JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 JPET ThisFast article Forward. has not been Published copyedited andon formatted.June 27, The 2006 final versionas DOI:10.1124/jpet.106.104463 may differ from this version. JPET #104463 PiP Title Page Functional selectivity and classical concepts of quantitative pharmacology Jonathan D. Urban, William P. Clarke, Mark von Zastrow, David E. Nichols, Brian Kobilka, Harel Weinstein, Jonathan A. Javitch, Bryan L. Roth, Arthur Christopoulos Patrick M. Sexton, Keith J. Miller, Michael Spedding, Richard B. Mailman Downloaded from Curriculum in Toxicology (JDU, RBM) and Departments of Pharmacology, Psychiatry Medicinal Chemistry (BLR, RBM), and Neurology (RBM), University of North Carolina, Chapel Hill, NC Department of Pharmacology, University of Texas Health Science Center, San Antonio, jpet.aspetjournals.org TX (WPC) Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, CA (MvZ) at ASPET Journals on September 25, 2021 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayette, IN (DEN) Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA Department of Physiology and Biophysics, and Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY (HW) Center for Molecular Recognition, and Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, NY (JJ) Department of Pharmacology, Monash University, Clayton, Victoria, Australia (AC, PMS) Obesity Department, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ (KJM) Institute de Recherches Servier, Suresnes 92150, France MS) 1 Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 This article has not been copyedited and formatted. The final version may differ from this version. JPET #104463 PiP Running Title Page Document statistics Text Pages 44 Tables 0 Figures 6 References 112 Abstract words 244 Corresponding Author: Downloaded from Richard B. Mailman CB#7160, 7011 NC Neurosciences Hospital University of North Carolina School of Medicine jpet.aspetjournals.org Chapel Hill, NC 27599-7160 USA [email protected] Phone 919-966-2484 Fax 919-966-9604 Running Title: at ASPET Journals on September 25, 2021 Functional selectivity – new concept of receptor signaling Abbreviations 2-AG 2-arachidonylglycerol 5-HT Serotonin 2C-B-CB 1-aminomethyl-3,6-dimethoxy-4-bromobenzocyclobutane β2AR β2 adrenergic receptor AA arachidonic acid AFM atomic force microscopy DAG Diacylglycerol DAMGO D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin DHX dihydrexidine; trans-10,11-dihydroxy-5,6,6a,7,8,12b- hexahydrobenzo[a]phenanthridine DOB (-)-1-2, 5-dimethoxy-4-bromophenyl-2-aminopropane ECM electron cryomicroscopy GIRK G protein-coupled inward rectifying potassium channel. 2 JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 This article has not been copyedited and formatted. The final version may differ from this version. JPET #104463 PiP GPCR G protein-coupled receptor GTPγS Guanosine 5`-(γ-thio)triphosphate HGP Human genome project HTS High throughput screen IP inositol phosphates IUPHAR International Union of Basic and Clinical Pharmacology LSD D-lysergic acid diethylamide MALDI-TOF Matrix Assisted Laser Desorption Ionization - Time of Flight mass spectroscopy Downloaded from MAPK Mitogen-Activated Protein Kinase MEK MAP kinase kinase NC-IUPHAR Nomenclature committee of IUPHAR jpet.aspetjournals.org NDI nephrogenic diabetes insipidus N-propylDHX N-n-propyldihydrexidine PLA Phospholipase A PLC Phospholipase C at ASPET Journals on September 25, 2021 SB 242084 6-chloro-5-methyl-N-(6-(2-methylpyridin-3-yloxy)pyridin-3- yl)indoline-1-carboxamide SILAC Stable isotope labeling with amino acids in culture SNP Single nucleotide polymorphism TM Transmembrane helix 3 JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 This article has not been copyedited and formatted. The final version may differ from this version. JPET #104463 PiP Abstract The concept of intrinsic efficacy has been enshrined in pharmacology for a half- century, yet recent data reveal that many ligands can differentially activate signaling pathways mediated via a single G protein coupled receptor (GPCR) in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this Downloaded from phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate jpet.aspetjournals.org conformational states, as shown for the β2-adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate at ASPET Journals on September 25, 2021 differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, as this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts. 4 JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 This article has not been copyedited and formatted. The final version may differ from this version. JPET #104463 PiP Receptor pharmacology for the new millennium For the last half century, pharmacological theory has posited that ligands could be characterized by the nature of the functional effects elicited by their interaction with their target receptor. These effects are governed by two important properties: affinity, the property of attraction between a ligand and its receptor, and efficacy, the property that allows ligands, once bound, to produce a response (Kenakin, 1997). This concept has led to the classification of receptor ligands as full agonists, partial agonists, neutral antagonists, or inverse agonists. Perhaps the key idea that governs this classification is the notion of “intrinsic efficacy”, originally proposed by Furchgott (1966) as a measure Downloaded from of the stimulus per receptor molecule produced by a ligand. According to this notion, full agonists possess sufficiently high intrinsic efficacy such that they maximally stimulate all cellular responses linked to a given receptor. Partial agonists possess lower degrees of jpet.aspetjournals.org intrinsic efficacy (leading to submaximal responses), whereas inverse agonists reduce constitutive (ligand-independent, basal) receptor signaling. Neutral antagonists possess no intrinsic efficacy, but occupy the receptor to block the effects of full, partial, or inverse agonists. This idea has also led to the assumption, dearly held in pharmacology, at ASPET Journals on September 25, 2021 that the ability of the ligand to impart (or reduce) stimulus once that ligand is bound to the receptor is an inherent property of the ligand-receptor complex. Intrinsic efficacy is thus differentiated from the more operational term “intrinsic activity” (Ariens, 1954), that simply refers to the maximal effect (Emax) of a ligand relative to a reference agonist in a given experimental system. In classical pharmacological terms, therefore, intrinsic efficacy has been viewed as a system-independent parameter that is constant for each ligand at a given receptor, irrespective of where that receptor is expressed. Any differences in the expression of agonism by a ligand between cell and tissue types were assumed simply to reflect differences in receptor density and/or the strength of stimulus-response coupling. In other words, the classification of compounds on the basis of intrinsic efficacy only allowed for variations in the quantity of the stimulus that was imparted to the cell, but not the quality. A full agonist would be expected to activate all of the signaling pathways linked to a receptor to the same degree as the endogenous ligand for that receptor. In contrast, a ligand that antagonizes one signaling pathway via a specific receptor should antagonize 5 JPET Fast Forward. Published on June 27, 2006 as DOI: 10.1124/jpet.106.104463 This article has not been copyedited and formatted. The final version may differ from this version. JPET #104463 PiP every pathway coupled to that receptor to the same extent. One consequence of this theory was the weight it added to Ehrlich’s idea of a therapeutic “magic bullet”. It led to a focus on discovery of new receptor ligands with