Equilibrium and Non-equilibrium Ensemble Methods for Accurate, Precise and Reproducible Absolute Binding Free Energy Calculations

Abstract

Free energy calculations for protein-ligand complexes have become widespread in recent years owing to several conceptual, methodological and technological advances. Central among these is the use of ensemble methods which permits accurate, precise and reproducible predictions and are necessary for uncertainty quantification. Absolute binding free energies (ABFEs) are challenging to predict using alchemical methods and their routine application in drug discovery has remained out of reach until now. Here, we apply ensemble alchemical ABFE methods to a large dataset comprising 219 ligand-protein complexes and obtain statistically robust results with high accuracy (< 1 kcal/mol). We compare equilibrium and non-equilibrium methods for ABFE predictions at large scale and provide a systematic critical assessment of each method. The equilibrium method is more accurate, precise, faster, computationally more cost-effective and requires a much simpler protocol, making it preferable for large scale and blind applications. We find that the calculated free energy distributions are non-normal and discuss the consequences. We recommend a definitive protocol to perform ABFE calculations optimally. Using this protocol, it is possible to perform thousands of ABFE calculations within a few hours on modern exascale machines.