2003 ORPHAN & NUCLEAR RECEPTORS - 3rd International Conference

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Overview

The application of drugs directed towards known nuclear receptor targets has produced some of the most commercially viable pharmaceuticals. Therefore, research and development has intensified toward identifying new ligands for orphan receptors as well as acquiring a greater understanding of known nuclear receptors and their interactions. As research progresses, additional areas are revealed in which nuclear receptors play an important role, such as metabolic and endocrine pathways, oncology or skin diseases.

This conference provides the latest information on the metabolism of orphan and nuclear receptors along with novel ligands, agonists and ligand screening technologies.

An international assembly of experts highlights the major achievements and challenges on topics such as:
• GPCR targeted libraries
• Biological membrane microarrays
• Chemokine receptors
• Crystal structures of ligand binding domains of PPAR
• PPARg modulators with novel biological activities
• PPAR and LXR in inflammation models
• Novel ligands for LXR and CAR
• Nuclear receptors as oncology targets
• The therapeutic potential of ERR
• Assay technologies for FXR antagonists

Discuss the newest developments in the research on nuclear hormone receptors with the experts. Register early - space is limited.

Pre-Conference Workshop

Monday, October 6, 2003

This workshop will address important key issues such as:
• Identifying Modulators • Structural Insights • A Bayesian Model of G -Protein Coupling • GPCR - Targeted Libraries • Fabrication of Membrane Microarrays

Agenda

Pre-Conference Workshop

G Protein Coupled Receptors - Opportunities in Drug Development
Monday, October 6, 2003

8:00 Registration, Poster / Exhibit Set-Up, Coffee and Pastries
8:25 Chairperson’s Opening Remarks
Joydeep Lahiri, Ph.D., Manager, Biochemical Sciences, Corning, Inc.

8:30 Chemokine Receptors in Drug Discovery and Development
Dr. James Onuffer, Department of Immunology, Berlex Biosciences

The “chemotactic cytokine” (chemokine) receptor family is the largest subfamily of peptide-binding GPCRs described thus far. Since their discovery in the late 1980’s, the physiological roles of chemokine receptors have expanded beyond host defense. Chemokine receptors are now considered
important targets for intervention is several disease indications. The opportunities and challenges facing drug development programs targeting chemokine receptors are being realized as antagonists move from discovery to the clinic.

9:10 Structural Insights into Adrenergic Receptor Signaling
Brian K. Kobilka, M.D., Professor of Medicine, and Molecular and Cellular Physiology, Stanford University Medical Center

Abstract not available at time of print.

9:50 A Naive Bayes Model of G-Protein Coupling
Jack Cao, Ph.D., Principal Scientist, Department of Molecular Sciences, Astrazeneca R&D Montreal

The problem of the G-protein coupling by GPCRs is important in understanding the signaling of the receptors and in the development of cell-based bioassays for drug screening. In this talk, I will present a Bayesian network approach to modeling the interaction between GPCRs and G-proteins. The G-protein coupling predicted by the model indicates it may serve as an alternative to the experimental undertaking.

10:30 Refreshment Break, Poster / Exhibit Viewing

11:00 Strengths and Weaknesses of Different Cell Based Assays in Identifying Modulators of G-Protein Coupled Receptors as Exemplified for GPR63
Evi Kostenis, Ph.D., Head of in vitro Pharmacology, 7TM Pharma and Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen

We have recently reported that sphingosine-1-phosphate (S1P), dihydro-S1P and dioleoylphosphatidic acid (DOPA) are agonists for the orphan receptor GPR63.
All three phospholipids mobilize intracellular Calcium in CHO cells transiently transfected with GPR63. Calcium signals required cotransfection of a chimeric Gaq/i protein in a Fluorometric Imaging Plate Reader TM assay but did not require overexpressed G proteins in an aequorin assay, using a green fluorescent protein-aequorin fusion protein as a bioluminescent Ca2+ reporter. GPR63 expression in CHO cells confers proliferative responses to S1P in a pertussis toxin-insensitive manner. GPR63-mediated responses have been tested in a variety of cellular assays (GTPgS binding assays, second messenger assays, MAP kinases, internalization, beta arrestin translocation, etc) and the suitability (advantages and drawbacks) of the various assay technologies for ligand identification of orphan receptors will be discussed here.

11:40 Novel G-Protein-Coupled Receptor-Targeted Libraries: Multi-Step Design and Synthesis Approach
Nikolay Savchuk, Ph.D., Chemical Diversity Labs, Inc.

