Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

Intracellular Allosteric Antagonist of the Olfactory Receptor OR51E2.

Olfactory receptors are members of class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on the discovery of novel ligands for the olfactory receptor family 51 subfamily E2 (OR51E2). We performed an artificial intelligence-based virtual drug screen of a ∼2.2 million small molecule library. Cell-based functional assay identified compound 80 (C80) as an antagonist and inverse agonist, and detailed pharmacological analysis revealed C80 acts as a negative allosteric modulator by significantly decreasing the agonist efficacy, while having a minimal effect on receptor affinity for agonist. C80 binds to an allosteric binding site formed by a network of nine residues localized in the intracellular parts of transmembrane domains 3, 5, 6, 7, and H8, which also partially overlaps with a G protein binding site. Mutational experiments of residues involved in C80 binding uncovered the significance of the C2406.37 position in blocking the activation-related conformational change and keeping the receptor in the inactive form. Our study provides a mechanistic understanding of the negative allosteric action of C80 on agonist-ctivated OR51E2. We believe the identification of the antagonist of OR51E2 will enable a multitude of studies aiming to determine the functional role of this receptor in specific biologic processes. SIGNIFICANCE STATEMENT: OR51E2 has been implicated in various biological processes, and its antagonists that can effectively modulate its activity have therapeutic potential. Here we report the discovery of a negative allosteric modulator of OR51E2 and provide a mechanistic understanding of its action. We demonstrate that this modulator has an inhibitory effect on the efficacy of the agonist for the receptor and reveal a network of nine residues that constitute its binding pocket, which also partially overlaps with the G protein binding site.

Allostery: The Good, the Bad, and the Ugly.

With the advent of functional screening, more allosteric molecules are being discovered and developed as possible therapeutic entities. Allosteric proteins are unique because of two specific properties: 1) separate binding sites for allosteric modulators and guests and 2) mandatory alteration of receptor conformation upon binding of allosteric modulators. For G protein-coupled receptors, these properties produce many beneficial effects on pharmacologic systems that are described here. Allosteric discovery campaigns also bring with them added considerations that must be addressed for the endeavor to be successful, and these are described herein as well. SIGNIFICANCE STATEMENT: Recent years have seen the increasing presence of allosteric molecules as possible therapeutic drug candidates. The scientific procedures to characterize these are unique and require special techniques, so it is imperative that scientists understand the new concepts involved in allosteric function. This review examines the reasons why allosteric molecules should be considered as new drug entities and the techniques required to optimize the discovery process for allosteric molecules.

A Pharmacological perspective on the temporal properties of sweeteners.

Several non-caloric sweeteners exhibit a delay in sweetness onset and a sweetness linger after sampling. These temporal properties are thought to be the result of non-specific interactions with cell membranes and proteins in the oral cavity. Data and analysis presented in this report also support the potential involvement of receptor affinity and binding kinetics to this phenomenon. In general, affected sweeteners exhibit distinctly higher binding affinity compared to carbohydrate sweeteners, which do not have temporal issues. In addition, binding kinetic simulations illustrate much slower receptor binding association and dissociation kinetics for a set of non-caloric sweeteners presenting temporal issues, in comparison to carbohydrate sweeteners. So, the higher affinity of some non-caloric sweeteners, dictating lower use levels, and affecting binding kinetics, could contribute to their delay and linger in sweetness perception. Simple pharmacology principles could explain, at least in part, some of the temporal issues of sweeteners.

Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.

Biased Receptor Signaling in Drug Discovery.

