Leveraging a Separation\nof States Method for Relative\nBinding Free Energy Calculations in Systems with Trapped Waters

Methods for calculating the relative binding free energy\n(RBFE)\nbetween ligands to a target protein are gaining importance in the\nstructure-based drug discovery domain, especially as methodological\nadvances and automation improve accuracy and ease of use. In an RBFE\ncalculation, the difference between the binding affinities of two\nligands to a protein is calculated by transforming one ligand into\nanother, in the protein–ligand complex, and in solvent. Alchemical\nbinding free energy calculations are often used for such ligand transformations.\nSuch calculations are not without challenges, however; for example,\nit can be challenging to handle interfacial waters when these play\na crucial role in mediating protein–ligand binding. In some\ncases, the exchange of the interfacial waters with solvent water might\nbe very infrequent in the course of typical molecular simulations,\nand such interfacial waters can be considered trapped on the simulation\ntime scale. In these cases, RBFE calculation between two ligands,\nwhere one ligand binds with a trapped water while the other ligand\ndisplaces it, can result in inaccuracies if the surrounding water\nstructure is not sampled adequately for both ligands. So far, a popular\nchoice for treating the trapped waters in RBFE calculations is to\ncombine free energy calculations with enhanced sampling methods that\ninsert/delete waters in the binding site. Despite recent developments\nin the enhanced sampling methods, they can result in hysteresis in\nthe RBFE estimate, depending on whether the simulations were started\nwith or without the trapped waters. In this study, we introduce an\nalternative method, separation of states, to calculate the RBFE between\nligand pairs where the ligands bind to the protein with different\nnumbers/positions of trapped waters. The separation of states approach\ntreats the sampling of the trapped waters separately from the free\nenergy calculation of the ligand transformation. In our method, a\ntrapped water in protein’s binding site is decoupled from the\nsystem first, and the cavity created by its decoupling is stabilized.\nWe then grow a larger ligand into this cavity– a ligand that\nis known to displace the trapped water. In this study, we show that\nour method results in precise and accurate estimates of RBFEs for\nligand pairs involving the rearrangement of trapped water via RBFE\ncalculations for five such ligand pairs. We have optimized our simulation\nprotocol to be suited for large distributed computational resources\nand have automated our RBFE calculation workflow.