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TUESDAY, APRIL 25
12:00pm Conference Registration
1:30 KEYNOTE PRESENTATION:
Structural Organization of G-Protein Coupled Receptors; Implications for Drug Design and Function
Graeme Milligan, Ph.D., Professor of Molecular Pharmacology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland
It has rapidly become accepted that G-protein coupled receptors possess quaternary structure. I will present evidence that the alpha1b-adrenoceptor is present in single cells as a higher-order oligomer rather than as a dimer and that this has implications for its pharmacology, function and membrane delivery. A number of G-protein coupled receptors can exist as
hetero-dimers/oligomers. I will discuss how this can alter the cellular distribution and function of these G-protein coupled receptors and will describe implications of this for both current medicines and the drug discovery process.
ALLOSTERIC MODULATION: THE WAVE OF THE FUTURE?
Understanding GPCR Drug Allosterism
The Allosteric Agonist Site of the M1 Muscarinic
Receptor:A New Landscape for Drug Discovery
Chairperson: Patrick M. Sexton, Ph.D., Professor of Pharmacology, NHMRC Principal Research Fellow, Monash Centre for Drug Discovey, Department of Pharmacology, Monash University
Marlene A. Jacobson, PhD., Senior Research Fellow, Neuroscience West Point, Merck
The atypical antipsychotic, clozapine has a unique clinical profile in efficacy against positive
symptoms in treatment-refractory schizophrenic patients. The mechanism of clozapine's action is unclear as it exhibits promiscuous pharmacology on multiple GPCRs, including all five cloned muscarinic receptors. Recently, we showed that the major metabolite of clozapine, N-desmethylclozapine is a potent M1 muscarinic receptor allosteric agonist and is able to potentiate hippocampal NMDA currents via M1 receptor activation.Activation of the M1 muscarinic receptor can occur through orthosteric agonist, allosteric agonist, and allosteric potentiator sites.To probe and identify critical residues involved in these binding sites, site directed mutagenesis and chimeric receptor strategies were employed to map the interaction sites of N-desmethylclozapine and the M1 allosteric agonist AC42. Identification of amino acid
residues within the allosteric agonist binding site provides new information for modeling this
receptor domain and could facilitate the optimization and development of novel M1 receptor
allosteric agonists for treatment of psychiatric disorders.
3:00 Technology Watch (Sponsorship Available)
3:15 Networking Refreshment Break, Poster and Exhibit Viewing
4:00 Discovery of Allosteric Modulators to Peptide Receptors
Teresa Bennett, Ph.D., Associate Director, Drug Discovery, Novasite Pharmaceuticals
Allosteric modulators of GPCR function have received increasing credibility and attention as candidates for drug therapy due to their unique mechanisms of action that may offer advantages at both “undrugable” and well established targets. Novasite has successfully applied their automated flow
cytometry-based screening technology towards the discovery of allosteric modulators of peptide
GPCRs. Results will be presented which illustrate the screening technology and the successful characterization of initial hits as allosteric modulators.
4:30 Design and Synthesis of Orally Efficacious Melanin Concentrating Hormone Receptor Antagonists as Antiobesity Therapeutics
Anandan Palani, Ph.D., Associate Director, Chemical Research, Schering-Plough Research Institute
Obesity is currently recognized as one of the top 10 global health problems by the World Health Organization, affecting over one third of adults in the U.S. Morbidities related to obesity include hyperlibidaemia, type 2 diabetes, stroke, osteoarthritis and cancer, underscoring the fact that obesity is not merely an aesthetic issue. Melanin Concentrating Hormone (MCH) is a cyclic, nonadecapeptide found in the CNS of all vertebrates which regulates feeding behavior and energy homeostasis via interaction with the central melanocortin system. Recent studies have demonstrated that elevated levels of MCH in mice have been found to stimulate food intake and promote fat storage, while mice null for MCH or MCH-R1 exhibit a lean phenotype and are resistant to diet induced obesity. The receptor for MCH belongs to the GPCR superfamily, identified by 7-transmembrane regions embedded in the cellular wall. Antagonists of the MCH-1 receptor are expected to decrease food intake and weight gain in rodents, making MCH-R1an attractive target for obesity therapeutics. The discovery challenges and SAR development of a novel, orally active series of potent MCHR-1 antagonists will be discussed.