We will describe a multidisciplinary approach applied at Chemical Diversity for the development of the universal GPCR-targeted library of about 45,000 compounds for 125 receptors. First, we apply systems biology methodology for selecting the receptors relevant to particular disease areas and disease pathways. We use the unique database of about 650 GPCRs with known ligands, with signaling cascades and tissue -specific effector networks. Second, we carry out proprietary morphing algorithms and target-specific privileged structures for synthesis of active compounds, many of novel chemotypes.
Third, we use Kohonen neural networks and other in silico funneling methods for evaluation of receptor-specific activity of potential ligands, eADMET and novelty scoring. The potential ligands are produced de novo in multi-parallel library validation and synthesis pipeline. We validate the library bioactivity via internal screening, academic and industrial collaboration.

12:20 Biological Membrane Microarrays
Joydeep Lahiri, Ph.D., Manager, Biochemical Sciences, Corning, Inc.

Membrane bound proteins constitute one of the most important families of drug targets. Despite their importance, the power and utility of microarray technology has not been extended to membrane proteins because of significant technical challenges associated with their fabrication and use. This talk will outline issues related to the fabrication of membrane microarrays, describe the fabrication of microarrays of G protein-coupled receptors, and demonstrate assays for screening of ligands on these arrays.

1:00 End of Workshop

Main Conference

Monday, October 6, 2003

1:45 Registration, Poster / Exhibit Viewing

2:10 Chairperson’s Opening Remarks
Thomas B. Burris, Group Leader, Gene Regulation Research, Lilly Research Laboratories

PPAR and LXR

Nuclear receptors are outstanding targets for drug discovery not only because of their profound roles in human physiology and diseases but also because of their inherited structure to interact with small chemical molecules that are selected through evolution. In this meeting I will present crystal structures of the ligand binding domains of the human glucocorticoid receptor and proxisome proliferator activated receptors, two receptors that are
critical in glucose regulation and inflammations, with focus on stereo insights of these structures into ligand recognition and drug discovery.

2:50 Identification and Characterization of Selective PPARg Modulators that Display Novel Biological Activities
Yang Li, Ph.D., Department of Biology, Tularik, Inc.

PPARg, a member of the nuclear receptor superfamily, is a ligand-activated transcription factor that has been shown to play a key role in the regulation of energy homeostasis, and adipocyte differentiation and function.
Furthermore, PPARg was identified as the molecular target for the thiazolidinedione class of anti-diabetic agents and has been implicated in the insulin sensitization of peripheral tissues by these drugs. In the course of our studies, we have identified and characterized multiple classes of novel PPARg ligands, and will discuss the distinctive pharmacological and biochemical properties of these compounds, as well as their potential utilities for treating metabolic diseases.

3:25 The Role of PPAR and LXR in the Regulation of Epidermal Differentiation and Inflammation
Kenneth Feingold M.D., Professor of Medicine, University of California at San Francisco

PPAR alpha, beta/delta, and gamma and LXR are present in
the epidermis. Studies have shown that activators of these receptors stimulates epidermal differentiation and development. This results in an improvement in permeability barrier function, a key function of the epidermis. In addition, PPAR and LXR activators inhibit inflammation in
models of irritant and allergic contact dermatitis. These results suggest that PPAR and LXR activators may be useful in the treatment of a variety of cutaneous disorders.

4:00 Refreshment Break, Poster / Exhibit Viewing

4:30 Tissue-Selective Liver X Receptor Agonists: Local Manager of Cholesterol Homeostasis
Ching Song, Ph.D., Nuclear Receptor Research, Anagen Therapeutics, Inc.

LXRs are intracellular cholesterol-sensors and play a major role in the cholesterol mobilization. LXR ligands regulate cholesterol efflux pathway in a variety of cell types. To avoid hypertriglyceridemia, we developed LXR agonists that are active in brain and artery wall yet show little activity in liver. We will report here that one leading compound, when administered orally to corresponding transgenic mouse models, decelerated arterial atherosclerosis progression, accelerated regression and reduced interstitial b-amyloid plaque formation in brain, possibly by mobilizing local cholesterol in the microenvironment.

Coup-TF and ERR

COUP-TFII gene is an orphan nuclear receptor that belongs to the steroid receptor superfamily. Its developmental expression patterns suggest that it participate in mesenchymal-epithelial interactions during organogenesis.
Inactivation of COUP-TFII gene in mice results in early embryonic lethality. To assess COUP-TFII function during later development, we used chimera analysis and showed that COUP-TFII plays cell autonomous function in formation of the endothelium of the vein and smooth muscle cells of the
artery. To further assess the role of COUP-TFII during vasculature development, conditional COUP-TFII null mutants are being generated and characterized.