A great deal of experimental evidence suggests that ligands can stabilize different receptor active states that go on to interact with cellular signaling proteins to form a range of different complexes in varying quantities. In pleiotropically linked receptor systems, this leads to selective activation of some signaling pathways at the expense of others (biased signaling). This article summarizes the current knowledge about the complex components of receptor systems, the evidence that biased signaling is used in natural physiology to fine-tune signaling, and the current thoughts on how this mechanism may be applied to the design of better drugs. Although this is a fairly newly discovered phenomenon, theoretical and experimental data suggest that it is a ubiquitous behavior of ligands and receptors and to be expected. Biased signaling is simple to detect in vitro and there are numerous methods to quantify the effect with scales that can be used to optimize this activity in structure-activity medicinal chemistry studies. At present, the major hurdle in the application of this mechanism to therapeutics is the translation of in vitro bias to in vivo effect; this is because of the numerous factors that can modify measures of bias in natural physiologic systems. In spite of this, biased signaling still has the potential to justify revisiting of receptor targets previously thought to be intractable and also furnishes the means to pursue targets previously thought to be forbidden due to deleterious physiology (as these may be eliminated through biased signaling).

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Preclinical Testing of Nalfurafine as an Opioid-sparing Adjuvant that Potentiates Analgesia by the Mu Opioid Receptor-targeting Agonist Morphine.

Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria). Recent data suggest that some functionally selective, or biased, KOR-targeting agonists might retain the therapeutic effects of KOR activation without inducing undesirable side effects. Nalfurafine, used safely in Japan since 2009 for uremic pruritus, is one such functionally selective KOR-targeting agonist. Here, we quantify the bias of nalfurafine and several other KOR agonists relative to an unbiased reference standard (U50,488) and show that nalfurafine and EOM-salvinorin-B demonstrate marked G protein-signaling bias. While nalfurafine (0.015 mg/kg) and EOM-salvinorin-B (1 mg/kg) produced spinal antinociception equivalent to 5 mg/kg U50,488, only nalfurafine significantly enhanced the supraspinal analgesic effect of 5 mg/kg morphine. In addition, 0.015 mg/kg nalfurafine did not produce significant conditioned place aversion, yet retained the ability to reduce morphine-induced conditioned place preference in C57BL/6J mice. Nalfurafine and EOM-salvinorin-B each produced robust inhibition of both spontaneous and morphine-stimulated locomotor behavior, suggesting a persistence of sedative effects when coadministered with morphine. Taken together, these findings suggest that nalfurafine produces analgesic augmentation, while also reducing opioid-induced reward with less risk of dysphoria. Thus, adjuvant administration of G protein-biased KOR agonists like nalfurafine may be beneficial in enhancing the therapeutic potential of MOR-targeting analgesics, such as morphine.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

G-Protein/Receptor inhibitors as blockers of receptor signaling.

This paper describes the behavior of binding and functional receptor systems where an antagonist of the receptor/G protein binding reaction is added as a blocker of agonist-induced receptor function. For agonist radioligands, the reduction of G protein receptor interaction leads to a possible change in the binding affinity of the agonist radioligand to the receptor. Reciprocally, the allosteric cooperativity between the agonist and the G protein binding site antagonist (quantified by the factor γB) affects the potency of the G protein antagonist modulator; this model presents the various profiles that would be expected for modulators that reduce (γB = 0.01), have no effect on (γB = 1) and increase (γB = 100) the affinity of the agonist for the receptor. It will be seen that modulators that increase the affinity of the receptor for the agonist are the most potent antagonists and may attain a profile of some special negative allosteric modulators referred to as PAM antagonists. In all cases, these modulators will be inverse agonists of constitutive receptor activity. This model presents a strategy for the discovery of PAM antagonists for therapeutic blockade of physiological signaling.

Theoretical Aspects of GPCR-Ligand Complex Pharmacology.

Over the past 50 years in pharmacology, an understanding of seven transmembrane (7TMR) function has been gained from the comparison of experimental data to receptor models. These models have been constructed from building blocks composed of systems consisting of series and parallel mass action binding reactions. Basic functions such as the the isomerization of receptors upon ligand binding, the sequential binding of receptors to membrane coupling proteins, and the selection of multiple receptor conformations have been combined in various ways to build receptor systems such as the ternary complex, extended ternary complex, and cubic ternary complex models for 7TMR function. Separately, the Black/Leff operational model has furnished an extremely valuable method of quantifying drug agonism. In the past few years, incorporation of the basic allosteric nature of 7TMRs has led to additional useful models of functional receptor allosteric mechanisms; these models yield valuable methods for quantifying allosteric effects. Finally, molecular dynamics has provided yet another new set of models describing the probability of formation of multiple receptor states; these radically new models are extremely useful in the prediction of functionally selective drug effects.

