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A systematic investigation of high-resolution G-protein-coupled receptor (GPCR) structures uncovers a conserved inter-helical network of non-covalent contacts that defines the GPCR fold, and provides insights into the molecular determinants of different GPCR conformations. Comparative analysis of GPCRs Understanding the conformational states of G-protein-coupled receptors (GPCRs) has been an area of intense research in recent years. GPCRs are membrane proteins that act as sensors for a broad range of extracellular signals, including photons, ions, small organic molecules, and even entire proteins. Approximately one-third of all known drugs target GPCRs, including small molecule therapeutics used to treat cardiac malfunction, asthma and migraines. This Review reports a systematic analysis of all known high-resolution GPCR structures and provides insights into the molecular determinants of different GPCR conformations. Understanding how these membrane proteins function at the molecular level should facilitate the development of subtype-selective, highly efficacious therapeutics. G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand–target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo . The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology. An automated approach designing drug ligands to multi-target profiles (with a 75% prediction success rate) is experimentally validated by the invention of novel ligands tailored to the complex and physiologically-relevant goal of identifying drugs that can specifically target profiles of multiple proteins. Designing multifunctional drugs This paper describes a new approach to the problem of designing drugs that interact with multiple targets, which may be desirable either to achieve exquisite selectivity over other drug targets, or to obtain a drug with a particular polypharmacological profile. The authors have developed an automated, adaptive design approach to the generation of analogues and prioritizing them against a set of objectives. They tested experimentally 800 ligand–target predictions of prospectively designed ligands; 75% were confirmed correct, and the predicted target engagement was confirmed in vivo .

Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2-Å-resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family. The X-ray crystal structure of the human G-protein-coupled receptor protease-activated receptor 1 (PAR1) bound to the antagonist vorapaxar is solved, revealing an unusual method of drug binding that should facilitate the development of improved PAR1-selective antagonists. Thrombin receptor structure The X-ray crystal structure of the human protease-activated receptor 1 (PAR1) bound to vorapaxar, a PAR1 antagonist, has been determined at 2.2 Å resolution. PAR1, also known as the thrombin receptor, is a G protein-coupled receptor that mediates cellular responses to the coagulation protease thrombin and related proteases. Vorapaxar was recently shown to prevent myocardial infarction in at-risk patients, and knowledge of the PAR structure will be relevant to the design of PAR1 antagonists with better drug properties.

Both kynurenic acid and 2-acyl lysophosphatidic acid have been postulated to be the endogenous agonists of GPR35. However, controversy remains whether alternative endogenous agonists exist. The molecular targets accounted for many nongenomic actions of thyroid hormones are mostly unknown. Here we report the agonist activity of multiple tyrosine metabolites at the GPR35. Tyrosine metabolism intermediates that contain carboxylic acid and/or catechol functional groups were first selected. Whole cell dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor were then used to characterize their agonist activity in native HT-29. Molecular assays including β-arrestin translocation, ERK phosphorylation and receptor internalization confirmed that GPR35 functions as a receptor for 5,6-dihydroxyindole-2-carboxylic acid, 3,3′,5′-triiodothyronine, 3,3′,5-triiodothyronine, gentisate, rosmarinate and 3-nitrotyrosine. These results suggest that multiple tyrosine metabolites are alternative endogenous ligands of GPR35 and GPR35 may represent a druggable target for treating certain diseases associated with abnormality of tyrosine metabolism.

Knowing the structures of G-protein-coupled receptors, says Lindau keynote speaker Brian Kobilka, should help with drug development. But how is this progressing?

G protein-coupled Receptor Kinase 6 (GRK6) belongs to a family of kinases that phosphorylate GPCRs. GRK6 levels were found to be altered in Parkinson's Disease (PD) and D 2 dopamine receptors are supersensitive in mice lacking GRK6 (GRK6-KO mice). To understand how GRK6 modulates the behavioral manifestations of dopamine deficiency and responses to L-DOPA, we used three approaches to model PD in GRK6-KO mice: 1) the cataleptic response to haloperidol; 2) introducing GRK6 mutation to an acute model of absolute dopamine deficiency, DDD mice; 3) hemiparkinsonian 6-OHDA model. Furthermore, dopamine-related striatal signaling was analyzed by assessing the phosphorylation of AKT/GSK3β and ERK1/2. GRK6 deficiency reduced cataleptic behavior, potentiated the acute effect of L-DOPA in DDD mice, reduced rotational behavior in hemi-parkinsonian mice and reduced abnormal involuntary movements induced by chronic L-DOPA. These data indicate that approaches to regulate GRK6 activity could be useful in modulating both therapeutic and side-effects of L-DOPA.