eBriefing

Elucidating GPCR Functional Selectivity for Drug Development

Elucidating GPCR Functional Selectivity for Drug Development
Reported by
Laura K. Fogli

Posted November 12, 2014

Overview

G protein-coupled receptors (GPCRs) are the largest class of targets in drug development and the targets for 40% of available drugs. Upon their discovery, GPCRs were thought to relay extracellular signals only by associating with intracellular G proteins (GTP-binding proteins). But these receptors are now known to signal through other pathways, notably those mediated by arrestins. Arrestins are multifunctional signaling modulators first described for their role in GPCR desensitization, the termination of GPCR activity through phosphorylation. Independent of G proteins, arrestins can recognize active GPCR conformations and induce downstream signaling. Some GPCR ligands induce G protein signaling, while others induce arrestin-mediated signaling at the same receptor. Ligand bias toward particular signal transduction pathways at the same GPCR is known as functional selectivity. On September 30, 2014, the Academy's Biochemical Pharmacology Discussion Group held a symposium titled Elucidating GPCR Functional Selectivity: Novel Opportunities for Drug Development to examine ligand bias and its implications for the design of better targeted and more effective drugs.

John A. Allen, a principal scientist at Pfizer, outlined the challenges involved in this kind of research. By understanding functional selectivity, scientists can identify suitable molecules for drug development that will engage the desired pathways while avoiding adverse events. However, the process of profiling molecules with potential as therapeutics must involve multiple in vitro signaling assays early in preclinical development to reveal bias for specific downstream outcomes. Furthermore, biased ligand binding activity observed in vitro may not occur in vivo, indicating the need for in vivo readouts for distinct signaling pathways. Terry Kenakin of the University of North Carolina School of Medicine also highlighted the differences between in vitro and in vivo ligand activity, describing three common methods used to quantify biased receptor signaling and explaining why it is difficult to use these equations to predict an agonist's in vivo therapeutic response. Signaling is driven by both a ligand's ability to bind its receptor, known as affinity, and its ability to induce a cellular response upon binding, known as efficacy. Cells express varying levels of the same receptors, so each biased ligand exhibits tissue-specific behavior that depends on its affinity for its receptor. Affinity and efficacy determine how receptor availability and receptor–ligand interaction impact the functional selectivity of compounds in vivo.

Laura Bohn of Scripps Research Institute described her work to move drug candidates from in vitro studies to animal models. Bohn's research focuses on therapeutic targeting of the kappa opioid receptor (KOR), the receptor for morphine and other opioid analgesic drugs. Evidence suggests G protein signaling through this receptor is associated with pain and itch relief, while β-arrestin-mediated signaling may induce depression and dysphoria. Bohn is therefore looking for KOR ligands that selectively activate G protein signaling. After testing G protein-biased KOR agonists in mice, she showed that the compounds elicited the expected antinociceptive and anti-itch effects without negative behavioral changes. These data suggest it is possible for biased agonists discovered in vitro to be translated into therapies that improve on existing drugs.

JoAnn Trejo from the University of California, San Diego, focused on the role of receptor protein modification in regulating GPCR coupling with G protein subtypes. The PAR1 receptor for the coagulation factor thrombin is modified by N-linked glycosylation at various sites, two located on the second extracellular loop that mediates tethered ligand interaction. Trejo's group investigated the functional significance of this glycosylation by studying the signaling pathways of a mutated form of the receptor and by using pharmacological agents to block glycosylation of the endogenous receptor in cultured human endothelial cells. The researchers discovered that N-linked glycosylation of the extracellular loop does not impact receptor trafficking or surface expression, but it does drive PAR1 to selectively couple with Gα12/13 proteins. Downstream, this signaling bias has an impact on cytoskeletal responses (specifically stress fiber formation) and cellular proliferation. Trejo's presentation highlighted a novel mechanism through which active receptors can be modified to promote distinct G protein signaling pathways, allowing us to better understand how to target GPCRs to achieve therapeutic benefit.

