Perspectives in Pharmacology Allosteric Modulators of G Protein-Coupled Receptors: Future Therapeutics for Complex Physiological Disorders

G protein-coupled receptors (GPCRs) are one of the most important classes of proteins in the genome, not only because of their tremendous molecular diversity but because they are the targets of nearly 50% of current pharmacotherapeutics. The majority of these drugs affect GPCR activity by binding to a similar molecular site as the endogenous cognate ligand for the receptor. These “orthosterically” targeted drugs currently dominate the existing pharmacopeia. Over the past two decades, novel opportunities for drug discovery have risen from a greater understanding of the complexity of GPCR signaling. A striking example of this is the appreciation that many GPCRs possess functional allosteric binding sites. Allosteric modulator ligands bind receptor domains topographically distinct from the orthosteric site, altering the biological activity of the orthosteric ligand by changing its binding affinity, functional efficacy, or both. This additional receptor signaling complexity can be embraced and exploited for the next generation of GPCR-targeted therapies. Despite the challenges associated with detecting and quantifying the myriad of possible allosteric effects on GPCR activity, allosteric ligands offer the prospect of engendering a facile stimulus-bias in orthosteric ligand signaling, paving the way for not only receptor-selective but also signaling pathway-selective therapies. Allosteric modulators possess specific advantages when considering the treatment of multifactorial syndromes, such as metabolic diseases or age-related cognitive impairment, because they may not greatly affect neurotransmitter or hormone release patterns, thus maintaining the integrity of complex signaling networks that underlie perception, memory patterns, or neuroendocrinological axes while introducing therapeutically beneficial signal bias. Heptahelical GPCRs are ubiquitously expressed throughout eukaryotic organisms and can account for as much as 3 to 4% of the genome (Foord, 2002). By detecting ligands in the extracellular milieu, they transmit environmental information from outside a cell to the interior. At the same time, changes in the expression of GPCRs or receptor-associated regulatory proteins under varying physiological conditions This research was supported in part by the Intramural Research Program of the National Institutes of Health National Institute on Aging; the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK055524] (L.M.L.); and the Research Service of the Ralph H. Johnson Veterans Affairs Medical Center (L.M.L.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.156380. ABBREVIATIONS: GPCR, G protein-coupled receptor; AM, allosteric modulator; PAM, positive allosteric modulator; NAM, negative allosteric modulator; ATCM, allosteric ternary complex model; NMS, N-methylscopolamine; AchR, acetylcholine receptor; mAChR, muscarinic acetylcholine receptor; mGluR, metabotropic glutamate receptor; CTC, cubic ternary complex; CB1, cannabinoid 1; PTH, parathyroid hormone; CPPHA, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] phenyl}-2-hydroxybenzamide; WIN55212, (R)-( )-[2,3-dihydro-5-methyl-3-(4morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone; McN-A-343, 4-(m-chlorophenyl-carbamoyloxy)-2butynyltrimethylammonium chloride; AC-42, 4-N-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride; Org27569, 5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl-1H-indole-2-carboxamide; VU0090157, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate; VU0029767, (E)-2-(4-ethoxyphenylamino)-N -((2-hydroxynaphthalen-1-yl) methylene) acetohydrazide; BQCA, benzyl quinolone carboxylic acid; VU10010, 3-amino-N-(4-chlorobenzyl)-4,6-dimethylthieno[2,3-b] pyridine-2carboxamide 2,2,2-trifluoroacetate; CP55940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; LY2033298, 3-amino-5-chloro-N-cyclopropyl-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxamide; ABP-280, 280-kDa actin-binding protein. 0022-3565/09/3312-340–348 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 2 U.S. Government work not protected by U.S. copyright 156380/3523426 JPET 331:340–348, 2009 Printed in U.S.A. 340 at A PE T Jornals on A ril 3, 2017 jpet.asjournals.org D ow nladed from can preprogram cells to respond in certain ways to external stimuli (Maudsley et al., 2004). GPCRs have evolved to allow cellular systems to sense their environment through selective recognition of almost every type of agent, e.g., photons, odorants, lipids, amino acids, carbohydrates, and complex polypeptides. Because of this unparalleled flexibility and their involvement in most of the physiological processes in an organism, it is not surprising that GPCRs have proven to be effective pharmacological drug targets. Appreciation of the functional complexity of GPCR biology in recent years has increased exponentially. It is now accepted that GPCRs interact with many proteins to exert their full range of activities (Brady and Limbird, 2002: Maudsley et al., 2004, 2007) and that, as with other transmembrane receptors (e.g., ligand-gated ion channels), they are subject to modulation by ligands that can act