In silico and vitro mapping of allosteric sites in GPCRs

Abstract

Allosteric modulators are considered to be promising candidates in G protein-coupled receptor (GPCR) drug development by displaying subtype selectivity and more specific receptor modulation. In this project, we have tested a probe confined dynamic mapping protocol that allows the prediction of allosteric sites at both the GPCR extracellular and intracellular sides, as well as at the receptor-lipid interface. The specific probes derived from GPCR allosteric ligand structures performed better in allosteric site mapping compared to commonly used cosolvents. The M₂ muscarinic, β₂ adrenergic and P₂Y₁ purinergic receptors were selected for the protocol retrospective validation. The protocol was next validated prospectively to locate the binding site of the UCB compound at the D₂ dopamine receptor.

We applied that probe confined dynamic mapping protocol to identify the binding site of GSK2239633A and Z5367428075, two selective CC-chemokine receptor type 4 (CCR4) negative allosteric modulators, using CCR4 homology and AlphaFold models. By comparing the performance across five computational models, we identified conserved (K310^8.49 and Y304^7.53) and non-conserved (M243^6.36) residue hotspots for GSK2239633A binding. We also compared three computational models and identified non-conserved (Y98^2.63) residue hotspots for Z5367428075 binding. They were all validated by mutagenesis and bioluminescence resonance energy transfer assay. Further analysis of 3D models and MD simulations highlighted the pair of residues 6.36 and 7.56 that might account for antagonist selectivity among chemokine receptors.

To figure out the impact of lipid during the allosteric site mapping, we analyse interactions in the allosteric sites of the PAR2, C5aR1, and GCGR receptors in three lipid compositions using molecular dynamics simulations. We show that besides classical hydrogen bonds weak polar interactions contribute to the stability of allosteric modulators at the extrahelical sites in the middle of the membrane. The allosteric cavities are detectable in various membrane compositions. The availability of polar atoms for interactions in such cavities can be assessed by water molecules from simulations. Although ligand-lipid interactions are weak, lipid tails play a role in ligand binding pose stability and the size of allosteric cavities.

In the developed for mapping the allosteric sites at lipid interface, multiple harmonic wall potentials were applied to enhance sampling of probe molecules in a selected area of a GPCR while preventing membrane distortion in molecular dynamics simulations. Based on that protocol, we propose an improved protocol that allows efficient prediction of allosteric sites at the receptor-lipid interface. The CB₁ cannabinoid receptors were selected for protocol validation. We show that our protocol together with the sequence analysis is specific in identifying an allosteric site of a compound and works in various receptor conformations.

Overall, we tested and improved a computational protocol to map the allosteric binding sites at the different locations of various GPCRs. The protocol provides fast and efficient prediction of key amino acid residues surrounding allosteric sites in membrane proteins and facilitates structure-based design of allosteric modulators.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Irina Tikhonova (Supervisor), Bianca Plouffe (Supervisor) & Fiona Furlong (Supervisor)