in biological systems. The T in vivo evaluation of the pharmacokinetic and findings can lead to new areas of research and novel pharmacodynamic properties of new chemical entities. targets for drug development. We work on project teams within the drug discovery In addition to our large libraries, we have 2 small group of the Department of Molecular Therapeutics and libraries that we built: a transcription factor library and support chemistry efforts within the Scripps Research a nuclear receptor library. These libraries are important Institute Molecular Screening Center. We help bridge new tools for investigators who study the effects of pro- medicinal chemistry and pharmacology by evaluating teins and signaling pathways on gene expression. These the metabolic fate and identifying the liabilities of libraries are also a mechanism for studies of the spec- compounds. Pharmacokinetic studies provide basic ificity of new potential drugs and chemical probes that parameters, including peak plasma concentration, bio- modulate gene expression. availability, exposure, half-life, clearance, volume of A third area of expertise we provide is the genera- distribution, and tissue distribution. Research interests tion of mutagenesis screens. Determining the regions 372 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE or residues in a protein that are important for its bio- United States. The prognosis for these patients is poor, logical function can be a key component in dissecting and treatment options are limited. We will focus on how the protein interacts with other factors. Chemical defining the role Jun N-terminal kinase signaling plays mutagenesis of a gene permits an unbiased approach in tumor maintenance and cell dispersal and whether to identifying these biologically critical residues of inhibition of this kinase has therapeutic potential in this the protein. We perform the mutagenesis and provide devastating disease. screening sets for investigators to examine the effect In addition, we are involved in the Scripps-Pfizer the mutation has on a chosen biological end point. collaboration that was started in 2007. Since then, sev- Last, we counsel researchers on how to validate eral assays have been designed for high-throughput their screens and counterscreen the rank-order hits. screening of targets of therapeutic interest to Pfizer. These tasks are extremely important to prove the sta- PUBLICATIONS tistical significance of a finding and to ascertain the Jiang, R., Duckett, D., Chen, W., Habel, J., Ling, Y.Y., LoGrasso, P., Kamenecka, specificity of the finding for that precise biological T.M. 3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors. Bioorg. Med. Chem. Lett. 17:6378, 2007. function or pathway. 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. PUBLICATIONS In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1. Amelio, A.L., Miraglia, L.J., Conkright, J.J., Mercer, B.A., Batalov, S., Cavett, V., Mol. Cancer Ther. 6:450, 2007. Orth, A.P., Busby, J., Hogenesch, J.B., Conkright, M.D. A coactivator trap identi- fies NONO (p54nrb) as a component of the cAMP-signaling pathway. Proc. Natl. Rech, J.C., Yato, M., Duckett, D., Ember, B., LoGrasso, P.V., Bergman, R.G., Ell- Acad. Sci. U. S. A. 104:20314, 2007. man, J.R. Synthesis of potent bicyclic bisarylimidazole c-jun N-terminal kinase Madoux, F., Li, X., Chase, P., Zastrow, G., Cameron, M.D., Conkright, J.J., Griffin, inhibitors by cyclic C-H bond activation. J. Am. Chem. Soc. 129:490, 2007. 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. 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. Discovery Biology: Kinases 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 Probe and Drug Discovery: The kinase inhibitors of biologic interest and thera- Lead Identification Department W peutic potential. Protein kinases are important components of signal transduction pathways, and P. Hodder, A. Abovich, P. Baillargeon, P. Chase, M. Crisp, deregulation of kinase activity in humans can lead to L. DeLuca, R. Einsteder, K. Emery, F. Madoux, B. Mercer, disease. Kinases have become one of the most impor- D. Minond, M. Petrillo, A. Porto, S. Saldanha, L. Scampavia, tant target classes for drug development. We are opti- M. Spaargaren, T. Spicer, V. Fernandez-Vega mizing a novel class of kinase inhibitors for treatment of Parkinson’s disease. Although the cause of Parkinson’s he Lead Identification Department is responsible disease is unknown, a strong correlation exists between for developing and executing high-throughput loss of primary dopaminergic neurons within the sub- T screening (HTS) assays and for supporting down- stantia nigra and progression to the diseased state. stream medicinal chemistry and related “hit-to-lead” Our current goal is to develop an inhibitor of the efforts (Fig. 1). The anchors of the department are 2 Jun N-terminal family