independently or cooperatively with the endogenous cognate ligands. This latter aspect of GPCR signaling, i.e., allosteric receptor modulation, will be the primary subject of this review as this capacity to synergize with the endogenous agents and their more subtle mode of activity makes allosteric modulators (AMs) prototypes for the next generation of therapeutics to treat complex disorders that affect multiple aspects of peripheral and central nervous tissue function (Christopoulos, 2002; Leach et al., 2007; Lewis et al., 2008; Conn et al., 2009). Orthosteric and Allosteric Modulation of Heptahelical GPCRs Modern classification of heptahelical GPCRs subdivides the superfamily into five major functional groups—glutamate receptors, rhodopsin-like receptors, adhesion family receptors, frizzled/taste receptors, and secretin-like receptors (Schiöth and Fredriksson, 2005)—that collectively mediate the cellular sensation of the most environmentally derived and endogenous somatic compounds. The rhodopsin-like class of GPCRs includes some of the most studied proteins in nature. Many of the GPCRs in this class have been subjected to extensive structure-function analysis by site-directed mutagenesis. Most rhodopsin-like GPCRs possess a distinctive orthosteric binding site (i.e., domain involved in docking of the endogenous ligand with the receptor), either deep within the helical bundle for small ligands (e.g., biogenic amines) or superficially across the extracellular loops and surface helical regions for larger ligands (e.g., small neuropeptides). This orthosteric binding site facilitates high-affinity ligand binding and allows transduction of the stimulus to the interior of the cell. It has been shown for most endogenous ligands that binding to this site initiates the majority of signaling activity associated with receptor-ligand engagement. Orthosteric ligand GPCR activation classically transmits a signal, mediated by conformational rearrangement, across the plasma membrane to the intracellular domains of the receptor. By contrast, AMs do not directly engage the orthosteric site. The binding of an AM may cause a conformational change in the receptor protein that is transmitted to the orthosteric site (and vice versa), in essence creating a “new” GPCR with its own set of binding and functional properties. In addition, AMs may engender collateral efficacy by biasing the stimulus, thus leading to signaling-pathway-selective allosteric modulation (either enhancement or blockade). In the context of pharmaceutical development, AMs are generally thought of as exogenous compounds, often small molecules that bind a region of the receptor that is distant from the native orthosteric site. However, it is important to consider that GPCRs interact with numerous intracellular proteins (e.g., heterotrimeric G proteins) that also affect receptor conformation. Thus, in the broad context, allosteric modulation of GPCR function can arise from the association of accessory proteins with the internal face of the receptor (Maudsley et al., 2005) or through AM interaction with intracellular binding sites (Espinoza-Fonseca and Trujillo-Ferrara, 2006). To understand the role of AMs in controlling GPCR responses to ligand-induced conformational changes, we shall first consider dynamic models of GPCR function. Modeling Allosteric Modulation In classical dynamic models of GPCR function, the receptor transmits the orthosteric ligand signal by functioning as a ligand-activated guanine nucleotide exchange factor for juxtamembrane heterotrimeric G proteins. G protein activation is initiated through ligand-driven changes in the tertiary structure of the transmembrane heptahelical receptor core (Ballesteros and Palczewski, 2001; Shapiro et al., 2002). These conformational changes are transmitted to the intracellular transmembrane loops and carboxyl terminus and alter the ability of the receptor to catalyze the rapid exchange of GDP for GTP on the heterotrimeric G protein -subunit. The GTP-bound -subunit then can stimulate its cognate downstream effectors, e.g., phospholipase C or adenylate cyclase, conveying information about the presence of the stimulus in the extracellular environment. In this basic conceptualization, the GPCR functions as a switch, existing in either an “off” or “on” state. Evidence that GPCR behavior is more complex originated with the finding that -adrenergic receptors exhibit two affinity states for agonists, the relative proportions of which are modulated by the presence of guanine nucleotides (DeLean et al., 1980). The model advanced to explain these phenomena predicted that, in the presence of GDP, agonist binding promotes the formation of a long-lived ternary complex among agonist (H), GPCR (R), and heterotrimeric G protein (G) that exhibits high agonist binding affinity. In the absence of the G protein or when the presence of GTP allows for receptor-catalyzed G protein activation, the H-R-G complex is dissociated, and the receptor resides in a low-affinity (H-R) state. However, even this simplistic model accommodates a wide variety of orthosteric effects. Ligands can act as positive agonists (stimulating G protein turnover), inverse agonists (reducing constitutive G protein activation by the unliganded receptor), partial