0026-895X/05/6805-1484–1495$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 5 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 16162/3061208 Mol Pharmacol 68:1484–1495, 2005 Printed in U.S.A. Allosteric Modulation of the Cannabinoid CB1 Receptor Martin R. Price, Gemma L. Baillie, Adele` Thomas, Lesley A. Stevenson, Morag Easson, Richard Goodwin, Adele` McLean, Lorraine McIntosh, Gillian Goodwin, Glenn Walker, Paul Westwood, Julia Marrs, Fiona Thomson, Phillip Cowley, Arthur Christopoulos, Roger G. Pertwee, and Ruth A. Ross School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland (M.R.P., G.L.B., A.T., L.A.S., R.G.P., R.A.R.); Organon Research, Newhouse, Lanarkshire, Scotland (M.E., R.G., A.M., L.M., G.G., G.W., P.W., J.M., F.T., P.C.); and Department of Pharmacology, University of Melbourne, Parkville, Australia (A.C.) Downloaded from Received June 28, 2005; accepted August 18, 2005 ABSTRACT 3 3 We investigated the pharmacology of three novel compounds, when the radioligand was [ H]CP 55,940 or [ H]SR 141716A. molpharm.aspetjournals.org Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4- However, the estimated cooperatively factor for the interaction piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5- between modulator and radioligand was greater than 1 when fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phe- determined against [3H]CP 55,940 and less than 1 when deter- nyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole- mined against [3H]SR 141716A. [3H]CP 55,940 dissociation 2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic kinetic studies also validated the allosteric nature of the Org acid salt), at the cannabinoid CB1 receptor. In equilibrium bind- compounds, because they all significantly decreased radioli- ing assays, the Org compounds significantly increased the gand dissociation. These data suggest that the Org com- 3 binding of the CB1 receptor agonist [ H]CP 55,940 [(1R,3R,4R)- pounds bind allosterically to the CB1 receptor and elicit a 3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypro- conformational change that increases agonist affinity for the pyl)cyclohexan-1-ol], indicative of a positively cooperative allo- orthosteric binding site. In contrast to the binding assays, how- at ASPET Journals on December 17, 2015 steric effect. The same compounds caused a significant, but ever, the Org compounds behaved as insurmountable antago- incomplete, decrease in the specific binding of the CB1 recep- nists of receptor function; in the reporter gene assay, the tor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4- guanosine 5Ј-O-(3-[35S]thio)triphosphate binding assay and the chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- mouse vas deferens assay they elicited a significant reduction carboximide hydrochloride], indicative of a limited negative in the Emax value for CB1 receptor agonists. The data presented binding cooperativity. Analysis of the data according to an clearly demonstrate, for the first time, that the cannabinoid CB1 allosteric ternary complex model revealed that the estimated receptor contains an allosteric binding site that can be recog- affinity of each Org compound was not significantly different nized by synthetic small molecule ligands. Mammalian tissues express at least two types of cannabi- view, see Howlett et al., 2002). CB1 receptors are found noid receptor, CB1 and CB2, both G protein-coupled (for re- predominantly at central and peripheral nerve terminals where they mediate inhibition of transmitter release. Endog- A.C. is a Senior Research Fellow of the National Health and Medical enous ligands for these receptors also exist. These “endocan- Research Council of Australia, M.P. is funded by the National Institute on nabinoids” are all eicosanoids, prominent examples including Drug Abuse, and G.B. is a Biotechnology and Biological Sciences Research arachidonoylethanolamide (anandamide) and 2-arachidonoyl Council/Organon Case award student. A.T. and L.S. are funded by GW Phar- maceuticals (Salsbury, Wiltshire, UK). glycerol, both of which are synthesized on demand, removed Article, publication date, and citation information can be found at from their sites of action by tissue uptake processes and http://molpharm.aspetjournals.org. doi:10.1124/mol.105.016162. metabolized by intracellular enzymes (Pertwee and Ross, ABBREVIATIONS: CB, cannabinoid; GPCR, G protein-coupled receptor; TCM, ternary complex model; CNS, central nervous system; WIN 55,212, (ϩ)-WIN 55,212-2 [(R)-(ϩ)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtha- lenylmethanone]; CP 55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; SR 141716A, N-(pip- eridin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; BSA, bovine serum albumin; DTT, di- thiothreitol; Gpp(NH)p, guanosine 5Ј-(␤,␥-imido)triphosphate; GTP␥S, guanosine 5Ј-O-(3-thio)triphosphate; Org 27569-0, 5-chloro-3-ethyl-1H- indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide; Org 27759-0, 3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94- dimethylamino-phenyl)-ethyl]-amide; Org 29647-1, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt; DMSO, dimethyl sulfoxide. 