Cambridge Healthtech Institute’s 2^nd Annual

GPCR-Targeted Drug Design

New Structural, Pharmacological and Biophysical Insights and Tools

April 3-4, 2018 | Hilton Bayfront | San Diego, California

G protein-coupled receptors (GPCRs) represent a highly medically relevant class of proteins because of the central role they play in a range of physiologically critical processes from vision to heart and breathing functions to CNS signaling. This meeting will focus on discovery and development of small molecules with drug potential that target GPCRs. Many of the medicines on the market today act on only a small percentage of the GPCRs in our body and were discovered over a decade ago without knowledge of the specific receptor(s) they target. The genomic knowledge of more GPCRs coupled with the explosion of X-ray crystal structures now available for all classes of GPCRs as well as new biophysical techniques to study the more intractable ones, has renewed interest within the pharmaceutical industry in discovering new medicines that target GPCRs. This conference convenes leading researchers from industry as well as academics to share insights on GPCR structure and function. Discuss with fellow medicinal chemists, structural biologists, pharmacologists and translational researchers, the impact of new GPCR knowledge and tools in today’s target-focused era of drug discovery.

Final Agenda

Tuesday, April 3

7:00 am Registration and Morning Coffee

RECEPTOR CONFORMATIONAL STATES AND BIASED LIGANDS

8:00 Welcome Remarks

Anjani Shah, PhD, Conference Director,Cambridge Healthtech Institute

8:05 Chairperson’s Opening Remarks

Andrew Alt, PhD, Director, Center for Chemical Genomics, University of Michigan

8:10 FEATURED PRESENTATION: β-arrestin Desensitization Cycles and Biosensor Assays

Martin Lohse, PhD, Chairman, Max Delbrück Center, Berlin, Germany
Optical analyses are providing new aspects in the analysis of GPCRs. Most notably, their activation and signaling can be monitored in real time and in intact cells by FRET microscopy. Motility and interactions can be studied by single molecule microscopy. These methods can also be used to investigate downstream signaling and to discover bias in G-protein- vs. beta-arrestin-mediated signals. They have allowed the discovery of multiple active states of beta-arrestins, suggesting that beta-arrestins undergo an activation/deactivation cycle. Differential effects of various ligands indicate that they may differ in their physiological effects by selectively stimulating distinct kinase pathways.

9:10 Fighting the Opioid Epidemic: Allosteric Modulators of Opioid Receptors and the Utility of Adding NAMs to the Therapeutic Arsenal

Andrew Alt, PhD, Director, Center for Chemical Genomics, University of Michigan

It is now emerging that determining drug target binding kinetics, next to traditional potency measures, may improve the success rate of a candidate drug moving through the clinical development. Our work provides new insights in ligand-GPCR interactions and underlines the importance of measuring binding kinetics of both drug candidates and competing endogenous ligands.Positive allosteric modulators of opioid receptors (opioid PAMs) have been proposed as a novel therapeutic approach for achieving analgesia with improved side-effect and addiction liability profile compared to traditional orthosteric opioid receptor agonists such as morphine or oxycodone. Newly discovered negative allosteric modulators (NAMs) of opioid receptors will be introduced, which may offer advantages over competitive antagonists for the acute treatment of opioid overdose.

9:40 Coffee Break

10:05 GPCR-Targeted Lead Optimization: The Importance of the Assay in Fitting Data to Models

Terry Kenakin, PhD, Professor, Department of Pharmacology, University of North Carolina School of Medicine

I will compare the muscarinic receptor Gq protein activation profiles of five exemplar molecules (slow binding agonists, partial agonists, inverse agonists, PAM-Agonists and Beta-PAMs) in calcium and IP1 assays to illustrate how quantitative comparisons to pharmacological models can both identify mechanisms of action and also convert descriptive findings to predict data for therapeutic systems. Using these models optimally allows the identification of consistent and simple scales of activity that can guide medicinal chemistry.

10:35 New Tools for GPCR Thermo-Stabilization

Vsevolod 'Seva' Katritch, PhD, Assistant Professor, The Bridge Institute, University of Southern California

Engineering of GPCR constructs with improved thermostability is a key for successful structural and biochemical studies of this transmembrane protein family. Here we introduce a computational approach to effective prediction of stabilizing mutations in GPCRs, named CompoMug, which employs sequence-based analysis, structural information, and a derived machine learning predictor. Tested experimentally on the serotonin 5-HT2C receptor target, CompoMug resulted in 10 new stabilizing mutations and enabled structure determination for the 5-HT2C receptor complexes in inactive and active-like states.