|5:00 A LMW CCR5 Antagonist in Combination with CsA Prolongs Graft Survival in a Life
Supporting Kidney TX Model in Cynomolgus Monkeys
||Gebhard Thoma, Ph.D., Novartis Institutes for BioMedical Research
The chemokine receptor CCR5 plays an important role in inflammatory and autoimmune disorders as well as in transplant rejection by affecting the trafficking of effector T cells and monocytes to diseased tissues. Antagonists of CCR5 are believed to be of potential therapeutic value for these disorders and HIV infection. Here we report on the discovery and the SAR of a new series of highly potent and selective competitive CCR5 antagonists. One of them was tested in a model of life-supporting renal allograft rejection in cynomolgus monkeys as monotherapy or in combination with a non-effective dose of cyclosporine (CsA). It was not efficacious as monotherapy but when administered in combination with the non-effective dose of CsA graft survival was statistically significantly prolonged. No PK interaction with CsA was observed demonstrating that the graft prolongation was compound related.
Walking Tour of the Scripps Institution of Oceanography and Reception at the
Birch Aquarium at Scripps
5:30 - 9:30
Refer to details below
WEDNESDAY, APRIL 26
CRITICAL TOOLS: NOVEL APPROACHES
8:00am GPCR Screening Methodologies for Drug Discovery
Chairperson: Marlene A. Jacobson, PhD., Senior Research Fellow, Neuroscience West
Point, Merck Research Labs
8:10 The Inside Out Approach to Modulating GPCR Actions
Annette Gilchrist, Ph.D., President, Cue BIOtech
Many biologically active molecules convey their signals via heterotrimeric G-proteins. At Cue BIOtech, we focus on identifying allosteric modulators of receptor action rather than receptor activation. Using a proprietary approach we identify small molecules
that modulate not only a given GPCR, but also the downstream G-protein. This allows for more selective inhibition of the critical pathways and physiological responses associated with disease. We will discuss the rationale of our approach, and provide data for
successful screening of the thrombin receptor, PAR-1.
8:40 System to Produce Membrane Proteins for Structural and Functional Applications
Julia Fletcher, M.S., Scientist, Transcription and Translation, Invitrogen Corporation
9:10 Emerging Concepts for GPCR Screening
Sofia Ribeiro, Ph.D., Scientist III, Department of Molecular Pharmacology, Berlex Biosciences Pharmaceuticals
G-protein coupled receptors constitute a large family of receptors with nearly 30% of marketed drugs designed to modulate GPCR activity. Screening of large compound libraries against GPCRs is achieved by several methods: competition binding with a known radioligand and/or functional activation or inhibition of cAMP, calcium mobilization and
GTPS signals. The increase demands for faster and cheaper screens has lead to several new methodologies and strategies for GPCR screening including: transients versus stable cells, in silico selection of sub-libraries of the libraries, GPCR targeted libraries, new homogeneous assays (IP one, CellKey systems) to mention a few. An overview of these strategies and selected results will be discussed.
9:40 Networking Coffee Break, Poster and Exhibit Viewing
10:10 A Primary Screening Technology for Antagonists and Agonists Targeting G-Protein Coupled Receptors
Brian F. O’Dowd, Ph.D., Professor, Departments of Pharmacology, University of Toronto
We describe a novel and universal drug strategy designed as a primary or secondary screen to uniquely discover both antagonist and agonist compounds targeting G-protein coupled receptors
(GPCRs). The incorporation of a small nuclear localization sequence (NLS, a 5 amino acid substitution) resulted in the dramatic receptor translocation from the cell surface to the nucleus in a
ligand-independent manner. Placement of the NLS in a critical location enabled blockade of this GPCR-NLS translocation from the cell surface by either antagonist or agonist treatments, both compounds achieving this result in a sensitive dose-dependent manner. GPCR-NLS translocation occurred regardless of the identity of the G-protein coupling and thus the strategy is ideally suited for identification of compounds targeting
GPCRs, including orphan GPCRs. The GPCR-NLS trafficking was directly visualized by fusion to optically detectable molecules. Quantification of this effect was most effectively evaluated by determining the density of cell surface receptors, by enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, we introduce a cellular assay for drugs targeting GPCRs suitable for screening without requiring prior identification of an agonist or knowledge of G-protein coupling.
