G protein-coupled receptors (GPCRs) mediate responses to a wide range of stimuli, from small molecule neurotransmitters and hormones to peptides and photons of light. Their wide expression profile and roles in major physiological functions have made them prime targets for marketed drugs [1].

Most drugs targeting GPCRs, whether agonists or antagonists, interact with the same binding site as that for the endogenous ligand for the receptor—the "orthosteric" binding site. The location of this site varies across the receptor family: In Class A monoamine receptors, such as muscarinic acetylcholine or dopamine receptors, it is usually located within the transmembrane domain bundle [2]. For Class C receptors, such as the gamma amino butyric acid (GABAb) or metabotropic glutamate (mGlu) receptors, the orthosteric binding site is formed by the large N-terminal domain [3].

Historically, high-throughput screens (HTSs) for GPCRs were run using radioligand binding assays employing a radiolabeled version of the endogenous ligand or, more often, a synthetic competitive antagonist. Such an assay format inherently biased the resultant hits toward compounds that interacted with the same site as the radioligand. However, the advent of functional assays as the screening paradigm of choice in the past decade has seen an increase in the detection of compounds that interact with allosteric sites on GPCRs. Allosteric ligands (from the Greek allos, meaning other, and stereos, meaning shape) can bind to sites on GPCRs that are topographically distinct from the

GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions,

Edited by Annette Gilchrist

Copyright © 2010 John Wiley & Sons, Inc.

orthosteric site such that the receptor is able to accommodate two ligands simultaneously. Allosteric binding sites have been identified on many GPCRs, including adenosine [4], muscarinic acetylcholine [5], dopamine [6],chemokine [7], calcium-sensing receptors [8], and mGlu [9] receptors. Upon binding, allosteric ligands are able to alter the conformation of the orthosteric site to modify orthosteric agonist activity as described below.

Allosteric modulators possess a number of advantages as potential drugs. By virtue of being the binding site for the endogenous agonist or transmitter, orthosteric binding sites are likely to be subject to evolutionary pressure to remain conserved across receptor families; however, allosteric binding sites are unlikely to be subject to such pressure and are likely to display divergence within a receptor family. This offers the prospect of selectively targeting individual receptor subtypes where it has not yet been possible to design orthosteric drugs of sufficient selectivity to avoid unwanted side effects, for example, subtype-selective drugs for muscarinic acetylcholine receptors.

As will be seen below, allosteric modulators display an inherent saturability to their effects, which makes them potentially safer as drugs than agonist molecules. Finally, by virtue of their subtly enhancing or reducing the activity of the endogenous ligand at a GPCR, allosteric modulators are capable of maintaining the temporal and spatial resolution of receptor signaling [10]. This is in contrast to direct agonist molecules, which will cause blanket activation of receptors so long as the drug is exposed to the receptor population.

In the late 1990s, the pharmaceutical industry switched from using radioli-gand binding screening assays to functional assays as a result of the availability of high-throughput generic signaling assays. This switch enabled allosteric ligands to be more routinely identified; any compound that perturbed the action of an agonist (or activated the receptor in its own right) could be detected, irrespective of the location of its binding site on the receptor. In the past 5 years, the pharmaceutical industry has seen the approval for market of its first two allosteric modulators of GPCRs: Cinacalcet, a positive allosteric modulator of the calcium-sensing receptor, was approved in 2004 for hyperparathyroidism [11] , and in 2007, maraviroc, a negative allosteric modulator of the chemokine receptor CCR5, was approved as an HIV entry inhibitor [12]. Many companies are now actively pursuing allosteric modulators of GPCRs as novel therapies for a whole range of disease indications.

A key part of the drug discovery process is the identification of compounds, which display activity at the receptor of interest. This typically occurs over a number of stages. In a HTS, compounds are typically assessed at the receptor of interest at a single concentration ("single shot"). Subsequently, at the stage of hit deconvolution or lead optimization, fewer compounds are tested in full concentration-response curve format. Finally, a small number of late -stage compounds may be assessed in more detailed mechanism of action studies. This chapter seeks to critically discuss the methods used to screen for and characterize allosteric modulators of GPCRs—many of the issues (such as choice of assay format) apply to all stages of the screening process, whereas others (such as novel analytical methods) are primarily aimed at later-stage characterization of compounds.

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