The thrombin receptor PAR1 is glycosylated at several sites, two of which are on the surface of the second extracellular loop (ECL2). Study of a mutated receptor lacking these sites (PAR1 NA ECL2) revealed that glycosylation of ECL2 impacts G protein signaling, biasing PAR1 to couple with Gα12/13 subunits rather than with Gα. This signaling bias has an effect on downstream cellular functions, including stress fiber formation and cellular proliferation. (Image courtesy of JoAnn Trejo)

The work presented by Bohn and Trejo demonstrated that GPCR ligands can selectively induce signaling pathways in vivo and that this biased GPCR activity can result from the ligand itself or be induced by modifications to the receptor. Jonathan A. Javitch of Columbia University and New York State Psychiatric Institute took a structural approach to the understanding of functional selectivity by discussing how GPCR signaling is biased by the location and orientation of the receptor–ligand interaction. Classical approaches to GPCR drug development targeted the orthosteric binding site, the primary active binding site for endogenous ligands. But a major focus of current research is allosteric binding at alternative sites on the receptor. Allosteric modulators typically bind with lower affinity but can be highly selective among receptor subtypes, because unlike orthosteric ligands they do not target evolutionarily conserved binding regions. Javitch discussed his collaborative work to characterize bitopic ligands, which are linked molecules that simultaneously bind orthosteric and allosteric sites. He described the role of bitopic ligands in the study of GPCR dimerization, the association of two receptors to form one functional unit. Focusing on the dopamine receptor, Javitch designed a functional complementation assay to probe for communication among GPCR protomers. His data show that bitopic ligand binding to one protomer can cause conformational changes that modulate the binding of another ligand to a second protomer, demonstrating the complexity of GPCR structural relationships and cross-talk between signaling pathways.

Bryan L. Roth of the University of North Carolina School of Medicine continued the discussion of GPCR structure by giving an overview of his work on serotonin receptors. Serotonin receptor agonists are used in the treatment of migraines and Parkinson's disease but can cause valvular heart disease. Ergotamine in particular displays extreme bias toward arrestin-mediated signaling at one of the serotonin receptors, which may play a role in the severe cardiac side effect. Roth found differences in the structural motifs and ligand orientations of the binding sites of biased and unbiased serotonin receptors. These data help explain the striking difference in downstream signaling. His studies emphasize the importance of molecular structure in determining biased agonism.

In the keynote presentation, Robert J. Lefkowitz from Duke University Medical Center gave a historical overview of the β-arrestin protein family. Beginning with the discovery of the arrestins as mediators of receptor desensitization, Lefkowitz described the years of research that contributed to our understanding of the arrestins as multifunctional adapter proteins and signaling modulators. Building on the previous talks, he gave several more examples of the therapeutic benefits of biased agonism and pointed out the differences between ligand bias (differential signaling due to ligand-specific receptor conformations) and system bias (differential signaling due to unequal transducer protein coupling). Research into GPCR functional selectivity has demonstrated that agonist-mediated signaling bias is probably a result of both mechanisms. To fully understand signaling bias, we need to analyze high-resolution crystal structures of the ternary (receptor–ligand–transducer) complexes involved. Lefkowitz shared his research on the crystal structure of β-arrestin 1 bound to an activating GPCR phosphopeptide. His work highlights how crystallography and 3D modeling can elucidate arrestin signaling interactions.

The day ended with two talks focused on the clinical implications of GPCR ligand bias. Marc G. Caron from Duke University Medical Center presented his work on dopamine receptor signaling. In mouse models with either β-arrestin 2 or the downstream signaling protein GSK3β conditionally deleted in neurons, Caron's group has demonstrated that β-arrestin-dependent signaling downstream of the dopamine D2 receptor is a key mechanism of action for anti-psychotic drugs. Dopamine signaling has various consequences in different parts of the brain, so D2 receptor ligands that are functionally selective for anti-psychotic pathways may serve as more effective medications with fewer side effects.