10:40 Focused Library and Lead Optimization Strategies for the Development of CRTH2 Antagonists for Anti-Inflammatory Therapy
Thomas Högberg, Ph.D., Director, Medicinal Chemistry, 7TM Pharma
The identification of small focused libraries for a given target facilitates the screening and hit validation required. A knowledge-based process (Site-Directed Drug Discovery®) for structure-guided design of small molecule ligands for 7TM receptors has been devised to produce small diverse libraries. The process involves analysis of physicogenetic relationships of binding sites of 7TM receptors and design of pharmacophores by incorporation of target and ligand information. The pharmacophore queries are used for in silico screening and construction of customized small libraries that will be illustrated for CRTH2. Initial hits obtained have been optimized into selective orally active functional antagonists.
11:10 Constitutively Active GPCRs in Drug Discovery:A Look Forward
Alan Kopin, M.D., Professor of Medicine, Director, Molecular Pharmacology Research Center, Tufts-New England Medical Center, Tufts University School of Medicine; President, Katama Bay Biosciences, Inc.
Constitutively active GPCRs can be engineered through introduction of single point mutations. These receptors can be utilized as hypersensitive screens to detect intrinsic activity (agonist or inverse agonist) of candidate compounds. We illustrate that our approach, initially applied to Class A receptors, is relevant for Class B GPCR as well as Class C heterodimer drug discovery.
11:40 G-Protein Coupled Receptor Assay Using Dual Luciferase Stable Cell Lines
Brad Swanson, Ph.D., Senior Research Scientist, Promega Corporation
Bioluminescent reporter technologies are uniquely suited for high-throughput screening due to their inherent high sensitivity, wide dynamic range, and low susceptibility to compound interference. Improvement of data quality and reduction of false positives caused by cytotoxic compounds can be achieved by incorporating a control reporter (e.g. a second luciferase) and ratiometric measurement. Introducing both reporters into the cell on the same plasmid backbone can result in aberrant expression from cross interference between promoters and response elements. Therefore we have developed a strategy of generating dual luciferase stable cell lines for GPCR assays using a two plasmid system. Plasmid one features both a firefly luciferase gene regulated by the response element of interest (e.g. CRE or NFAT-RE) and a hygromycin selectable marker. The second plasmid expresses the target GPCR (e.g. muscarinic receptor 3) and a Renilla luciferase-neomycin selectable marker fusion. Destabilized
luciferase reporters provide significant benefit by reducing assay time, which limits the duration of cytotoxic compound interactions. This dual luciferase assay system enables rapid screening and improves data quality.
12:00pm Lunch on Your Own (Sponsorship Available)
OLIGOMERS: FUNCTIONAL CROSSTALK
1:45 Crosstalk Between GPCRs; Functional Consequences of Dimerization
Chairperson: Annette Gilchrist, Ph.D., President, Cue BIOtech
|2:00 FEATURED PRESENTATION:
Galanin Receptor Subtypes as Antidepressant Drug Targets
Tamas Bartfai, Ph.D., Chair and Professor, Molecular & Integrative Neurosciences Department; Director, The Harold L. Dorris Neurological Research Institute; The Scripps Research Institute
We have tagged GALR1 at its C-terminus with CFP and YFP respectively for the FRET studies in transiently transfected CHO cells. Ratio imaging analysis and emission spectral analysis revealed substantial homodimerization of GalR1. In addition, internalization of GalR1 after 1h of agonist stimulation with the GalR1 agonist galanin (1-29) was observed with time lapse fluorescence imaging, whereas stimulation with the GalR2 specific agonist galanin (2-11) did not lead to internalization. Treatment of GalR1 transfected cells with the non-selective adenylyl cyclase activator forskolin influenced the rate of internalization when administered together with
galanin. These results indicate that GalR1 can act as a dimer on the cell surface and that receptor desensitization and internalization was observed after stimulation with the agonist
galanin. Internalization and dimerization of GalR1 is shown, contributing to the regulation of galanergic signaling.
|2:30 Umami and Other Taste Modalities Mediated by GPCRs
Nigel R.A. Beeley, Ph.D., Vice President, Discovery,
Several GPCRs have been identified as mediators of human taste including the heteromeric T1R1/T1R3 umami receptor, the heteromeric
T1R2/T1R3 sweet receptor and a family of 25 T2R bitter receptors. Appropriate expression of these in HEK293 cells results in viable assay systems that can be used to identify interesting ligands as well as, in the case of the T2Rs, identifying receptor/bitter tastant pairs. We have also been able to map the interactions of selected ligands with subunits of the T1R1/T1R3 and the T1R2/T1R3 complexes by using human/rodent and other chimeras.
