GPCR Binding Technologies: An Overview.

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes and thus have long been attractive drug targets. With the crystal structures of more than 50 different human GPCRs determined over the past decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-electron microscopy (cryo-EM) structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects in which crystal or cryo-EM structures have been complemented with NMR investigations and discuss the impact of this integrative approach on GPCR biology and drug discovery.

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.

Nuclear Receptors (NRs) are multi-domain proteins, whose natural regulation occurs via ligands for a classical, orthosteric, binding pocket and via intra- and inter-domain allosteric mechanisms. Allosteric modulation of NRs via synthetic small molecules has recently emerged as an interesting entry to address the need for small molecules targeting NRs in pathology, via novel modes of action and with beneficial profiles. In this review the general concept of allosteric modulation in drug discovery is first discussed, serving as a background and inspiration for NRs. Subsequently, the review focuses on examples of small molecules that allosterically modulate NRs, with a strong focus on structural information and the ligand binding domain. Recently discovered nanomolar potent allosteric site NR modulators are catapulting allosteric targeting of NRs to the center of attention. The obtained insights serve as a basis for recommendations for the next steps to take in allosteric small molecular targeting of NRs.

Covalent allosteric modulation: An emerging strategy for GPCRs drug discovery.

G protein-coupled receptors (GPCRs) form one of the largest drug target families, reflecting their involvement in numerous pathophysiological processes. In this Review, we analyse drug discovery trends for the GPCR superfamily, covering compounds, targets and indications that have reached regulatory approval or that are being investigated in clinical trials. We find that there are 516 approved drugs targeting GPCRs, making up 36% of all approved drugs. These drugs act on 121 GPCR targets, one-third of all non-sensory GPCRs. Furthermore, 337 agents targeting 133 GPCRs, including 30 novel targets, are being investigated in clinical trials. Notably, 165 of these agents are approved drugs being tested for additional indications and novel agents are increasingly allosteric modulators and biologics. Remarkably, diabetes and obesity drugs targeting GPCRs had sales of nearly US $30 billion in 2023 and the numbers of clinical trials for GPCR modulators in the metabolic diseases, oncology and immunology areas are increasing strongly. Finally, we highlight the potential of untapped target-disease associations and pathway-biased signalling. Overall, this Review provides an up-to-date reference for the drugged and potentially druggable GPCRome to inform future GPCR drug discovery and development.

Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists

Allosteric modulators of human A2B adenosine receptor

The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which aequorin is a prototypic example. These biosensors have the advantage that they provide an intense luminescent signal in response to elevations in intracellular Ca2+. This exquisite sensitivity, the high signal-to-noise ratios, and the ability to target expression to discrete subcellular sites (in order to detect Ca2+ microdomains) have made photoproteins a principal choice in a wide range of GPCR drug discovery programs. Photoproteins are also finding increasing use in detecting activation of other molecular target classes such as ligand-gated ion channels and transporters. This review focuses upon the use of calcium photoproteins principally for use in GPCR drug discovery and HTS.

Deep learning (DL) methods have drastically advanced structure-based drug discovery by directly predicting protein structures from sequences. Recently, these methods have become increasingly accurate in predicting complexes formed by multiple protein chains. We evaluated these advancements to predict and accurately model the largest receptor family and its cognate peptide hormones. We benchmarked DL tools, including AlphaFold 2.3 (AF2), AlphaFold 3 (AF3), Chai-1, NeuralPLexer, RoseTTAFold-AllAtom, Peptriever, ESMFold, and D-SCRIPT, to predict interactions between G protein-coupled receptors (GPCRs) and their endogenous peptide ligands. Our results showed that structure-aware models outperformed language models in peptide binding classification, with the top-performing model achieving an area under the curve of 0.86 on a benchmark set of 124 ligands and 1240 decoys. Rescoring predicted structures on local interactions further improved the principal ligand discovery among decoy peptides, whereas DL-based approaches did not. We explored a competitive tournament approach for modeling multiple peptides simultaneously on a single GPCR, which accelerates the performance but reduces true-positive recovery. When evaluating the binding poses of 67 recent complexes, AF2 reproduced the correct binding modes in nearly all cases (94%), surpassing those of both AF3 and Chai-1. Confidence scores correlate with structural binding mode accuracy, which provides a guide for interpreting interface predictions. These results demonstrated that DL models can reliably rediscover peptide binders, aid peptide drug discovery, and guide the selection of optimal tools for GPCR-targeted therapies. To this end, we provided a practical guide for selecting the best models for specific applications and an independent benchmarking set for future model evaluation.