5:40 Estrogen-Related Receptors: Novel Ligands, Disease Linkage, and Drug Discovery
Trish Willy, Ph.D., Research Scientist II, X-Ceptor Therapeutics, Inc.

Current data suggest that ERRs may be potential therapeutic targets for the treatment of breast cancer, osteoporosis, and metabolic disorders. We are working to understand the biological roles of ERRs and their influence on estrogen signaling pathways in normal and disease states. Utilizing
high-throughput biochemical and cell-based screens, we have identified synthetic ERR-selective compounds that efficiently antagonize the constitutive activity of ERRalpha. Additionally, we have identified several novel ERR target genes that are involved in multiple and distinct signaling pathways. We are currently using our ERR-selective compounds and ERR target genes to elucidate the biological roles and therapeutic potential of these orphan receptors.

6:15 End of Day One

Tuesday, October 7, 2003

8:55 Chairperson’s Opening Remarks
Ching Song, Ph.D., Nuclear Receptor Research, Anagen Therapeutics, Inc.

Orphan Receptors and Xenobiotics

The nuclear receptors, LXRa and LXRb, function as physiological “cholesterol sensors.” Their role in regulation of lipid metabolism and transport indicate that these receptors may be an important node for modulation by pharmaceutical agents. CAR is another member of the nuclear receptor superfamily, which appears to function as a receptor for xenobiotics and mediate some forms of drug-drug interactions. In this presentation, I will discuss both of these receptors in terms of recent advances we have made in identification of novel ligands, receptor variants, and new physiological roles. Natural product ligands as well as a role for LXR in glucose metabolism will be presented.

9:35 Insights From a Three-Dimensional Model Into Ligand Binding to Nuclear Orphan Receptor NRI Subfamily
K.C. Cheng, Ph.D., Senior Principal Scientist, Schering-Plough Research Institute

Two orphan nuclear receptors, constitutive active (or androstane) receptor (CAR) and pregnane X receptor (PXR), are among the most important mediators of ligand-activated transcriptional induction of liver microsomal cytochrome P-450 drug metabolizing enzymes. This talk will present our recent work on modeling of the ligand binding domain of human CAR (hCAR). The results from this study indicate that structural modeling will be a useful tool for understanding ligand binding to hCAR and for design of drugs free of hCAR
mediated enzyme induction.
In collaboration with: Xiao, Li; Madison, Vincent; Cui,
Xiaoming; Morrison, Richard A.; White, Ronald E.

10:10 Nuclear Receptor Humanized Mice in Safer Drug Development
Wen Xie, M.D., Ph.D., Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh

The drug metabolizing enzymes and transporters are induced by numerous drugs, yet the inducibility shows clear species specificity. The orphan nuclear receptor PXR has been established as a species-specific xeno-sensor that regulates the expression of these enzymes and transporters. We have created “humanized” mice in which the mouse PXR was genetically replaced with its human counterpart. Displaying a human drug response profile, these mice represent a unique tool to dissect the drug-induced xenobiotic response, and should aid the development of safer drugs.

10: 45 Refreshment Break and Poster / Exhibit Viewing

11:15 Characterization of the Sterol 27-hydroxylase null Mouse Reveals Novel Ligands for the Xenobiotic Receptor PXR
Joyce J. Repa, Ph.D., Assistant Professor, Departments of Physiology and Internal Medicine, Touchstone Center for Diabetes Research, UT Southwestern Medical Center

The sterol 27-hydroxylase (cyp27a1) knockout mouse lacks a critical enzyme of bile acid biosynthesis. As a result this mouse harbors very low levels of bile acids, the ligands of FXR, and has proven to be a valuable animal model to study FXR signaling and gene regulation. During the characterization of this mouse strain, it was noted that hepatic expression of target genes for the xenobiotic receptor PXR were dramatically increased. Several bile acid
intermediates accumulate in the livers of these mice, and they were tested as potential ligands of PXR. Three of these potentially toxic sterols are potent activators of the mouse PXR. Interestingly, the human PXR is far less responsive to these bile acid intermediates than rodent PXRs, which may explain to some extent, why the phenotype of the cyp27a1 null mouse differs from that seen in humans harboring mutations in the CYP27A1 gene and exhibiting the genetic disorder, Cerebrotendinous Xanthomatosis (CTX). These findings may lead to novel therapeutic approaches to the treatment of CTX.

