Quantitative Predictions of Binding Free Energy Changes in Drug-Resistant Influenza Neuraminidase

Quantitatively predicting changes in drug sensitivity associated with residue mutations is a major challenge in structural biology. By expanding the limits of free energy calculations, we successfully identified mutations in influenza neuraminidase (NA) that confer drug resistance to two antiviral drugs, zanamivir and oseltamivir. We augmented molecular dynamics (MD) with Hamiltonian Replica Exchange and calculated binding free energy changes for H274Y, N294S, and Y252H mutants. Based on experimental data, our calculations achieved high accuracy and precision compared with results from established computational methods. Analysis of 15 µs of aggregated MD trajectories provided insights into the molecular mechanisms underlying drug resistance that are at odds with current interpretations of the crystallographic data. Contrary to the notion that resistance is caused by mutant-induced changes in hydrophobicity of the binding pocket, our simulations showed that drug resistance mutations in NA led to subtle rearrangements in the protein structure and its dynamics that together alter the active-site electrostatic environment and modulate inhibitor binding. Importantly, different mutations confer resistance through different conformational changes, suggesting that a generalized mechanism for NA drug resistance is unlikely. The capacity of the influenza virus to rapidly mutate and render resistance to a handful of FDA approved neuraminidase (NA) inhibitors represents a significant human health concern. To gain an atomic-level understanding of the mechanisms behind drug resistance, we applied a novel computational approach to characterize resistant NA mutations. These results are comparable in accuracy and precision with the best experimental measurements presently available. To the best of our knowledge, this is the first time that a rigorous computational method has attained the level of certainty needed to predict subtle changes in binding free energies conferred by mutations. Analysis of our simulation data provided a thorough description of the thermodynamics of the binding process for different NA-inhibitor complexes, with findings that in some cases challenge current views based on interpretations of the crystallographic data. While we did not find a generalized mechanism of NA resistance, we identified key differences between oseltamivir and zanamivir that discriminate their responses to the three mutations we considered, namely H274Y, N294S and Y252H. It is worth noting that our approach can be broadly applied to predict resistant mutations to existing and newly developed drugs in other important drug targets.