Jonathan D. Violin, cofounder of Trevena Inc., presented clinical data that supports the idea that biased ligands make better drugs. For the treatment of acute heart failure, Trevena has developed a compound called TRV027 that targets imbalances in the renin–angiotensin system by directly binding the angiotensin II receptor. The ligand blocks G protein signaling while enhancing β-arrestin recruitment, leading to increased cardiac function. Violin also discussed the development of an opioid analgesic (TRV130) that selectively induces G protein-coupled signaling at the kappa opioid receptor. TRV130 exploits the same mechanism as the drug candidates Bohn is investigating to achieve better pain management while preventing side effects. The drugs Violin described show promise in early clinical trials, lending support to hopes that understanding GPCR functional selectivity can lead to improved health care.

TRV027 is a novel β-arrestin-biased ligand of the angiotensin II receptor that may find use as a more effective treatment for acute heart failure. By blocking G protein signaling, this compound reduces blood pressure in rats with the same efficacy as the full angiotensin receptor blocker (ARB) telmisartan. Rather than simultaneously blocking other beneficial pathways associated with this receptor, TRV027 enhances β-arrestin-mediated signaling, improving cardiac contractility and function. (Image courtesy of Jonathan D. Violin)


 
Use the tabs above to find multimedia from this event.

Presentations available from:
John A. Allen, PhD (Pfizer)
Marc G. Caron, PhD (Duke University Medical Center)
Jonathan A. Javitch, MD, PhD (Columbia University; New York State Psychiatric Institute)
Terry Kenakin, PhD (University of North Carolina School of Medicine)
Bryan L. Roth, MD, PhD (University of North Carolina School of Medicine)
JoAnn Trejo, PhD (University of California, San Diego)
Jonathan D. Violin, PhD (Trevena Inc.)


The Biochemical Pharmacology Discussion Group is proudly supported by


  • American Chemical Society
  • Boehringer Ingelheim
  • Merck
  • Pfizer
  • WilmerHale

Mission Partner support for the Frontiers of Science program provided by Pfizer

Resources

Journal Articles

Allen JA, Yost JM, Setola V, et al. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A. 2011;108(45):18488-93.

Beaulieu JM, Zhang X, Rodriguiz RM, et al. Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. Proc Natl Acad Sci U S A. 2008;105(4):1333-8.

Bohn LM, Lefkowitz RJ, Gainetdinov RR, et al. Enhanced morphine analgesia in mice lacking β-arrestin 2. Science. 1999;286(5449):2495-8.

DeWire SM, Violin JD. Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology. Circ Res. 2011;109(2):205-16.

Fenalti G, Giguere PM, Katritch V, et al. Molecular control of delta-opioid receptor signalling. Nature. 2014;506(7487):191-6.

Galandrin S, Bouvier M. Distinct signaling profiles of beta1 and beta2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol Pharmacol. 2006;70(5):1575-84.

Han Y, Moreira IS, Urizar E, et al. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat Chem Biol. 2009;5(9):688-95.

Kenakin T. Being mindful of seven-transmembrane receptor 'guests' when assessing agonist selectivity. Br J Pharmacol. 2010;160(5):1045-7.

Kenakin T, Christopoulos A. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov. 2013;12(3):205-16.

Lane JR, Donthamsetti P, Shonberg J, et al. A new mechanism of allostery in a G protein-coupled receptor dimer. Nat Chem Biol. 2014;10(9):745-52.

Luttrell LM, Lefkowitz RJ. The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci. 2002;115(Pt 3):455-65.

Raehal KM, Schmid CL, Groer CE, et al. Functional selectivity at the mu-opioid receptor: implications for understanding opioid analgesia and tolerance. Pharmacol Rev. 2011;63(4):1001-19.

Raehal KM, Walker JK, Bohn LM. Morphine side effects in β-arrestin 2 knockout mice. J Pharmacol Exp Ther. 2005;314(3):1195-201.