of kinases; our aim is to protect fully automated robotic platforms. One supports screen- the primary dopaminergic neurons from cell death, thus ing of 384- and 1536-well microtiter plates in a vari- slowing or halting the progression of the disease. Work- ety of biochemical and cell-based assay formats. The ing closely with scientists in other disciplines neces- other is used to manage and distribute the more than sary for lead optimization (chemistry, pharmacology, 600,000 compounds used for drug discovery at Scripps and drug metabolism), we were successful in securing Research and 300,000 compounds for the Molecular funding from the National Institute of Neurological Dis- Libraries Probe Production Centers Network. The facility orders and Stroke for this research. also contains an assay development laboratory, equipped We are also investigating the role of MAP kinases with bacterial culture, protein purification, compound in primary brain cancers. In 2008, brain tumors will characterization, and tissue culture laboratories as well be diagnosed in approximately 20,000 patients in the as semi-automated equipment for liquid handling and TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 373

Fig. 1. The uHTS laboratory of the Lead Identification Department hous- es 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 related collaborations (Table 1) and have contributed to laboratory information management system, which tracks the discovery more than 10 novel leads (chemical HTS assay data and compound usage and quality. Addi- probes) of G protein–coupled receptors, metallopro- tionally, we are involved in developing metallo-β-lacta- teinases, nuclear receptors/transcription factors, and mase class B1 chemical probes. kinases (http://molscreen.florida.scripps.edu/). THE SCRIPPS RESEARCH INSTITUTE MOLECULAR DISCOVERY AND DEVELOPMENT OF CLASS B SCREENING CENTER METALLO-β -LACTAMASE INHIBITORS Established in July 2005, the Scripps Research The diversity of bacterial β-lactamases continues to Institute Molecular Screening Center is a national outpace the development of useful β-lactam–based antibi- resource for small-molecule screening and the devel- otics. Although the development of class B β-lactamase opment of chemical probes. It is 1 of 9 members in inhibitors has been an active area of past research, an the Molecular Libraries Probe Production Centers Net- array of potent, class-specific small-molecule inhibitors work, a translational research initiative sponsored by has yet to be fully characterized in the clinically relevant National Institutes of Health (NIH) and part of the NIH VIM-2 metallo-β-lactamase system. Additionally, VIM- Roadmap. The mission of the Scripps center is to screen 2 inhibitors that are effective inhibitors of other class B the NIH library of more than 300,000 individual com- β-lactamases will be of great interest. Such compounds pounds against peer-reviewed targets; the goal is to dis- will be useful as tools for characterizing gram-negative cover proof-of-concept probes. The results are available pathogens or as adjuvant in antibiotic therapy. to the scientific community through the PubChem Web One of our goals is to develop HTS-ready assays suit- site of the National Center for Biotechnology Informa- able for rapid identification of compounds that modulate tion: http://pubchem.ncbi.nlm.nih.gov. Currently, the the activity of Ambler molecular class B (Bush-Jakoby- Lead Identification department serves as the HTS core Medeiros group 3) metallo-β-lactamases, specifically within the Scripps screening center; our responsibilities VIM-2 and IMP-1 enzymes. In preliminary research are to develop biological and biochemical assays, per- efforts, we have developed HTS-ready fluorescence- and form HTS campaigns, manage the resulting data, act absorbance-based VIM-2 and IMP-1 inhibition assays. In as steward of the NIH screening library, and provide collaboration with K.B. Sharpless, Department of Chem- assay support for the development of probes. istry, we have screened a diverse click-chemistry library OTHER SCREENING ACTIVITIES or compounds designed specifically to inhibit metallo-β- Since the inauguration of the ultra-HTS (uHTS) oper- lactamases. Currently we are developing several novel ation in November of 2005, we have also been actively scaffolds that appear to be specific inhibitors. screening the Scripps collection of compounds against drug discovery targets not only from the MLPCN but also PUBLICATIONS Chung, C.C., Ohwaki, K., Schneeweis, J.E., Stec, E., Varnerin, J.P., Goudreau, from scientists at Scripps Research and from outside P.N., Chang, A., Cassaday, J., Yang, L., Yamakawa, T., Kornienko, O., Hodder, P., partners. So far, members of the department have ini- 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 tiated and successfully completed more that 50 uHTS- inhibitors of coenzyme a production. Assay Drug Dev. Technol. 6:361, 2008. 374 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE

