Iterated Virtual Screening-Assisted Antiviral and Enzyme Inhibition Assays Reveal the Discovery of Novel Promising Anti-SARS-CoV-2 with Dual Activity.

Unfortunately, COVID-19 is still a threat to humankind and has a dramatic impact on human health, social life, the world economy, and food security. With the limited number of suggested therapies under clinical trials, the discovery of novel therapeutic agents is essential. Here, a previously identified anti-SARS-CoV-2 compound named Compound 13 (1,2,5-Oxadiazole-3-carboximidic acid, 4,4'-(methylenediimino) bis,bis[[(2-hydroxyphenyl)methylene]hydrazide) was subjected to an iterated virtual screening against SARS-CoV-2 Mpro using a combination of Ligand Designer and PathFinder. PathFinder, a computational reaction enumeration tool, was used for the rapid generation of enumerated structures via default reaction library. Ligand designer was employed for the computerized lead optimization and selection of the best structural modification that resulted in a favorable ligand-protein complex. The obtained compounds that showed the best binding to Mpro were re-screened against TMPRSS2, leading to the identification of 20 shared compounds. The compounds were further visually inspected, which resulted in the identification of five shared compounds M1-5 with dual binding affinity. In vitro evaluation and enzyme inhibition assay indicated that M3, an analogue of Compound 13 afforded by replacing the phenolic moiety with pyridinyl, possesses an improved antiviral activity and safety. M3 displayed in vitro antiviral activity with IC50 0.016 µM and Mpro inhibition activity with IC50 0.013 µM, 7-fold more potent than the parent Compound 13 and potent than the antivirals drugs that are currently under clinical trials. Moreover, M3 showed potent activity against human TMPRSS2 and furin enzymes with IC50 0.05, and 0.08 µM, respectively. Molecular docking, WaterMap analysis, molecular dynamics simulation, and R-group analysis confirmed the superiority of the binding fit to M3 with the target enzymes. WaterMap analysis calculated the thermodynamic properties of the hydration site in the binding pocket that significantly affects the biological activity. Loading M3 on zinc oxide nanoparticles (ZnO NPs) increased the antiviral activity of the compound 1.5-fold, while maintaining a higher safety profile. In conclusion, lead optimized discovery following an iterated virtual screening in association with molecular docking and biological evaluation revealed a novel compound named M3 with promising dual activity against SARS-CoV-2. The compound deserves further investigation for potential clinical-based studies.

Evolution of H5-Type Avian Influenza A Virus Towards Mammalian Tropism in Egypt, 2014 to 2015.

Highly pathogenic avian influenza viruses (HPAIV) of the H5-subtype have circulated continuously in Egypt since 2006, resulting in numerous poultry outbreaks and considerable sporadic human infections. The extensive circulation and wide spread of these viruses in domestic poultry have resulted in various evolutionary changes with a dramatic impact on viral transmission ability to contact mammals including humans. The transmitted viruses are either (1) adapted well enough in their avian hosts to readily infect mammals, or (2) adapted in the new mammalian hosts to improve their fitness. In both cases, avian influenza viruses (AIVs) acquire various host-specific adaptations. These adaptive variations are not all well-known or thoroughly characterized. In this study, a phylogenetic algorithm based on the informational spectrum method, designated hereafter as ISM, was applied to analyze the affinity of H5-type HA proteins of Egyptian AIV isolates (2006-2015) towards human-type cell receptors. To characterize AIV H5-HA proteins displaying high ISM values reflecting an increased tendency of the HA towards human-type receptors, recombinant IV expressing monobasic, low pathogenic (LP) H5-HA versions in the background of the human influenza virus A/PR/8/1934(H1N1) (LP 7+1), were generated. These viruses were compared with a LP 7+1 expressing a monobasic H5-HA from a human origin virus isolate (human LP-7271), for their receptor binding specificity (ISM), in vitro replication efficiency and in vivo pathogenicity in mammals. Interestingly, using ISM analysis, we identified a LP 7+1 virus (LP-S10739C) expressing the monobasic H5-HA of AIV A/Chicken/Egypt/S10739C/2015(H5N1) that showed high affinity towards human-type receptors. This in silico prediction was reflected by a higher in vitro replication efficiency in mammalian cell cultures and a higher virulence in mice as compared with LP-7271. Sequence comparison between the LP-S10739C and the LP-7271 H5-HA, revealed distinct amino acid changes. Their contribution to the increased mammalian receptor propensity of LP-S10739C demands further investigation to better deduce the molecular determinant behind the reported high morbidity of 2014 to 2015 HPAI H5N1 virus in humans in Egypt. This study provides insights into the evolution of Egyptian H5 HPAIVs and highlights the need to identify the viral evolution in order to recognize emerging AIV with the potential to threaten human and animal populations.