Identifying ligands at orphan GPCRs: current status using structure-based approaches

Abstract

GPCRs are the most successful pharmaceutical targets in history. Nevertheless, the pharmacology of many GPCRs remains inaccessible as their endogenous or exogenous modulators have not been discovered. Tools that explore the physiological functions and pharmacological potential of these 'orphan' GPCRs, whether they are endogenous and/or surrogate ligands, are therefore of paramount importance. Rates of receptor deorphanization determined by traditional reverse pharmacology methods have slowed, indicating a need for the development of more sophisticated and efficient ligand screening approaches. Here, we discuss the use of structure-based ligand discovery approaches to identify small molecule modulators for exploring the function of orphan GPCRs. These studies have been buoyed by the growing number of GPCR crystal structures solved in the past decade, providing a broad range of template structures for homology modelling of orphans. This review discusses the methods used to establish the appropriate signalling assays to test orphan receptor activity and provides current examples of structure-based methods used to identify ligands of orphan GPCRs. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Figure 1

A putative pipeline for unlocking the pharmacology of orphan GPCRs by combining targeted HTS and SBDD. Combining both in vitro screening and hit validation with VLS at ligand‐optimized homology models is one rational approach to unlocking orphan GPCRs. First, an appropriate signalling assay must be identified, and then, a small library is screened for low affinity hits. This surrogate screen informs the composition of a ligand training set that can be used for the iterative ligand‐guided optimization of a homology model. Ideally, a subset of active and inactive ligands is set aside for naïve challenge of the best model to validate performance. VLS is then performed and hits validated in vitro by at least one assay readout. Additional site‐directed mutagenesis data and structure–activity relationships can further feed into repeated iterations of homology model optimization and VLS.

Figure 2

Assays for the identification of G protein signalling pathways using the constitutive activity of an orphan GPCR. (A) Yeast GPCR signalling pathway. The yeast pheromone receptor, Ste2, couples to the endogenous yeast heterotrimeric G proteins (Gα = Gpa1) to drive Gβγ‐mediated (MAPK) pathway activation and Ste12‐mediated transcription via the pheromone‐response element (PRE). MEK: MAPK kinase; MEKK: MEK kinase. (B) Modified yeast G protein signalling assay: chimeric versions of Gpa1 bearing the final five amino acids of each of the human Gα proteins have been introduced into yeast strains lacking each of the yeast GPCR, Ste2, the regulator of G protein signalling, Sst2, and the cell cycle arrest protein, Far1. Upon transformation with the orphan GPCR of interest, constitutive coupling to a specific G protein chimera drives the MAPK module to stimulate transcription of HIS3 (facilitates selection and expansion in HIS3‐deficient media) and the lacZ reporter via FUS1. (C) Mammalian reporter assays for transcription factors downstream of each of the major G protein families. Constitutive coupling via Gα_s leads to stimulation of AC and generation of cAMP, which stimulates transcription of luciferase that lies downstream of a cAMP response element (CRE). Likewise, Gα_i activity is reported via the serum response element (SRE) upon Gβγ signalling to ERK1/2 MAPK; Gα_q via nuclear factor of activated T‐cells (NFAT) and Gα₁₂ via serum response factor (SRF) response element.

Figure 3

Orphan GPCRs ranked according to their closest crystal structure template. Each orphan GPCR (left) was ranked according to the crystal structure with which it shares the highest TM sequence identity, independent to GPCR subgroup (expressed as a percentage; listed in Table 1). The minimum identity required for VLS at a homology model in the absence of further refinement is 35% (dotted line). The homologous crystal structure is shown in the bottom right‐hand corner of each box. Red: α subgroup of rhodopsin‐like GPCRs; orange: β‐subgroup; yellow: γ‐subgroup; green: δ‐subgroup; blue: unclassified.

Figure 4

Structures of GW9508, TAK‐975 and novel FFA1 agonists identified by virtual screening.