T able 1. Summary of collaborations in the development of HTS and HTS assays. Target class Target name (Abbreviation) Collaborator, affiliation Antibacterial Pseudomonas aeruginosa R. Miller, Pfizer, Groton, Connecticut ATPase p97 R. Deshaies, California Institute of Technology, Pasadena, California VIM-2 β-Lactamase P. Hodder, Scripps Research, Jupiter, Florida IMP-1

5HT1a M. Teitler, Albany Medical College, Albany, New York 5HT1e GalR2 S. Brown, Scripps Research, La Jolla, California GPR7 O. Civelli, University of California, Irvine, California

S1P1

S1P2 H. Rosen, Scripps Research, La Jolla, California S1P G protein–coupled receptor 3 S1P4 AGTRL-1 (APJ) L. Smith, Burnham Institute for Medical Research, Orlando, Florida GLP-1 P. LoGrasso, Scripps Research, Jupiter, Florida GPR119 P. McDonald. Scripps Research, Jupiter, Florida m Opioid heterodimers L. Devi, Mt. Sinai School of Medicine, New York, New York NPY- Y1 C. Wahlestedt, Scripps Research, Jupiter, Florida NPY- Y2 RBBP9 B. Cravatt, Scripps Research, La Jolla, California Hydrolase b-gluc J. Kelly, Scripps Research, La Jolla, California Aquaporins (AQP) M. Yeager, Scripps Research, La Jolla, California Ion channel TRPML3 S. Heller, Stanford University, Stanford, California TRPN1 JAK2 R. Levine, G. Gilliland, Sloan Kettering, New York, New York JNK3 P. LoGrasso, Scripps Research, Jupiter, Florida Kinase ROCK2 T. Schröter, Scripps Research, Jupiter, Florida PKA FAK P. Hodder, Scripps Research, Jupiter, Florida ADAMTS4 MMP13 G. Fields, Florida Atlantic University, Boca Raton, Florida Metalloproteinase MMP8 Falciparum M18 metalloprotease D. Gardiner, Queensland Institute of Medical Research, Queensland, Australia IDE M. Leissring, Mayo Clinic, Jacksonville, Florida NADPH oxidase Nox-1 G. Bokoch, Scripps Research, La Jolla, California SHP-1 P. Griffin, Scripps Research, Jupiter, Florida Estrogen receptor K. Nettles, Scripps Research, Jupiter, Florida Nuclear receptor RAR P. Griffin, Scripps Research, Jupiter, Florida SF1 (NR5A1) X. Li, Orphagen Pharmaceuticals, San Diego, California RORa (NR1F1) Phosphotransferase TPT1 H. Harding, New York University, New York, New York Proliferation/viability Jurkat E6.1 cells P. Hodder, Scripps Research, Jupiter, Florida EphB4-ephrinB2 P. Kuhn, Scripps Research, La Jolla, California Protein/protein HCV core homodimer D. Strosberg, Scripps Research, Jupiter, Florida NS5B/CYPB Hsp70 R. Morimoto, Northwestern University, Chicago, Illinois Protein misfolding AL-09 M. Ramirez-Alvarado, Mayo Clinic, Rochester, Minnesota PERK D. Ron, New York University, New York, New York Protein/RNA HIV Rev-RRE RNA J. Williamson, Scripps Research, La Jolla, California Reductase msrA H. Weissbach, Florida Atlantic University, Boca Raton, Florida Stem cell proliferation Notch H. Petrie, Scripps Research, Jupiter, Florida PPARg/Src1 PPARg/Src2 P. Griffin, Scripps Research, Jupiter, Florida PPARg/Src3 NF-kB J. Reed, Burnham Institute for Medical Research, La Jolla, California Transcription factor STAT1 D. Frank, Dana-Farber Cancer Institute, Boston, Massachusetts STAT3 KLF5 V. Yang, Emory University, Atlanta, Georgia AHR M. Denison, University of California, Davis, California Ubiquitin proteolysis WEE1 N. Ayad, Scripps Research, Jupiter, Florida TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 375

Lauer-Fields, J.L., Minond, D., Chase, P.S., Baillargeon, P.E., Saldanha, S.A., GPCR that has been implicated in type 2 diabetes and Stawikowska R., Hodder, P., Fields, G.B. High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate. Bioorg. obesity and that signals and functions in an analogous Med. Chem. Lett., in press. manner to GLP-1R. In collaboration with P. Kenny, Lauer-Fields, J.L., Spicer, T.P., Chase, P.S., Cudic, M., Burstein, G.D., Nagase, Molecular Therapeutics, we are also developing small- H., Hodder, P., Fields, G.B. Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluores- molecule inhibitors of a GPCR previously shown to be cence resonance energy transfer substrate. Anal. Biochem. 373:43, 2008. involved in drug dependence that may lead to a novel 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 therapy for substance abuse. by functional ultra-high-throughput screening. Mol. Pharmacol. 73:1776, 2008. As part of the collaboration between Scripps and Roth, J., Madoux, F., Hodder, P., Roush, W.R. Synthesis of small molecule inhibi- Pfizer, Inc., that was initiated in 2007, we are designing tors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on iso- quinolinone scaffolds. Bioorg. Med. Chem. Lett. 18:2628, 2008. and developing 3–4 assays per year for GPCR targets of therapeutic interest to Pfizer. 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. In Vivo Pharmacology Screen. 13:17, 2008.