Emergent Concepts of Receptor Pharmacology.

Pharmacology, the chemical control of physiology, emerged as an offshoot of physiology when the physiologists using chemicals to probe physiological systems became more interested in the probes than the systems. Pharmacologists were always, and in many ways still are, bound to study drugs in systems they do not fully understand. Under these circumstances, null methods were the main ways in which conclusions about biologically active molecules were made. However, as understanding of the basic mechanisms of cellular function and biochemical systems were elucidated, so too did the understanding of how drugs affected these systems. Over the past 20 years, new ideas have emerged in the field that have completely changed and revitalized it; these are described herein. It will be seen how null methods in isolated tissues gave way to, first biochemical radioligand binding studies, and then to a wide array of functional assay technologies that can measure the effects of molecules on drug targets. In addition, the introduction of molecular dynamics, the appreciation of the allosteric nature of receptors, protein X-ray crystal structures, genetic manipulations in the form of knock-out and knock-in systems and Designer Receptors Exclusively Activated by Designer Drugs have revolutionized pharmacology.

Is the Quest for Signaling Bias Worth the Effort?

The question of whether signaling bias is a viable discovery strategy for drug therapy is discussed as a value proposition. On the positive side, bias is easily identified and quantified in simple in vitro functional assays with little resource expenditure. However, there are valid pharmacological reasons why these in vitro bias numbers may not accurately translate to in vivo therapeutic systems making the expectation of direct correspondence of in vitro bias to in vivo systems a problematic process. Presently, in vitro bias is used simply as a means to identify unique molecules to be advanced to more complex therapeutic assays but from this standpoint alone, the value proposition lies far to the positive. However, pharmacological attention needs to be given to the translational gap to reduce inevitable and costly attrition in biased molecule progression.

Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors.

This paper discusses the process of determining the activity of candidate molecules targeting Gq-protein activation through G-protein-coupled receptors for possible therapeutic application with two functional assays; calcium release and inositol phosphate metabolism [inositol monophosphate (IP1)]. While both are suitable for detecting ligand activity (screening), differences are seen when these assays are used to quantitatively measure ligand parameters for therapeutic activity. Specifically, responses for Gq-related pathways present different and dissimulating patterns depending on the functional assay used to assess them. To investigate the impact of functional assays on the accuracy of compound pharmacological profiles, five exemplar molecules [partial agonist, antagonist, inverse agonist, positive allosteric modulator (PAM) agonist, and positive ß-PAM] targeting either muscarinic M1 or ghrelin receptors were tested using two functional assays (calcium release and IP1) and the results were compared with theoretical pharmacological models. The IP1 assay is an equilibrium assay that is able to determine the correct (i.e., internally consistent) pharmacological profiles of all tested compounds. In contrast, the nonequilibrium nature of calcium assays yields misleading classification of most of the tested compounds. Our study suggests that the use of an equilibrium assay, such as IP1, is mandatory for the optimal use of pharmacological models that can both identify mechanisms of action and also convert descriptive-to-predictive data for therapeutic systems. Such assays allow the identification of consistent and simple scales of activity that can guide medicinal chemistry in lead optimization of candidate molecules for therapeutic use.

N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and ß-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

Allosteric Modulation: An Alternate Approach Targeting the Cannabinoid CB1 Receptor.