Rajagopal S, Ahn S, Rominger DH, et al. Quantifying ligand bias at seven-transmembrane receptors. Mol Pharmacol. 2011;80(3):367-77.

Schmid CL, Streicher JM, Groer CE, et al. Functional selectivity of 6'-guanidinonaltrindole (6'-GNTI) at kappa-opioid receptors in striatal neurons. J Biol Chem. 2013;288(31):22387-98.

Shukla AK, Manglik A, Kruse AC, et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature. 2013;497(7447):137-41.

Soergel DG, Subach RA, Burnham N, et al. Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: A randomized, double-blind, placebo-controlled, crossover study in healthy volunteers. Pain. 2014;155(9):1829-35.

Soergel DG, Subach RA, Cowan CL, et al. First clinical experience with TRV027: pharmacokinetics and pharmacodynamics in healthy volunteers. J Clin Pharmacol. 2013;53(9):892-9.

Soto AG, Trejo J. N-linked glycosylation of protease-activated receptor-1 second extracellular loop: a critical determinant for ligand-induced receptor activation and internalization. J Biol Chem. 2010;285(24):18781-93.

Violin JD, Soergel DG, Boerrigter G, et al. GPCR biased ligands as novel heart failure therapeutics. Trends Cardiovasc Med. 2013;23(7):242-9.

Wacker D, Wang C, Katritch V, et al. Structural features for functional selectivity at serotonin receptors. Science. 2013;340(6132):615-9.

Zhou L, Bohn LM. Functional selectivity of GPCR signaling in animals. Curr Opin Cell Biol. 2014;27:102-8.

Zhou L, Lovell KM, Frankowski KJ, et al. Development of functionally selective, small molecule agonists at kappa opioid receptors. J Biol Chem. 2013;288(51):36703-16.

At the Academy

At the Other Site: Allosteric Modulation of G Protein-coupled Receptors

Organizers

John A. Allen, PhD

Pfizer
website | publications

John A. Allen earned a PhD in physiology and biophysics from the University of Illinois College of Medicine, studying cell and molecular mechanisms regulating Gs/adenylyl cyclase/cAMP signaling. He completed postdoctoral training at the University of North Carolina School of Medicine, researching mechanisms of antipsychotic drug action and the neuropharmacology of serotonin and dopamine receptors. In 2011 he joined Pfizer Neuroscience, where his research aims to identify and advance therapeutic targets to treat neurological diseases, including profiling GPCRs for preclinical drug development. His recent work has looked at functional selectivity at dopamine receptors and determined structural mechanisms enabling ligand bias. Allen is a member of the Society for Neuroscience and ASPET. He has received research awards including a Young Investigator Award from the American College of Neuropsychopharmacology and a NARSAD Young Investigator Award.

Mercedes Beyna, MS

Pfizer
publications

Mercedes Beyna is a scientist in the Neuroscience Research Unit at Pfizer. Her research focuses on target identification and assay development in the areas of psychiatric and neurodegenerative disorders. Captivated by neuroscience, she has worked in the field for over ten years, in both academic and industrial laboratory settings. Before joining pharmaceutical R&D, Beyna held lab manager and senior lab technician positions at New York University (NYU). She holds an undergraduate degree in biology from Binghamton University and a Master's degree in biology from NYU. She enjoys developing interesting and educational symposia as the Pfizer lead in the Biochemical Pharmacology Discussion Group at the New York Academy of Sciences.

Bryan L. Roth, MD, PhD

University of North Carolina School of Medicine
website | publications

Bryan L. Roth is the Michael Hooker Distinguished Professor of Pharmacology at University of North Carolina School of Medicine. Roth received postdoctoral training in molecular pharmacology at the National Institutes of Health and psychiatry training at Stanford University. His research interests include chemical and synthetic biology, particularly applied to G protein-coupled receptors. Roth serves on the boards of many pharmacology and chemistry journals and is currently the deputy editor of the Journal of Clinical Investigation. Roth also directs the National Institute of Mental Health's small molecule screening program—now in its 16th year. Over the past five years, this program (NIMH-PDSP) has facilitated work on more than 500 projects and contributed to more than 500 publications and patents, assisting investigators worldwide. Roth hold an MD and PhD in biochemistry from St. Louis University School of Medicine. He was elected to the Institute of Medicine in 2014.