Schürer, S.C., Brown, S.J., Gonzales-Cabrera P.J., Schaeffer, M.T., Chapman, J., A.S. Pachori, M. Ganno, S. Khan, S. Clapp, D. Hansen 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 he In Vivo Pharmacology group at Scripps Florida screening. ACS Chem. Biol. 3:486, 2008. is an integrated group of investigators involved T in preclinical studies in support of drug discovery efforts at Scripps Research in both Florida and Califor- Discovery Biology: nia. We develop appropriate animal models of diseases for ongoing projects such as studies in hypertension, G Protein–Coupled Receptors glaucoma, Parkinson’s disease, diabetes, and heart failure. These models are then used to test the efficacy of P. McDonald, D. Obradovich, A. Smith, E. Sturchler, new compounds for a particular therapeutic area. An S. Tabrizifard important aspect of establishing the efficacy of a com- protein–coupled receptors (GPCRs) are the largest pound is to determine if the compound is altering its and most versatile family of cell-surface recep- intended target. We initially evaluate the role of a par- G tors. The ubiquitous cell-surface distribution and ticular target in primary cell culture and then evaluate involvement of these proteins in almost all biological the target in vivo. The cell culture experiments are also processes explain why the largest percentage of cur- used to screen novel compounds for the effects of the rently marketed therapeutic drugs target these receptors. compounds on targets. We focus on developing biochemical and cell-based In addition, we evaluate the pharmacodynamic functional assays to monitor GPCR activity that involve properties of new chemical entities in vivo by doing high-throughput and high-content technologies. Using dose-response and time-course studies to determine a multidisciplinary approach that involves collabora- the effects of the compounds on the intended target. tions with disciplines such as lead identification, chem- For these evaluations, we use standard techniques such istry, drug metabolism and pharmacokinetics, and in as Western blotting, enzyme-linked immunosorbent vivo pharmacology, we aim to identify and develop assays and immunohistochemistry. We also collabo- small-molecule modulators of GPCRs for the treatment rate with the drug metabolism and pharmacokinetics of metabolic diseases such as type 2 diabetes mellitus laboratory to monitor plasma and tissue concentra- and obesity. tions of chemical entities; the results help us further We have developed a series of novel cell-based refine and develop the disease models. Finally, we eval- assays for the glucagon-like peptide 1 receptor (GLP-1R) uate the compounds for toxicity. to promote a drug discovery program for this clinically In the past year, we have successfully developed validated target of type 2 diabetes. In parallel with the animal models of hypertension to test the efficacy of GLP-1R assays, we have developed similar assays for Rho kinase inhibitors as novel antihypertensive agents. 2 other closely related receptors, GLP-2R and glucagon We have screened several novel Rho kinase inhibitors receptor, which serve as counterscreens for selectivity for their efficacy and toxicity and established the need against GLP-1R. We are also working on an orphan for isoform-selective inhibitors to avoid toxicity issues. 376 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE

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 Fig. 1. HD Desktop experiment view. will help us not only expand our portfolio but also discover new approaches for some of the biggest unmet need to measure and validate the calibration of the instru- needs of patients. ments. 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 Omics Informatics the standardization and validation of proteomics data. We have developed an automated method for deter- B.D. Pascal mining calibrated mass drift on high-resolution instru- ments. On a daily basis, the quality of the data collection he Omics Informatics group at Scripps Florida is monitored by routine analysis of a tryptic digest of a addresses support and software needs of various β-casein standard. The resulting binary files are collected, laboratories. Our goals are to identify and integrate T moved into the laboratory information management sys- existing solutions where possible and to build new solu- tem, and then processed through an automated work- tions only when necessary. A primary specialization is flow, which conducts file conversions and peak quality the analysis and management of mass spectrometric data assessments and sends the files to a compute cluster for and development of proteomics software research tools peptide identification search. The results are then parsed, that enable proteomics researchers to validate, visual- and the difference between the observed and calculated ize, and share their data. mass is stored in a database; the data are made