Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding...

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 R173^5.34 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.