Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive Human Adenosine A3 Receptor

A structure-based drug design pipeline that considers both thermodynamic and kinetic binding data of ligands against a receptor will enable the computational design of improved drug molecules. For unresolved GPCR-ligand complexes, a workflow that can apply both thermodynamic and kinetic binding data in combination with alpha-fold (AF)-derived or other homology models and experimentally resolved binding modes of relevant ligands in GPCR-homologs needs to be tested. Here, as test case, we studied a congeneric set of ligands that bind to a structurally unresolved G protein-coupled receptor (GPCR), the inactive human adenosine A3 receptor (hA3R). We tested three available homology models from which two have been generated from experimental structures of hA1R or hA2AR and one model was a multistate alphafold 2 (AF2)-derived model. We applied alchemical calculations with thermodynamic integration coupled with molecular dynamics (TI/MD) simulations to calculate the experimental relative binding free energies and residence time (τ)-random accelerated MD (τ-RAMD) simulations to calculate the relative residence times (RTs) for antagonists. While the TI/MD calculations produced, for the three homology models, good Pearson correlation coefficients, correspondingly, r = 0.74, 0.62, and 0.67 and mean unsigned error (mue) values of 0.94, 1.31, and 0.81 kcal mol–1, the τ-RAMD method showed r = 0.92 and 0.52 for the first two models but failed to produce accurate results for the multistate AF2-derived model. With subsequent optimization of the AF2-derived model by reorientation of the side chain of R1735.34 located in the extracellular loop 2 (EL2) that blocked ligand's unbinding, the computational model showed r = 0.84 for kinetic data and improved performance for thermodynamic data (r = 0.81, mue = 0.56 kcal mol–1). Overall, after refining the multistate AF2 model with physics-based tools, we were able to show a strong correlation between predicted and experimental ligand relative residence times and affinities, achieving a level of accuracy comparable to an experimental structure. The computational workflow used can be applied to other receptors, helping to rank candidate drugs in a congeneric series and enabling the prioritization of leads with stronger binding affinities and longer residence times.