avail- HD DESKTOP able through a Web-based interface (Fig. 2, page 377). Scientists at Scripps Florida are using hydrogen- deuterium exchange mass spectrometry to characterize PUBLICATIONS Chalmers, M.J., Busby, S.A., Pascal, B.D., Southern, M.R., Griffin, P.R. A two- protein dynamics and protein-protein or protein-ligand stage differential hydrogen deuterium exchange method for the rapid characteriza- interactions. The Deuterator software, released last year, tion of protein/ligand interactions. J. Biomol. Tech. 18:194, 2007. addresses some of the data analysis bottlenecks by 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 providing a platform to automate and visualize centroid deuterium levels from H/D exchange MS data. BMC Bioinformatics 8:156, 2007. calculations. Despite these efforts, the task of assembling and visualizing the resulting data is still a man- ual operation left to the end user. The current software, Drug Discovery Biology: HD Desktop, leverages the existing code base and stores all data in a relational database. Novel rendering and Cell Biology analysis tools have been presented in an integrated user interface (Fig. 1). T. Schröter, A.M.W. Handy, E. Griffin, J.R. Pocas, K. Clarke, C. Hahmann MASS SPECTROMETRY CALIBRATION SOFTWARE Although every mass spectrometry laboratory will ith more than 500 members, protein kinases vary in instrument manufacturer and experimental are important drug discovery targets for a designs, most proteomics laboratories have a common Wwide variety of therapeutic indications. These TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE 377

For the cancer program, in 2007, we successfully finished a collaboration with scientists at Poniard Phar- maceuticals, 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. Fig. 2. Data flow of mass spectrometry calibration software. 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 kinases control signal transduction pathways, and in fluorescence resonance energy transfer and enzyme-coupled luciferase high- humans, deregulation of their activity can lead to throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. Biomol. Screen. 13:17, 2008. 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 Chemical Informatics Program retinal ganglion cells and, ultimately, loss of vision. S.C. Schürer, D. Vidovic´, C. Chung Inhibition of ROCK activity increases outflow, lowers intraocular pressure, and preserves retinal neurons. We e collaborate with scientists in the Scripps concentrate on developing biochemical and cell-based Research Institute Molecular Screening Center, functional assays to monitor ROCK activity via both high- W and we also received a grant from the Colum- content and high-throughput screening technologies. bia University Molecular Library Screening Center. Both Working closely with researchers in high-through- of these centers are part of the national Molecular Librar- ies Screening Centers Network. We are also involved in put screening, medicinal chemistry, pharmacology, and drug discovery efforts within the Translational Research drug metabolism and pharmacokinetics, we identified Institute. We have developed a platform of industry-stan- small-molecule lead compounds from a high-through- dard software tools for analysis, visualization, hypothesis put screening of the Scripps collection of more than building, and modeling of large and focused experimental 700,000 compounds. During lead optimization, we screening data sets. Our platform enables us to generate screened thousands of new ROCK inhibitors for the and evaluate a large variety of structural, pharmacophore, biochemical activity against the enzyme and the close and physicochemical 2- and 3-dimensional descriptors. family members protein kinase A, Akt1, and MRCKα. The platform includes computational chemistry tools for Hundreds of these compounds were chosen, and their 3-dimensional pharmacophore-alignment ligand-based cell-based activity against ROCK was tested by using quantitative structure-activity relationships, ligand-protein target modulation and functional assays. Changes in docking (in a variety of approaches and scoring func- myosin light-chain phosphorylation were measured by tions), homology modeling, molecular modeling and using a 96-well immunocytochemical assay and infrared dynamics, and statistical tools. imaging, and changes in the formation of stress fibers We have also developed various interactive reporting and neurite protection were evaluated by using a high- and visualization protocols that are used in collaborative content imaging system. research. Our platform provides broad chemical informat- 378 TRANSLATIONAL RESEARCH INSTITUTE 2008 THE SCRIPPS RESEARCH INSTITUTE ics and computational chemistry capabilities. Examples Screening Informatics Program of research projects in which these technologies play a key role include analysis of data on toxic effects in cells