Deorphanization of the large group of G protein-coupled receptors (GPCRs) for which an endogenous activating ligand has not yet been identified (orphan GPCRs) has become increasingly difficult. A specialized technique that has been successfully applied to deorphanize some of these GPCRs involves two-electrode voltage-clamp recordings of currents through ion channels, which are activated by GPCRs heterologously expressed in Xenopus oocytes. The ion channels that couple to GPCR activation in Xenopus oocytes can be endogenous calcium-activated chloride channels (CaCCs) or heterologously expressed G protein-coupled inwardly rectifying potassium channels (GIRKs). We will describe a general approach for expression of GPCRs in Xenopus oocytes and characterization of these using electrophysiological recordings. We will focus on the detection of GPCR activation by recordings of currents through CaCCs that are activated by calcium release from the endoplasmic reticulum and thus the G(q) signaling pathway.

Subtleties in GPCR drug discovery: a medicinal chemistry perspective

The first structure-activity relationship study of oxytocin as a positive allosteric modulator for the µ opioid receptor

Differential Pharmacology and Binding of mGlu2 Receptor Allosteric Modulators

Releasing the technical ‘shackles’ on GPCR drug discovery: opportunities enabled by detergent-free polymer lipid particle (PoLiPa) purification

The M₁ muscarinic acetylcholine receptor (mAChR) is predominantly expressed in the brain where it plays a major role in mediating cognitive processes such as learning and memory. As a result, it has been implicated in diseases where such processes are impaired, such as Alzheimer’s disease and schizophrenia. Drug discovery efforts aimed at developing selective ligands for this receptor, both as therapeutics and as experimental tools, have largely failed as they focused on targeting the acetylcholine (ACh) binding site, which is identical in all five mAChR subtypes. The discovery of benzyl quinolone carboxylic acid (BQCA), the first positive allosteric modulator (PAM) with high selectivity for the M₁ mAChR, has lead to a renaissance in selective targeting of this receptor family. In chapter 2 we exploit the unique “two-state” pharmacology of BQCA to investigate allosteric modulation at a chemogenetically modified M1 mAChR, developed as an alternative means to achieve selective receptor targeting in vivo. This study demonstrates that such an approach may not be valid, as chemogenetic modification of the M₁ mAChR leads to changes in the allosteric behaviour of BQCA that are not reminiscent of its behaviour at the native receptor. As a consequence, caution must be exercised when interpreting studies of allosteric modulation using chemogenetically modified receptors in vivo. Despite the unique pharmacology of BQCA, the molecular mechanisms of its binding and function and the structural basis of its M₁ mAChR selectivity remain poorly defined. Such knowledge would enable the design of novel M₁ mAChR PAMs with improved pharmacological profiles. Chapters 3 and 4 comprise studies focussed on identifying the amino acid residues that form the allosteric binding pocket at the M₁ mAChR and/or play a role, either directly or indirectly, in the transmission of cooperativity with the orthosteric (ACh) binding site. Deeper mechanistic insights into allosteric modulation at the M₁ mAChR are further afforded by the use of benzoquinazolinone 12, a high affinity structural derivative of BQCA. The experimental findings are contextualised using molecular models, and collectively, the results suggest that many of the key residues that form the allosteric binding pocket at the M₁ mAChR are structurally conserved in other mAChR subtypes. The findings in this thesis challenge the common assumption that allosteric ligands achieve subtype selectivity through binding to allosteric sites that are less conserved between subtypes and propose that the selectivity of BQCA and benzoquinazolinone 12 arises from selective cooperativity with ACh at the M₁ mAChR. The information herein may guide the rational design of M₁ mAChR positive and/or negative allosteric ligands with increased therapeutic potential.