Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational ‘assay.

Molecular Dynamics Simulations Suggest that Electrostatic Funnel Directs Binding of Tamiflu to Influenza N1 Neuraminidases

Oseltamivir (Tamiflu) is currently the frontline antiviral drug employed to fight the flu virus in infected individuals by inhibiting neuraminidase, a flu protein responsible for the release of newly synthesized virions. However, oseltamivir resistance has become a critical problem due to rapid mutation of the flu virus. Unfortunately, how mutations actually confer drug resistance is not well understood. In this study, we employ molecular dynamics (MD) and steered molecular dynamics (SMD) simulations, as well as graphics processing unit (GPU)-accelerated electrostatic mapping, to uncover the mechanism behind point mutation induced oseltamivir-resistance in both H5N1 “avian” and H1N1pdm “swine” flu N1-subtype neuraminidases. The simulations reveal an electrostatic binding funnel that plays a key role in directing oseltamivir into and out of its binding site on N1 neuraminidase. The binding pathway for oseltamivir suggests how mutations disrupt drug binding and how new drugs may circumvent the resistance mechanisms

Independent-Trajectories Thermodynamic-Integration Free-Energy Changes for Biomolecular Systems: Determinants of H5N1 Avian Influenza Virus Neuraminidase Inhibition by Peramivir

Free-energy changes are essential physicochemical quantities for understanding most biochemical processes. Yet, the application of accurate thermodynamic-integration (TI) computation to biological and macromolecular systems is limited by finite-sampling artifacts. In this paper, we employ independent-trajectories thermodynamic-integration (IT-TI) computation to estimate improved free-energy changes and their uncertainties for (bio)molecular systems. IT-TI aids sampling statistics of the thermodynamic macrostates for flexible associating partners by ensemble averaging of multiple, independent simulation trajectories. We study peramivir (PVR) inhibition of the H5N1 avian influenza virus neuraminidase flexible receptor (N1). Binding site loops 150 and 119 are highly mobile, as revealed by N1-PVR 20-ns molecular dynamics. Due to such heterogeneous sampling, standard TI binding free-energy estimates span a rather large free-energy range, from a 19% underestimation to a 29% overestimation of the experimental reference value (−62.2 ± 1.8 kJ mol−1). Remarkably, our IT-TI binding free-energy estimate (−61.1 ± 5.4 kJ mol−1) agrees with a 2% relative difference. In addition, IT-TI runs provide a statistics-based free-energy uncertainty for the process of interest. Using ∼800 ns of overall sampling, we investigate N1-PVR binding determinants by IT-TI alchemical modifications of PVR moieties. These results emphasize the dominant electrostatic contribution, particularly through the N1 E277−PVR guanidinium interaction. Future drug development may be also guided by properly tuning ligand flexibility and hydrophobicity. IT-TI will allow estimation of relative free energies for systems of increasing size, with improved reliability by employing large-scale distributed computing

On the Lower Susceptibility of Oseltamivir to Influenza Neuraminidase Subtype N1 than Those in N2 and N9

AbstractAiming to understand, at the molecular level, why oseltamivir (OTV) cannot be used for inhibition of human influenza neuraminidase subtype N1 as effectively as for subtypes N2 and N9, molecular dynamics simulations were carried out for the three complexes, OTV-N1, OTV-N2, and OTV-N9. The three-dimensional OTV-N2 and OTV-N9 initial structures were represented by the x-ray structures, whereas that of OTV-N1, whose x-ray structure is not yet solved, was built up using the aligned sequence of H5N1 isolated from humans in Thailand with the x-ray structure of the N2-substrate as the template. In comparison to the OTV-N2 and OTV-N9 complexes, dramatic changes were observed in the OTV conformation in the OTV-N1 complex in which two of its bulky side chains, N-acethyl (−NHAc) and 1-ethylproxy group (−OCHEt2), were rotated to adjust the size to fit into the N1 catalytic site. This change leads directly to the rearrangements of the OTV’s environment, which are i), distances to its neighbors, W-178 and E-227, are shorter whereas those to residues R-224, E-276, and E-292 are longer; ii), hydrogen bonds to the two nearest neighbors, R-224 and E-276, are still conserved in distance and number as well as percentage occupation; iii), the calculated ligand/enzyme binding free energies of −7.20, −13.44, and −13.29kcal/mol agree with their inhibitory activities in terms of the experimental IC50 of 36.1–53.2nM, 1.9–2.7nM, and 9.5–17.7nM for the OTV-N1, OTV-N2, and OTV-N9 complexes, respectively; and iv), hydrogen-bond breaking and creation between the OTV and neighborhood residues are accordingly in agreement with the ligand solvation/desolvation taking place in the catalytic site