11:05 GPCR Allosteric Coupling Investigated by NMR and X-Ray Diffraction

Matthew Eddy, PhD, Postdoctoral Fellow, Laboratory of Raymond Stevens, University of Southern California and The Scripps Research Institute

Drug binding in human GPCRs is allosterically connected over 30 Å to the intracellular signaling surface. Using advanced techniques for stable isotope labeling, we probe this allosteric network with NMR spectroscopy in solution for a native GPCR and variant with strikingly different signaling properties. X-ray crystal structures of the same variant reveal local conformational rearrangements in a known signaling-related structural motif. In parallel, NMR data uncover large signaling-related changes in conformational dynamics. Information from both techniques paired together provides a comprehensive picture of changes in structure and dynamics underpinning GPCR allosteric coupling.

11:35 Luncheon Presentation: Reaching beyond Developing Stable GPCR Cell Lines

Lisa Minor, Scientific Consultant, Multispan, Inc.

Developing high quality assays is paramount for drug discovery screening. Multispan devoted significant effort in developing signaling and phenotypic assays using endogenous targets such as RXFP1 in THP-1, CGRP in SK-N-MC, AMPK in C2C12, and DNA-PK in HELA cells. We also developed stable cell line assays for CGRP, AM, and Amylin by studying and overcoming endogenous RAMP expression and designed a 32-GPCR panel comprising CNS and cardiovascular liability targets. In addition to radioligand binding, we established a FACS-based quantification of GPCR expression to benchmark target expression against physiological level in native cells.

12:20 pm Session Break

GPCRs IN CANCER AND OTHER DISEASES

1:15 Chairperson’s Remarks

JoAnn Trejo, PhD, MBA, Professor and Vice Chair, Department of Pharmacology, Associate Dean for Health Sciences Faculty Affairs, University of California, San Diego

1:20 GPCRs as Targets in Cancer

Paul A. Insel, MD, Distinguished Professor, Pharmacology and Medicine; Co-Director, Medical Scientist MD/PhD Training Program, University of California, San Diego

Emerging data suggest that GPCRs contribute to malignancy and certain GPCRs have higher expression in tumors compared to normal tissue. Using multiple approaches to assess GPCRs in human tumors, cancer cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment, we find that tumors, cancer cells and CAFs have higher expression of many GPCRs. Confirmatory, validation data exist for multiple such GPCRs in pancreatic cancer, a highly lethal cancer in need of new, effective therapies.

1:50 Illuminating the Onco-GPCRome

J. Silvio Gutkind, PhD, Professor, Department of Pharmacology; Associate Director of Basic Science, Moores Cancer Center, UCSD

Recent large cancer sequencing initiatives have revealed that more than 25% of all human malignancies harbor mutations in G proteins and GPCRs, and that certain GPCR families are aberrantly expressed in multiple human neoplasia. We will present new evidence supporting the potential clinical benefit of targeting GPCRs, G proteins, and their regulated signaling circuitry for cancer prevention and treatment. How GPCRs modulation can be exploited to increase the response to new immunotherapies will be discussed.

2:20 Anti-Leukemic Activity of Imipridone ONC212 via Selective Targeting of Orphan GPCR GPR132/G2A

Varun Vijay Prabhu, PhD, Associate Director, Research and Development, Oncoceutics, Inc.

Imipridones are a new class of anti-cancer small molecules that share a unique tri-heterocyclic core structure and selectively engage GPCRs. Experimental GPCR profiling using the PathHunter® β-Arrestin assay (DiscoverX) and multidose validation revealed that imipridone ONC212 selectively targets orphan GPCR GPR132/G2A at nanomolar concentrations. BIOSENS-ALL BRET assay (Domain) showed that ONC212 promotes Gq family activation downstream of GPR132. ONC212 was non-toxic to normal cells at therapeutic concentrations and demonstrated robust in vivo safety/efficacy in leukemia xenograft models.

2:50 Therapeutic Promise of Allosteric Modulators of Angiotensin II Receptor

Sadashiva Karnik, PhD, Professor, Molecular Cardiology, Lerner Research Institute, Cleveland Clinic

Novel allosteric modulators were discovered based on crystal structure and computer assisted drug development. These novel molecules showed high specificity and efficacy in pharmacological and signaling studies. In vivo evaluation in animal models are in progress. This will be the first report of allosteric chemotypes for any angiotensin receptor.

