Abstract
The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional in vitro studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.
Highlights
The molecular targets for about 50–60% of currently validated drugs are membrane proteins, such as G-protein coupled receptors (GPCRs); this class of proteins still features the main target in drug discovery programs (Hauser et al, 2017; Ribeiro-Oliveira et al, 2019)
A major bottleneck in structure determination of GPCRs by X-ray crystallography is obtaining of large amounts (>1–2 mg) of highly pure, homogeneous protein samples that are stable in detergent solutions when extracted from the lipid environment of the membranes
A number of protein engineering approaches have been used to overcome the problem of the intrinsic instability and conformational heterogeneity of GPCRs
Summary
The molecular targets for about 50–60% of currently validated drugs are membrane proteins, such as G-protein coupled receptors (GPCRs); this class of proteins still features the main target in drug discovery programs (Hauser et al, 2017; Ribeiro-Oliveira et al, 2019) To this purpose, a range of chemical, biochemical and biophysical techniques are available for the characterization of ligand binding and for screening libraries of compounds searching for potential drug candidates. A range of chemical, biochemical and biophysical techniques are available for the characterization of ligand binding and for screening libraries of compounds searching for potential drug candidates One of such techniques is surface plasmon resonance (SPR) spectroscopy, a label-free technique which enables measurement of real-time ligand-binding affinities and kinetics using relatively small amounts of membrane protein in a native or native-like environment (Olaru et al, 2015). An alternative method consists in capturing the detergent-solubilized receptor, engineered with a tag (such as multiple histidine residues or a short peptide sequence), with an appropriate antibody which has been previously covalently immobilized onto a chip through covalent bonding (Rich et al, 2011)
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