The cannabinoid CB1 receptor is a G protein coupled receptor and plays an important role in many biological processes and physiological functions. A variety of CB1 receptor agonists and antagonists, including endocannabinoids, phytocannabinoids, and synthetic cannabinoids, have been discovered or developed over the past 20 years. In 2005, it was discovered that the CB1 receptor contains allosteric site(s) that can be recognized by small molecules or allosteric modulators. A number of CB1 receptor allosteric modulators, both positive and negative, have since been reported and importantly, they display pharmacological characteristics that are distinct from those of orthosteric agonists and antagonists. Given the psychoactive effects commonly associated with CB1 receptor agonists and antagonists/inverse agonists, allosteric modulation may offer an alternate approach to attain potential therapeutic benefits while avoiding inherent side effects of orthosteric ligands. This review details the complex pharmacological profiles of these allosteric modulators, their structure-activity relationships, and efforts in elucidating binding modes and mechanisms of actions of reported CB1 allosteric modulators. The ultimate development of CB1 receptor allosteric ligands could potentially lead to improved therapies for CB1-mediated neurological disorders.

A Scale of Agonism and Allosteric Modulation for Assessment of Selectivity, Bias, and Receptor Mutation.

An index of agonism is described that can be used to quantify agonist receptor selectivity, bias, cell-based agonism, and the effects of receptor mutation on signaling. The parameter is derived from agonist concentration-response curves and comprises the maximal response to the agonist (max) and the EC50 in the form of Δlog(max/EC50). This parameter is derived from equations describing agonists as positive allosteric facilitators of receptor-signaling protein interaction. A similar index is also derived to quantify the potentiating effects of positive allosteric modulators, which can be used to quantify in situ positive allosteric modulator activity in vivo. These indices lend themselves to statistical analysis and are system-independent in that the effects of the system processing of agonist response and differences in assay sensitivity and receptor expression are cancelled. The various applications of the Δlog(max/EC50) scale are described for each pharmacologic application.

A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.

Detailed Characterization of the In Vitro Pharmacological and Pharmacokinetic Properties of N-(2-Hydroxybenzyl)-2,5-Dimethoxy-4-Cyanophenylethylamine (25CN-NBOH), a Highly Selective and Brain-Penetrant 5-HT2A Receptor Agonist.

Therapeutic interest in augmentation of 5-hydroxytryptamine2A (5-HT2A) receptor signaling has been renewed by the effectiveness of psychedelic drugs in the treatment of various psychiatric conditions. In this study, we have further characterized the pharmacological properties of the recently developed 5-HT2 receptor agonist N-2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH) and three structural analogs at recombinant 5-HT2A, 5-HT2B, and 5-HT2C receptors and investigated the pharmacokinetic properties of the compound. 25CN-NBOH displayed robust 5-HT2A selectivity in [3H]ketanserin/[3H]mesulergine, [3H]lysergic acid diethylamide and [3H]Cimbi-36 binding assays (Ki2C/Ki2A ratio range of 52-81; Ki2B/Ki2A ratio of 37). Moreover, in inositol phosphate and intracellular Ca2+ mobilization assays 25CN-NBOH exhibited 30- to 180-fold 5-HT2A/5-HT2C selectivities and 54-fold 5-HT2A/5-HT2B selectivity as measured by Δlog(Rmax/EC50) values. In an off-target screening 25CN-NBOH (10 µM) displayed either substantially weaker activity or inactivity at a plethora of other receptors, transporters, and kinases. In a toxicological screening, 25CN-NBOH (100 µM) displayed a benign acute cellular toxicological profile. 25CN-NBOH displayed high in vitro permeability (Papp = 29 × 10-6 cm/s) and low P-glycoprotein-mediated efflux in a conventional model of cellular transport barriers. In vivo, administration of 25CN-NBOH (3 mg/kg, s.c.) in C57BL/6 mice mice produced plasma and brain concentrations of the free (unbound) compound of ∼200 nM within 15 minutes, further supporting that 25CN-NBOH rapidly penetrates the blood-brain barrier and is not subjected to significant efflux. In conclusion, 25CN-NBOH appears to be a superior selective and brain-penetrant 5-HT2A receptor agonist compared with (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), and thus we propose that the compound could be a valuable tool for future investigations of physiologic functions mediated by this receptor.

International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.