1484 Allosteric Modulation of the Cannabinoid CB1 Receptor 1485 2002). There is ample evidence for a role of the endocannabi- have been presented to suggest that allosteric modulators noid system in a number of physiological processes, including may affect the efficacy of orthosteric ligands in addition to, or cardiovascular regulation, appetite control, learning, and independently of, effects on orthosteric ligand binding, neces- memory and pain processing (for review, see Ross, 2003; Di sitating the development of more complex models of allosteric Marzo et al., 2004). Within the brain, the distribution of CB1 modulation (Christopoulos and Kenakin, 2002, and refer- receptors is heterogeneous, accounting for several well docu- ences therein). mented pharmacological properties of CB1 receptor agonists. To date, the main principle underlying GPCR-based drug For example, the cerebral cortex, hippocampus, lateral cau- discovery has invariably been the optimization of lead mole- date-putamen, substantia nigra, pars reticulata, globus pal- cules toward the orthosteric site on the GPCR to obtain lidus, entopeduncular nucleus, and the molecular layer of the selectivity of action. However, allosteric modulators offer ad- cerebellum are all populated with particularly high concen- vantages not readily available with orthosteric ligands. For trations of CB1 receptors. CB1 receptor ligands have thera- example, allosteric drugs that affect affinity (but not efficacy) peutic potential in a range of disorders, including obesity, would be advantageous in that they would boost or dampen nicotine addiction, pain, inflammation, gastrointestinal dis- the effect of the endogenous ligand without disrupting the orders, multiple sclerosis, psychosis, schizophrenia, and os- inherent spatial and temporal patterns of physiological sig- teoporosis. naling (May and Christopoulos, 2003). CB1 receptor pharma- The traditional view of G protein-coupled receptor (GPCR) cology is the subject of intense academic and commercial Downloaded from signaling posits that agonists initiate signaling by binding to research effort directed at the treatment of obesity (Horvath, a site on the GPCR that has specifically evolved to recognize 2003), pain (Pertwee, 2001; Iversen and Chapman, 2002), the endogenous agonist for that receptor; this site has been inflammation (Rice et al., 2002), osteoporosis (Idris et al., defined as the orthosteric binding site (Neubig et al., 2003). It 2005), cancer (Bifulco and Di Marzo, 2002), multiple sclerosis is now recognized, however, that GPCRs may also contain (Pertwee, 2002), and cardiovascular disorders (Randall et al., allosteric binding sites for endogenous and/or synthetic li- 2002). There is ample evidence that the levels of endocan- molpharm.aspetjournals.org gands (Christopoulos and Kenakin, 2002). Allosteric sites are nabinoids are altered in these pathophysiological situations. topographically distinct from the orthosteric site; hence, the Allosteric enhancers of the cannabinoid CB1 receptor would structural features that determine ligand binding to alloste- thus offer the prospect of producing clinically useful com- ric sites are different from those of orthosteric ligands. In pounds that do not display the CNS side effects that are contrast to the direct effects on receptor function that are characteristic of direct receptor agonism. In this investiga- mediated by orthosteric ligands, allosteric drugs act by mod- tion, we provide the first evidence for an allosteric binding ulating receptor activity through conformational changes in site on the cannabinoid CB1 receptor, as revealed by the the receptor that are transmitted from the allosteric to the pharmacology and mode of action of a novel class of synthetic orthosteric site and/or to effector coupling sites (Christopou- small molecules (Fig. 2). at ASPET Journals on December 17, 2015 los, 2002). The simplest allosteric interaction can be described by the Materials and Methods allosteric ternary complex model (TCM; Fig. 1), where the effect of an allosteric ligand is to change the affinity of an Materials orthosteric