3:20 Selected Poster Presentation(s)

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing

4:30 Plenary Session Welcome Remarks from Event Director

Anjani Shah, PhD, Conference Director, Cambridge Healthtech Institute

4:35 Sponsored Plenary Keynote Introduction (Opportunity Available)

4:40 PLENARY KEYNOTE: Activity-Based Proteomics: Protein and Ligand Discovery on a Global Scale

Benjamin F. Cravatt, PhD, Professor and Co-Chair, Department of Molecular Medicine, The Scripps Research Institute
To address uncharacterized proteins, we have introduced chemical proteomic technologies that globally profile the functional state of proteins in native biological systems. Among these methods is activity-based protein profiling (ABPP), which utilizes chemical probes to map activity states of large numbers of proteins in parallel. I will discuss the application of ABPP to discover and functionally annotate proteins in mammalian physiology and disease, and the generation and implementation of advanced ABPP platforms for proteome-wide ligand discovery.

5:30 Welcome Reception in the Exhibit Hall with Poster Viewing

6:30 End of Day

Wednesday, April 4

7:30 am Continental Breakfast Breakout Discussions

ENDOSOMAL SIGNALING

8:30 Chairperson’s Remarks

Irina Kufareva, PhD, Project Scientist, The Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego

8:35 Ubiquitin-Mediated Inflammatory Signaling by GPCRs

JoAnn Trejo, PhD, MBA, Professor and Vice Chair, Department of Pharmacology, Associate Dean for Health Sciences Faculty Affairs, University of California, San Diego

Ubiquitination of 40 mammalian GPCRs has been reported, but despite the rich complexity of GPCR signaling, ubiquitination is attributed largely to GPCR degradation. We discovered that ubiquitination of GPCRs promotes p38 activation on endosomes via recruitment of TAB2, which co-associates with TAB1 that directly binds to p38a. TAB1-dependent p38 activation is critical for PAR1-mediated endothelial inflammatory responses. The mechanisms by which GPCR-induced p38 endosomal inflammatory signaling is regulated is not known and will be discussed.

9:05 Decoding the Layered Internet-like Architecture of a Cell’s Signaling Network:Precisionalized Drug Discovery in the Era of Network Medicine

Pradipta Ghosh, MD, Professor, Departments of Medicine and Cell and Molecular Medicine, UC San Diego

We posit that the architecture and the principles of information transfer in the intracellular communication are in many ways similar to those in the global communication network, the Internet, spurring the Intranet of Cells (IoC) metaphor. In particular, as in telecommunications, flexibility, evolvability and robustness in the IoC are gained at the expense of speed, and appear to be mediated by the layered protocol architecture at the signaling stations. Our IoC initiative is to organize the cell’s information and make it accessible and useful.

9:35 Coffee Break in the Exhibit Hall with Poster Viewing

NEW GPCR SCREENING AND BINDING ASSAYS AND TOOLS

10:30 Discovery of Small Molecule Protease-Activated Receptor 2 (PAR2) Antagonists

Dean G. Brown, PhD, Director of External Chemistry, Hit Discovery, Discovery Sciences, IMED Biotech Unit, AstraZeneca

We employed two screening strategies to identify antagonists at protease activated receptor (PAR2), one being a DNA-encoded library screen on PAR2 and the second a fragment screen using a stabilized PAR2 GPCR receptor. From these efforts, we identified two lead series of compounds, each of which bind to distinct and previously unknown allosteric sites. These results illustrate the power of integrating stabilized GPCR technologies into established screening paradigms.

11:00 Binding and Functional Analysis - Complementary Approaches in Safety Pharmacology using GPCRs

Thierry Jolas, PhD, Study Director, Eurofins Pharma Discovery Services

In vitro pharmacological profiling is an integral part of drug discovery and development, and provides critical information at multiple key decision points in the process. Using several examples, I will show how a combined approach of adopting both binding and functional assays may provide a more holistic assessment of test compounds activity.

11:30 Kinetic Drug Discovery for GPCRs

Sam Hoare, PhD, Founder and Chief Scientist, Pharmacology Data Analysis, Pharmechanics, LLC

Novel paradigms are needed for translating the raw data emerging from new molecular biosensor and reader technology-based assays into meaningful pharmacological activity parameters that can be used for structure-activity analysis. We have developed a new kinetic data analysis framework that, using standard curve-fitting software, yields values of the rate of onset of the response, and the total signal produced. Here we will show the resulting structure-activity kinetics for beta2 adrenoceptor signaling, and for biased agonism at the D2 dopamine receptor.

12:00 pm End of Conference