Jennifer Henry, PhD

The New York Academy of Sciences

Jennifer Henry is the former director of Life Sciences at the New York Academy of Sciences. Before joining the Academy, she was a publishing manager in the Academic Journals division at Nature Publishing Group. She also served for eight years as editor of Functional Plant Biology for CSIRO Publishing in Australia. She received her PhD in plant molecular biology from the University of Melbourne, specializing in the genetic engineering of transgenic crops. As director of Life Sciences, she developed scientific symposia across a range of life sciences, including biochemical pharmacology, neuroscience, systems biology, genome integrity, infectious diseases and microbiology. She also generated alliances with organizations interested in developing programmatic content.


Keynote Speaker

Robert J. Lefkowitz, MD

Duke University Medical Center
website | publications

Robert J. Lefkowitz is the James B. Duke Professor of Medicine and a professor of biochemistry at the Duke University Medical Center. He has been an investigator of the Howard Hughes Medical Institute since 1976. He has received numerous awards and honors for his research, including the National Medal of Science, the Shaw Prize, the Albany Prize, and the 2012 Nobel Prize in Chemistry. He was elected to the National Academy of Sciences in 1988, the American Academy of Arts and Sciences in 1983, and the Institute of Medicine in 1994. He is best known for his study of G protein-coupled receptors, a field he has pioneered for more than 45 years. Lefkowitz writes review articles in the areas of hormone and drug receptors and their regulation and is a consultant for several drug companies specializing in drugs that may affect signal transduction processes, including Norak, Lexicon Genetics, and Genentech. He holds an MD from Columbia University.


Speakers

John A. Allen, PhD

Pfizer
website | publications

Laura Bohn, PhD

Scripps Research Institute–Florida
website | publications

Laura Bohn is a professor of molecular therapeutics and neuroscience at Scripps Research Institute. Bohn holds a PhD in biochemistry and molecular biology from St. Louis School of Medicine. She received postdoctoral training in the Howard Hughes Medical Institute at Duke University Medical Center in the laboratory of Marc Caron. Bohn achieved tenure at Ohio State University College of Medicine before joining Scripps–Florida, where she is pursuing new therapies for the treatment of pain. She is a recipient of the Women in Neuroscience/Society for Neuroscience Career Development Award, the 2009 College of Drug Dependence Joseph Cochin Young Investigator Award, and the John J. Abel Award in pharmacology from the American Society for Pharmacological and Experimental Therapeutics and Pfizer. She has consulted for Trevena Inc., Purdue Pharma LP, and Mencuro Therapeutics Inc., and she collaborates with Eli Lilly & Company.

Marc G. Caron, PhD

Duke University Medical Center
website | publications

Marc G. Caron holds a PhD from the University of Miami. He is the James B. Duke Professor of Cell Biology at Duke University Medical Center. His long-standing research interests have been in the mechanisms and regulation of G protein-coupled receptors and the mechanisms of neurotransmission as controlled by neurotransmitter transporters. His recent efforts have been centered on using genetic approaches to develop animal models of abnormal neurobiological function, including disorders associated with aberrant serotonergic, dopaminergic, and reward mechanisms.