Nuclear Receptors and Cancer

Several members of the nuclear hormone receptor superfamily have been directly involved in the tumoral progression or inversely have shown tumor-suppressive potential through their activities at modulating cell proliferation, differentiation and apoptosis. We will discuss recent works focusing on the identification of ligands of promising chemo -preventive or -therapeutic use and their mechanisms of action.

12:25 A Subclass of RXR Ligands Induce Differentiation and Involution in Breast Cancer
Magnus Pfahl, Ph.D., CEO and CSO, MaxoCore Pharmaceuticals.

Many features of breast cancer are also displayed during the developmental cycle of the mammary gland. An essential feature of this developmental cycle is a process called “involution”, in which upon cessation of lactation stromal preadipocytes differentiate into adipocytes and proliferating epithelial cells undergo apoptosis. We have discovered a subclass of RXR ligands that induce critical features of involution. These compounds are highly effective against breast cancer in vivo. Their in vitro and in vivo activities will be discussed.

1:00 Luncheon, Sponsored by The Knowledge Foundation, Inc.

2:15 Chairperson’s Remarks
Steven Hayes, Senior Scientist, Department of Cell Biology, PanVera LLC

Screening Technologies

We present here a cell-based screening assay for the androgen receptor. Using a beta-lactamase reporter system and the cell-permeable, fluorescent, ratiometric substrate CCF4/AM, AR activity can be read out in live cells.
Complementary glucocorticoid and mineralocorticoid receptor assays employed as secondary screens allow compounds to be identified which specifically inhibit the mutated AR, and not other related steroid hormone receptors.

2:55 A Comparison of Assay Technologies for FXR Antagonists: Implications in Lead-Finding
J. Fraser Glickman, MPH, Ph.D., Laboratory Head, Basel Screening Operations, Novartis Institute for BioMedical Research

The technologies of AlphaScreen, Time -resolved fluorescence resonance energy transfer, and Time-resolved Fluorescence were used to develop assays for the farnesoid nuclear receptor, FXR. These assay technologies were compared in a high throughput screening campaign. The results suggest that different technologies yield different validated hits-as defined by subsequent cellular assays. The implications of assay design on lead finding will be
discussed.

3:30 Refreshment Break, Poster / Exhibit Viewing

4:00 Beyond the 48: Deep Mining of the Human Genome for Novel NR Proteins
Dr. Richard J. Fagan, Associate Director, Head of Target Discovery and Dr. Janet Allen, Director Discovery Biology, Inpharmatica, London

Inpharmatica has developed a drug discovery platform, PharmaCarta which links the worlds of genomics, protein annotation and structure prediction, cheminformatics analysis and hits and leads. Using advanced structure-based bioinformatics tools, we have been able to identify 16 proteins with previously unrecognised structural similarity to the ligand binding domain of the Nuclear Receptors, all clearly outside of the known 48 family members. A small focussed screening library was designed using the platform and hits which invoke ligand activated transcription have been identified for all screened to date. My talk will focus on the use of the platform to discover the 16 novels, the characteristics of the novels and some of the biology around a selected novel NR, and finally discussion of the NR family in simple eukaryotes.

4:35 ThermoFluor®, a Miniaturized HTS Thermodynamic Assay, and its Applications in Nuclear Receptors
Dionisios Rentzeperis, Ph.D., Research Fellow, 3-Dimensional Pharmaceuticals

Nuclear receptors are an attractive target class for drug discovery. ThermoFluor® is a HTS and hit profiling direct binding thermodynamic assay that provides several advantages over-existing technologies in discovering ligands for orphan nuclear receptors and measuring multi-ligand binding phenomena, a hallmark of nuclear receptor biology. We will present examples how we apply this technology in addressing key issues in the drug discovery effort for nuclear receptors.

5:15 End of Conference

Call for Posters

Call for Posters

Industry and academic scientists are encouraged to submit poster titles for this event. One-page abstracts (8 1/2” x 11” with 1-inch margins) must be submitted no later than September 15, 2003 for inclusion in conference documentation. Additional poster submissions will be accepted until October 1st, 2003 but may not be included in conference documentation.

Size of Posterboard: 3x4 feet / 90 (h)x 120 (w) cm

Note: If you are submitting a poster, you MUST be registered and paid in advance to ensure that a posterboard is reserved for you.

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