GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures

Abstract

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.

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Figure 1: Therapeutic use, efficacy and biased signalling of GPCR ligands.
Figure 2: Selected crystallography data on GPCR structural architectures and intermolecular interactions.
Figure 3: Conformational plasticity of β2AR.
Figure 4: NMR-observable conformational equilibria related to biased signalling of β2AR.
Figure 5: Effects on local conformational equilibria of β2AR from different experimental set-ups.
Figure 6: NMR-observable conformational equilibria related to biased signalling of MOR.
Figure 7: NMR affords a global view of A2AAR response to variable drug efficacy and inactivation of an allosteric centre.
Figure 8: NMR methods for studies of GPCR–ligand interactions.
Figure 9: NMR screening of biased ligands.

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Acknowledgements

This work is supported by The Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS) KAKENHI, grant numbers JP17H06097 (I.S.), JP18H04540 (T.U.) and JP17H04999 (Y.K.); the development of core technologies for innovative drug development based upon IT and the development of innovative drug discovery technologies for middle-sized molecules, from the Japan Agency for Medical Research and Development (AMED; I.S.); an American Cancer Society Postdoctoral Fellowship (M.T.E.); and NIH/NIGMS R01GM115825 (K.W.). K.W. is the Cecil H. and Ida M. Green Professor of Structural Biology at The Scripps Research Institute.

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Glossary

Allosteric modulation

Allosteric modulators are molecules that bind to sites on G protein-coupled receptors that are spatially distinct from the orthosteric binding pocket and modulate the affinity and/or efficacy of drugs bound to the orthosteric site. Allosteric modulators can be synthetic or endogenous compounds or metal ions in the cellular environment.

Biased signalling

G protein-coupled receptor agonists can activate both G protein and β-arrestin signalling pathways (as shown in Fig. 1). Agonists that activate predominantly one of the intracellular pathways are referred to as 'biased ligands'. Drugs functioning as agonists may produce unwanted side effects mediated through the activation of multiple signalling pathways. Such side effects can be minimized by designing biased ligands that selectively activate only the signalling pathway required to produce the desired therapeutic response.

Conformational equilibria

Solution NMR studies established that G protein-coupled receptors in near-physiological environments exist in multiple, locally different conformers that are simultaneously populated in function-related equilibria. It has been shown that the relative populations of these conformers are related to the efficacies and the bias of bound drugs.

Efficacy

The extent to which a G protein-coupled receptor (GPCR) ligand changes the receptor signalling intensity relative to its basal level. The efficacy is a key determinant of the therapeutic properties of a GPCR-targeting drug.

Motif

A polypeptide segment of two or several amino acids that are highly conserved among G protein-coupled receptors of a given class. Motifs have been identified as key components of activation centres, which are clusters of closely spaced amino acids in the 3D structure.

Transverse relaxation-optimized spectroscopy

(TROSY). An experiment that enables solution NMR studies of large macromolecules or supramolecular structures, in particular of membrane proteins reconstituted into micelles, bicelles or nanodiscs.

Labelling with stable isotopes

NMR spectroscopy with complex biomacromolecular systems is routinely based on labelling of proteins or other components with stable NMR-observable isotopes. Widely used stable isotopes in G-protein coupled receptor research are 2H, 13C, 15N and 19F.

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Shimada, I., Ueda, T., Kofuku, Y. et al. GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures. Nat Rev Drug Discov 18, 59–82 (2019). https://doi.org/10.1038/nrd.2018.180

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