Understanding the Structure–Activity Relationship through Density Functional Theory: A Simple Method Predicts Relative Binding Free Energies of Metalloenzyme Fragment-like Inhibitors

[Image: see text] Despite being involved in several human diseases, metalloenzymes are targeted by a small percentage of FDA-approved drugs. Development of novel and efficient inhibitors is required, as the chemical space of metal binding groups (MBGs) is currently limited to four main classes. The use of computational chemistry methods in drug discovery has gained momentum thanks to accurate estimates of binding modes and binding free energies of ligands to receptors. However, exact predictions of binding free energies in metalloenzymes are challenging due to the occurrence of nonclassical phenomena and interactions that common force field-based methods are unable to correctly describe. In this regard, we applied density functional theory (DFT) to predict the binding free energies and to understand the structure–activity relationship of metalloenzyme fragment-like inhibitors. We tested this method on a set of small-molecule inhibitors with different electronic properties and coordinating two Mn(2+) ions in the binding site of the influenza RNA polymerase PA(N) endonuclease. We modeled the binding site using only atoms from the first coordination shell, hence reducing the computational cost. Thanks to the explicit treatment of electrons by DFT, we highlighted the main contributions to the binding free energies and the electronic features differentiating strong and weak inhibitors, achieving good qualitative correlation with the experimentally determined affinities. By introducing automated docking, we explored alternative ways to coordinate the metal centers and we identified 70% of the highest affinity inhibitors. This methodology provides a fast and predictive tool for the identification of key features of metalloenzyme MBGs, which can be useful for the design of new and efficient drugs targeting these ubiquitous proteins.