Protocols for Metallo- and Serine-β-Lactamase Free Energy Predictions: Insights from Cross-Class Inhibitors.

While relative binding free energy (RBFE) calculations using alchemical methods are routinely carried out for many pharmaceutically relevant protein targets, challenges remain. For example, open-source tools do not support the easy setup and simulation of metalloproteins, particularly when ligands directly coordinate to the metal site. Here, we evaluate the performance of RBFE methods for KPC-2, a serine-β-lactamase (SBL), and two nonbonded metal parameter setups for VIM-2, a metallo-β-lactamase (MBL) with two active site zinc ions. We tested two different ways of modeling the ligand-zinc interactions. First, a restraint-based approach, in which FF14SB zinc parameters are combined with harmonic restraints between the zincs and their coordinating residues. The second approach uses an upgraded Amber force field (UAFF) for zinc-metalloproteins with adjusted partial charges and nonbonded terms of zinc-coordinating residues. Molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) simulations show that the crystallographically observed zinc coordination is not retained in MM simulations with either zinc parameter set for a series of known phosphonic acid-based inhibitors bound to VIM-2. These phosphonic acid-based inhibitors exhibit known cross-class affinity for SBLs and MBLs and serve as a benchmark for RBFE calculations for VIM-2, after validation with KPC-2. The KPC-2 free energy of binding estimates are within expected literature accuracies for the ligand series with a mean absolute error of kcal/mol and a Pearson's correlation coefficient of . For VIM-2, the UAFF approach has improved correlation from to , compared to the restraint approach. The presented strategies for handling ligands coordinating to metal sites highlight that simple metal parameter models can provide some predictive free energy estimates for metalloprotein-ligand systems, but leave room for improvement in their ease of use, modeling of coordination sites and as a result, their accuracy.