M.R. Southern, K. Hoda, Y. Cruz and animals; development of small-molecule modulators of a broad variety of targets, including metalloproteases, cientists in the Screening Informatics Program phosphatases, kinases, nuclear receptors, and sphingosine at Scripps Florida collaborate broadly with lipid receptors (for which we also modeled the receptor S researchers in the Scripps Research Institute structures); and image-based high-content assays, includ- Molecular Screening Center, part of the national Molecu- ing probing the inflammatory pathway at the stage of lar Libraries Screening Centers Network, and in drug NF-κB translocation and expression of E-selectin or vas- discovery efforts within the Translational Research cular adhesion molecule 1 and targeting the aggregation Institute. Our responsibilities include high- and low- of the protein huntingtin. throughput screening assays and downstream drug In collaboration with the screening informatics team, metabolism and pharmacology, medicinal chemistry, we play a key role in implementing work flow, proce- and probe development. We have an operational envi- dures, and business rules and in integrating discovery ronment for data management and quality assurance informatics with the operational informatics infrastruc- and a knowledge environment that facilitates efficient ture to facilitate discovery processes. Examples include optimization of probes. These activities take place at the Scripps Research compound registration system; both the Florida and the California sites. integration of the Molecular Libraries Screening Centers The software systems have been built primarily Network chemoinformatics server, which is hosted at by using the MDL Discovery Experiment Management Scripps Research, with PubChem; integration of images Framework from Symyx Technologies, Inc., Santa Clara, from image-based screening with the operational infra- California, and support specific work flows involving structure; and publication of screening data to PubChem. tasks such as chemical compound registration, plate To date, more than 5.5 million data points and more and sample registration, assay development, and entire than 120 assays obtained by using these protocols have screening campaigns. On top of these systems, we have been published. developed in-house software that is tightly coupled to Other chemoinformatics research efforts are focused provide additional functionality and to improve our effi- on structure-based comprehensive analyses of target-sim- ciency. Examples include robotic automation, plate map- ilarity relationships in the phosphatase and kinase gene ping operations, and structure search. A key component families, ligand-based and target “fishing,” and the devel- is our Assay Exploration Data Warehouse, which along opment of integrative applied chemoinformatics methods. with its Web-based front end is known to end users as ChemInfo. PUBLICATIONS ChemInfo is assay metric and structure centric, Guha, R., Schürer, S.C. Utilizing high throughput screening data for predictive toxicology models: protocols and application to MLSCN assays. J. Comput. Aided Mol. enabling exploration of assay data by compound, target, Des. 22:367, 2008. or assays. It integrates chemical descriptors, physical

Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P., properties, and data on drug metabolism and pharmaco- Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M., kinetics to facilitate probe optimization. Complicated Tsinoremas, N., Lograsso, P., Hodder, P. Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high- Venn-like queries are possible. ChemInfo contains indi- throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. vidual and aggregated assay data from our internal Biomol. Screen. 13:17, 2008. assays as well as from PubChem. The database has Xie, Y., Deng, S., Thomas, C.J., Liu, Y., Zhang, Y.Q., Rinderspacher, A., Huang, up to 30 users within Scripps Research. W., Gong, G., Wyler, M., Cayanis, E., Aulner, N., Többen, U., Chung, C., Pam- pou, S., Southall, N., Vidovic,´ 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. Identifica- PUBLICATIONS tion of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating Schröter, T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P., NFκB activity within two separate high-throughput screens of NFκB activation. Baillargeon, P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M., Bioorg. Med. Chem. Lett. 18:329, 2008. Tsinoremas, N., Lograsso, P., Hodder, P. Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput Xie, Y., Liu, Y., Gong, G., Rinderspacher, A., Deng, S.X., Smith, D.H., Többen, U., screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J. Biomol. Tzilianos, E., Branden, L., Vidovic,´ D., Chung, C., Schürer, S., Tautz, L., Landry, Screen. 13:17, 2008. D.W. Discovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine phosphatase. Bioorg. Med. Chem. Lett. 18:2840, 2008.