3:00 Family B Peptide Receptors; A Complex Road to Drug Discovery
Patrick M. Sexton, Ph.D., Professor of Pharmacology, NHMRC Principal Research Fellow, Monash Centre for Drug Discovery, Department of Pharmacology, Monash University
G-protein coupled receptors (GPCRs) are a major target class for the treatment of most clinically important diseases. Although much attention has been focused on the large Family A subclass, there is increasing interest in a subset of Family B GPCRs that are the receptors for peptide hormones such as calcitonin, CGRP, amylin, adrenomedullin, PTH, glucagon, GLP1, GLP2, and CRF. These receptors are attractive targets for treatment of many major diseases including diabetes, obesity, osteoporosis, migraine and hypertension and indeed have been validated in many cases using peptide based therapy (eg. calcitonin, amylin, PTH and exendin, the latter a GLP1 receptor agonist). However, the development of small molecule drugs for these receptors has been relatively slow. Most, if not all these receptors, exist as oligomers with good evidence for homo- and hetero- oligomerization of receptors within this class. In addition, a significant proportion of these receptors can also heterodimerize with receptor activity modifying proteins to form novel functional units. The complex nature of Family B receptors will be discussed in context of how this may impact on receptor function and development of drugs.
3:30 Networking Refreshment Break, Poster and Exhibit Viewing
4:00 Reciprocal Interactions in Endogenous GPCR+RTK Signaling Complexes
Kevin J. Catt, M.D., Ph.D., Chief, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health
The capacity of certain agonist-activated GPCRs to utilize the growthpromoting actions of receptor tyrosine kinases (RTKs) is exemplified by the manner in which the agonist-activated angiotensin AT1 receptor utilizes HB-EGF formation and other pathways to transactivate the EGFR and stimulate ERK phosphorylation. In addition to its regulation of cell growth, survival, and proliferation, such AT1R-mediated activation of the EGFR is relevant to its well-defined pathogenic actions in the cardiovascular system, and in renal disease, diabetes and cancer. Recent studies have revealed reciprocal signaling from RTKs to GPCRs, in which agonist activation of either species leads to complex formation and transphosphorylation between the two types of receptors. In hepatic C9 cells, agonist activation of the endogenous EGFR causes phosphorylation of the AT1R that is dependent on colocalization of the receptors in caveolin-rich domains of the plasma membrane. These observations demonstrate an essential scaffolding role of caveolin during agonist-induced transactivation and reciprocal signaling between the two major receptor families.
4:45 Can Chronic Inverse Agonist Treatment Enhance GPCR Signaling? Examples Using Heart Failure and Asthma
Richard A. Bond, Ph.D., Associate Professor, College of Pharmacy, University of Houston
A paradigm shift that has occurred with regard to the use of beta-adrenoceptor agonists and inverse agonists in the treatment of congestive heart failure. Chronic treatment with certain drugs from the previously contraindicated class known as 'beta-blockers' have now been shown to decrease mortality and increase cardiac contractility in heart failure patients. We are attempting to determine whether the unexpected reversal in heart failure upon chronic administration of beta blockers is a one-off event, or indicative of a more general pattern of GPCR regulation by inverse agonists that is applicable to other diseases such as asthma.
5:30 Close of Conference
Scientific Advisory Board
• Annette Gilchrist, Ph.D., President, Cue BIOtech
• Terry P.Kenakin Ph.D., Principal Research Investigator Molecular Discovery,
Assay Development, GlaxoSmithKline Research and Development
• Graeme Milligan, Ph.D., Professor of Molecular Pharmacology, Division of
Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Scotland
Conference Image adapted
from CSIRO Australia: Leifert et al., J
Biomol Screen in press
and CARA Therapeutics website: www.caratherapeutics.com/dimerScreen-technology.php
Tuesday, April 25
Walking Tour of the Scripps Institution of Oceanography and Reception at the
Birch Aquarium at Scripps
5:30 - 9:30
All Drug Discovery Chemistry delegates are cordially invited to join William Fenical, Ph.D., Professor of Oceanography, Director of the Center for Marine Biotechnology and Biomedicine and colleagues to tour their research laboratories at the Scripps Institution of Oceanography. A reception will follow at the Birch Aquarium, a public exploration center at the University of California. Buses leave promptly at
For more information regarding the agenda please contact:
Holly Groelle, Ph.D., Conference Director
Phone: 781-972-5455 Email: firstname.lastname@example.org
For sponsorship or exhibiting information, please contact:
Suzanne Carroll, Manager, Business Development