Jonathan A. Javitch, MD, PhD

Columbia University; New York State Psychiatric Institute
website | publications

Jonathan A. Javitch is the Lieber Professor of Experimental Therapeutics in Psychiatry and a professor of pharmacology at Columbia University College of Physicians and Surgeons. He also directs the Lieber Center for Schizophrenia Research and Treatment and is chief of the Division of Molecular Therapeutics at New York State Psychiatric Institute. He obtained an MS in biological sciences at Stanford University and completed the MD-PhD program at Johns Hopkins University School of Medicine, where as a graduate student with Dr. Solomon Snyder he demonstrated that a key step in the neurotoxicity of MPTP is the uptake of its metabolite MPP+ by the dopamine transporter. Javitch completed a medical internship and psychiatric residency at Columbia Presbyterian Hospital and New York State Psychiatric Institute. He did postdoctoral work on the structure of dopamine receptors with Dr. Arthur Karlin at Columbia University. His research focuses on the structure, function, and regulation of G protein-coupled receptors and neurotransmitter transporters.

Terry Kenakin, PhD

University of North Carolina School of Medicine
website | publications

Terry Kenakin began his career as a synthetic chemist, receiving a PhD in pharmacology at the University of Alberta, Canada. After a postdoctoral fellowship at University College London, UK, he worked in drug discovery at Burroughs-Wellcome, Glaxo Inc., GlaxoWellcome, and GlaxoSmithKline. Kenakin is now a professor of pharmacology at the University of North Carolina School of Medicine. He is studying the optimal design of drug activity assays systems, the discovery and testing of allosteric molecules for therapeutic application, and the quantitative modeling of drug effects. He also directs the pharmacology curriculum at the School of Medicine. He is a member of several editorial boards, editor-in-chief of the Journal of Receptors and Signal Transduction, and coeditor-in-chief of Current Opinion in Pharmacology. He has authored ten books on pharmacology.

Bryan L. Roth, MD, PhD

University of North Carolina School of Medicine
website | publications

JoAnn Trejo, PhD

University of California, San Diego
website | publications

JoAnn Trejo holds a PhD from the University of California, San Diego, where she is a professor in the School of Medicine. Her research aims to define the pathways that regulate vascular inflammation and breast cancer progression. She directs the San Diego Institutional Research and Academic Career Development Award (IRACDA), an NIH-sponsored postdoctoral training program that aims to develop a diverse group of highly trained biomedical scientists. She is also vice chair of Education in the Department of Pharmacology at UC San Diego. Trejo serves on the council of the American Society for Cell Biology (ASCB) and chairs Gordon Research Conferences. She is a recipient of the American Heart Association Established Investigator Award and the 2015 American Society for Biochemistry and Molecular Biology Ruth Kirchstein Diversity in Science Award.

Jonathan D. Violin, PhD

Trevena Inc.
website | publications

Jonathan D. Violin helped launch Trevena in 2008 from its scientific foundations at Duke University. He now leads its investor relations program. Before joining Trevena, he was a postdoctoral fellow in the laboratory of Robert Lefkowitz at Duke University Medical Center, where he studied biased ligands and β-arrestin functions for several GPCRs. His research helped establish how biased ligands elicit unique cellular pharmacology with potential to translate into differentiated therapeutics. Violin holds a PhD from the Department of Pharmacology at the University of California, San Diego, where he developed biosensors to study the spatial and temporal dynamics of kinase signaling. He also received an MBA with a concentration in health sector management from the Fuqua School of Business.

Laura K. Fogli

Laura K. Fogli is a PhD candidate at New York University School of Medicine, where she is studying pathology and immunology. Her doctoral research is on mechanisms of T cell-driven lung inflammation. She plans to pursue a career in medical writing.

Sponsors

Academy Friend

DiscoveRx

Grant Support

This activity is supported by an education grant from Lilly. For further information concerning Lilly grant funding visit www.lillygrantoffice.com.

This program is supported in part by a grant from Merck and Co., Inc.

This program is supported in part by a grant from Otsuka America Pharmaceutical, Inc.


The Biochemical Pharmacology Discussion Group is proudly supported by


  • American Chemical Society
  • Boehringer Ingelheim
  • Merck
  • Pfizer
  • WilmerHale

Mission Partner support for the Frontiers of Science program provided by Pfizer