In silico insights into the effect of mutation on peramivir resistance against influenza H7N9 virus and the development of potential inhibitors.

Abstract

Influenza A virus infections causes substantial population illness with consequent healthcare and economic problems. There has been an outbreak of novel influenza A (H7N9) virus strains on the Chinese mainland as of March 2013. As a result of their fast geographical spread and genomic variety, the ongoing circulation of H7N9 virus poses a pandemic threat. At present, available anti-influenza drugs are mainly directed at the viral M2 ion-channel (amantadine and rimantadine), neuraminidase (oseltamivir, zanamivir, laninamivir, and peramivir), or polymerase (baloxavir marboxil), and emerging anti-viral resistance against these inhibitors is a concern. The development of safe and effective anti-influenza drugs is essential to a balanced strategy against seasonal influenza. The use of computational approaches for designing and developing new antiinfluenza drugs has proven beneficial in response to resistance to current therapies. The use of computer-aided drug design (CADD) is crucial to the development of novel drugs. It has been shown over the years that CADD plays a vital role in the drug design process, accelerating the discovery of possible drug candidates at a lower cost. CADD approaches are able to investigate protein-ligand interactions at the atomic level, which provides insights that can be used to improve drug design. As a result, the studies presented in this thesis employed CADD approaches in order to explore molecular mechanisms of action of new therapeutic approaches designed to combat H7N9 viral infections. The aim of this study was to offer an indepth understanding of the effect of H7N9 mutation on neuraminidase inhibitor resistance and discover the fundamentals for the design and development of more potent anti-viral drugs. Clinical studies demonstrated that the peramivir resistance to extremely pathogenic influenza H7N9 viruses is caused R292K mutation. As such, we used numerous molecular dynamics methods to assess the effect of neuraminidase-R292K mutation towards peramivir resistance in influenza H7N9 viruses. We found that a R292K mutation caused peramivir orientation to be altered in the binding site of the peramivir-R292 mutant complex, consequently hampering the mutant's ability to bind peramivir. In contrast to its wildtype counterpart, R292K mutant decreased the interaction between neighboring amino acid residues, as evidenced by a high degree of flexibility in the radius of gyration. The mutation altered hydrogen bond-mediated interactions with peramivir and resulted in a greater accessibility of water molecules nearby the K292 mutated amino acid residue. Based on the energy binding calculations, it was determined that the R292K mutation caused a reduction of 17.28 kcal/mol in the peramivir binding affinity compared to the peramivir-wildtype complex. As a result, the peramivir was oriented differently in the binding site and the overall conformation of the peramivir-mutant complex changed.

Experimental investigations have been conducted into the mutation of E119 in neuraminidase. Contrary to this, there is insufficient information regarding the impact of E119V mutation towards peramivir at the intermolecular level. Therefore, a thorough understanding of the protein-ligand intermolecular interactions is crucial to understanding its inhibition. In the present study, we explored the intermolecular mechanism and dynamics associated with the susceptibility of peramivir to influenza H7N9 virus containing E119V mutation. We utilized molecular dynamic simulations and a wide range of post-molecular dynamic analysis for comprehensive insights into the impact of the E119V mutation and the conformational of the peramivir-E119V mutant complex. Based on the post-molecular dynamic analysis, the peramivir-E119V mutant complex showed relative stability. For the peramivir-wildtype complex, the calculated binding free energy (ΔGbind) is -49.09 ± 0.13 kcal/mol, while for E119V mutant it is -58.55 ± 0.15 kcal/mol. The increase in binding free energy by 9.46 kcal / mol is in accordance with other post-molecular dynamic analyses, which found that the E119V mutation increases protein stability. These findings could play a crucial role in developing new antiinfluenza drugs and controlling the avian influenza H7N9 virus. New and re-emerging diseases like influenza are challenging to treat due to the lengthy development process and high failure rate. To develop potential therapies against the H7N9 virus, we repurposed FDA-approved drugs using an in silico drug repurposing method. A total of 2,568 drugs were screened for potential inhibitors. A DrugBank database virtual screening identified the compounds promacta, tucitanib, and lurasidone as promising hits. The calculations of MM-GBSA suggest that tucitanib (-54.1 kcal/mol) and promacta (-56.2 kcal/mol) occupy the active site of neuraminidase with a higher binding affinity than the standard drug peramivir (- 49.09 kcal/ mol). Based on the results of Molecular dynamics (MD) simulation, the C-α atom backbones of the complexes of tucatinib and promacta neuraminidase remained stable during the simulation time. Absorption, distribution, metabolism, and excretion (ADME) analysis revealed that the hit compounds have a high gastrointestinal absorption (GI) and lack properties that allow them to cross the blood-brain barrier (BBB). Based on the in silico toxicity prediction, promacta is not cardiotoxic, while lurasidone and tucatinib are only weakly inhibitory. We, therefore, propose to test these compounds experimentally against influenza H7N9. To bring these compounds to clinical settings, further investigation, and validation of these potential H7N9 inhibitors are necessary. In summary, this study has established that the R292K mutation decreases peramivir binding affinity and distorts peramivir optimum position in the binding site of neuraminidase. In contrast, the E119V mutation contributed to relative stability of the peramivir-neuraminidase complex. Promacta and tucatinib could be used as lead compounds to combat the H7N9 influenza virus. Insights gained from this study will enhance future drug development and help in combating the avian influenza H7N9 virus. Nonetheless, the concept of multi-target drugs, quantum mechanics (QM) method such as density functional theory (DFT) and hybrid quantum mechanics/molecular mechanics (QM/MM) for effective design of neuraminidase inhibitors should be widely explored.