Allosteric modulation
Technology combination to address GPCR allosteric modulator drug-discovery pitfalls

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Allosteric modulators (AMs) represent a novel paradigm to therapeutically target G-protein-coupled receptors (GPCRs). However, their identification and characterization using standard functional assays remain elusive due to the ‘context-dependent phenomena’. Novel technological approaches such as combining a Fluorescence Resonance Energy Transfer (FRET)-based library filtering with a Bioluminescence Resonance Energy Transfer (BRET)-based multiparametric compound profiling can circumvent the limitations of current GPCR screening processes and simplify the discovery of biased AMs.

Introduction

The family of G-protein-coupled receptors (GPCRs) has proven to be highly valuable source of therapeutic targets with nearly 30% of marketed drugs acting through this receptor superfamily [1]. However, for numerous GPCRs the development of selective and druggable ligands remains challenging due to highly evolutionary-conserved orthosteric binding sites. To overcome this obstacle, a novel strategy consisting of targeting allosteric modulator (AM) sites has emerged. A second emerging concept, referred to as functional selectivity and based on the ability of individual GPCRs to elicit multiple cellular responses, can lead to the development of AMs biased for a specific signaling pathway responsible only for beneficial effects. Current technologies used for AM high-throughput screening (HTS) and signaling characterization are mainly based on the use of functional assays whose major limitations are the ‘context-dependent phenomena’. This article discusses the utility of combining novel technological approaches to efficiently discover AMs and thoroughly characterize them for their ability to activate/inhibit specific signaling pathways.

Section snippets

Allosteric modulators

AMs are a novel class of small molecule ligands that can be used to therapeutically modulate GPCRs [2, 3]. They exert a variety of pharmacological actions (Glossary box) and present significant advantages in terms of drug discovery and development. For instance, (1) AMs give access to more druggable chemistries for peptide, lipid or family C GPCRs; (2) subtype selectivity is easier to achieve with AMs due to a lower evolutionary pressure of their binding sites [4]; (3) PAMs and NAMs exert

Functional selectivity and ligand-biased signaling

A recent paradigm shift that will increasingly impact GPCR drug discovery stems from the observation that rather than functioning as simple switches, turning preselected linear signaling cascades ‘on’ or ‘off’, GPCRs are signaling hubs that can regulate alternative subsets of signaling modes depending on the receptor conformations stabilized by specific ligands. In addition to ‘selecting’ particular receptor/G-protein combinations which differ in their ability to stimulate particular effectors,

GPCR AM identification and characterization

Radioligand binding assays are rarely used for AM identification. Indeed, GPCR radioactive probes are most of the time orthosteric ligands and their competitive displacement results almost exclusively in the selection of molecules interacting with the orthosteric binding site. A few AMs have however been identified with this technique such as metabotropic glutamate receptor 2 (mGluR2) NAMs discovered by Woltering et al. [17], but their identification seems series-dependent as alternative mGluR2

Library filtering with FRET-based DTect-All™

Reducing the number of compounds to be functionally characterized can be achieved by a technology independent of the signaling pathways such as DTect-All™. This drug discovery platform consists of a Fluorescence Resonance Energy Transfer (FRET)-based binding assay, where the energy donor, the Green Fluorescent Protein (GFP), is fused to the amino terminal (Nter) part of the targeted GPCR, and the energy acceptor is a fluorophore (or a chromophore) linked to a probe binding the receptor [28, 29]

Multiparametric profiling with BRET-based biosensors

In the context of drug discovery programs, the novel concept of ‘ligand-based signaling’ raises the question of what are the most pertinent signaling pathways to be targeted for therapeutic efficacy or avoided to prevent undesirable effects. In recent years, observations made in animal models suggest that harnessing GPCR functional selectivity may represent a promising avenue in drug discovery [33, 34, 35]. These considerations raise important challenges regarding the development of the best

Combining technologies to discover biased allosteric modulators

It is obvious that DTect-All™ and BRET-based biosensor technologies can complement each other as shown in Fig. 6. In this integrative HTS strategy, the first step consists of the screening of a diverse compound library against a DTect-All™ assay to identify a list of binders. These binders are then tested against a second FRET-based binding assay to focus only on those specific binders interacting with the GPCR of interest. This counter-screen, performed using a second assay developed for a

Conflict of interest statement

S.S. and P.N. are employees of Domain Therapeutics.

Glossary

Orthosteric binding site
binding site of the endogenous ligand.
Allosteric modulator binding site(s)
binding site(s) topologically distinct from the orthosteric binding site.
Positive allosteric modulator (PAM)
ligand binding an allosteric binding site and enhancing orthosteric ligand affinity, efficacy, or both. A PAM, in absence of the endogenous ligand, is devoid of activity.
Negative allosteric modulator (NAM)
ligand binding an allosteric binding site and decreasing orthosteric ligand affinity,

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