agonists (exhibiting lower intrinsic efficacy than a full agonist), or classical antagonists (binding the orthosteric site without G protein activation). Along with the increasing complexity of orthosteric ligandreceptor interactions, the past decade has witnessed an increase in the number of potential therapeutic ligands that target GPCRs by binding to allosteric sites on the receptor. AMs may increase or decrease the ability of the orthosteric ligand to interact with the receptor and/or modulate its ability to stabilize the active conformation of the receptor. Although both modulatory processes may occur simultaneously, the most commonly observed AM effect is modulation of orthosteric ligand affinity. As with the ternary Allosteric Pharmacological Therapy for Complex Disorders 341 at A PE T Jornals on A ril 3, 2017 jpet.asjournals.org D ow nladed from complex model for orthosteric ligand-receptor interactions, models have been generated for AM interactions (May et al., 2004). One model designed to quantify AM activity is described as the allosteric ternary complex model (ATCM; Fig. 1A). The ATCM is the simplest mass-action scheme applied to allosteric interactions, and its properties at equilibrium have been used to derive quantitative models for AM activity simulation (Stockton et al., 1983; Ehlert, 1988; Christopoulos and Kenakin, 2002). The ATCM can be used to quantify AM activity in terms of ligand affinity for the unoccupied receptor and its cooperativity factor ( ). The cooperativity factor is a thermodynamic measure of the strength and direction of the allosteric change in affinity for one site when the other is occupied. Allosteric modulators can be broadly grouped as either positive AMs (PAMs, 1) or as negative AMs (NAMs, 1). For example, the binding of the orthosteric antagonist N-methylscopolamine (NMS) to the M2-muscarinic acetylcholine receptor (mAChR) is allosterically enhanced by alcuronium (Avlani et al., 2004) but is allosterically inhibited by gallamine, even though both AMs bind to a common allosteric site on the receptor (Lanzafame et al., 1997). Beyond effects on orthosteric ligand affinity, AMs can produce changes in the intrinsic efficacy of the receptor-orthosteric ligand complex. This property is exemplified by a series of modulators of cannabinoid CB1 receptors. The allosteric modulator Org27569 enhances the binding of the orthosteric agonist CP55940 at mouse CB1 receptors but significantly reduces the efficacy of the orthosteric agonist WIN552122 for inhibition of electrically evoked contractions in a mouse vas deferens preparation and the efficacy of CP55940 at human CB1 receptors in a reporter-gene assay (Price et al., 2005). To accommodate these effects, an allosteric two-state model has been proposed that provides an additional cooperativity factor governing the transition of the receptor between a resting (R) and an activated (R*) state in the presence of an allosteric ligand, the allosteric cubic ternary complex (CTC) model (Fig. 1B). Although most allosteric GPCR modulators are pharmacologically quiescent in the absence of an orthosteric ligand, it has been noted that some allosteric ligands, termed “agoallosteric” modulators, act as agonists in their own right (Knudsen et al., 2006). Such “allosteric agonists” further expand the number of possible receptor-ligand interactions because they have the potential to modulate orthosteric ligand pharmacology in addition to perturbing cellular signaling in their own right. Two mAChR ligands suggested to act this way are the functionally selective partial agonists, McNA-343 and AC-42. In addition to engendering partial agonist effects, they produce incomplete inhibition of the binding of the orthosteric antagonist NMS when present at saturating concentrations at rat M2 (McN-A-343) and human M1 (AC42) mAChRs while retarding NMS dissociation (Langmead et al., 2006; Valant et al., 2008). It is noteworthy that it is also possible for a ligand to bind to an allosteric site without altering orthosteric regulation of receptor function, in effect acting as a “neutral” antagonist at the allosteric site. Allosteric Receptor Modulation by GPCR Accessory Proteins GPCRs are naturally allosteric proteins that interact with numerous other proteins that alter their ligand-binding affinity or signaling properties. In effect, heterotrimeric G proteins are AMs, in that they alter ligand affinity by contacting the receptor at a topographically distant site from the orthosteric binding site. Numerous other proteins interact with the intracellular face or transmembrane regions of GPCRs. GPCR-interacting proteins include kinases (e.g., G proteincoupled receptor kinases and protein kinase A) (Fraser et al., 2000), arrestins (Pfister et al., 1985), the 4.1 family of cytoskeletal proteins (e.g., ABP-280) (Li et al., 2000), amyloid precursor like protein 1 (APLP1) (Weber et al., 2006), receptor activity-modifying proteins/calcitonin gene-related peptide-receptor component protein (McLatchie et al., 1998; Evans et al., 2000), and PDZ-domain containing proteins (Hall et al., 1998). These interacting proteins influence GPCR signaling by regulating downstream effectors and by participating in scaffolding, endocytosis, trafficking, or recy-