Identification and characterization of compounds with antiviral activity against influenza viruses.

摘要

Prophylactic and therapeutic antiviral drugs for influenza are available as an adjunct to vaccination. However, their effectiveness is already being limited because of the rapid emergence of drug resistant isolates due to the vast use of these drugs and the recombination potential of the viral genome. Moreover, an immediate pandemic threat (influenza H5N1) raises issues like the lack of surge capacity and availability of drugs as well as potential drug resistance.;Given the need for additional antiviral agents, our study intended to identify new compounds with potential anti-influenza activity. This was done by screening a library of 34,000 small molecules by means of a cell-based system that assayed the inhibition of virus-induced cell death. As a result, 330 primary hits were identified from which two lead compounds (QMV-13 and QMV-15) and three of their analogs (QMV-13B, QMV-15A, QMV-15B) were described as potential candidates for further characterization. First, we evaluated their cytotoxicity in a variety of primary and immortalized cell lines. Next, their antiviral activity against influenza A/WSN/33 was confirmed using different methods. Furthermore, replication of a cohort of influenza A and B laboratory-adapted, clinical isolates, and drug resistant strains was quantified in the presence of the leads, resulting in low IC50 values. Next, we tested the specificity of their antiviral activity against other RNA viruses such as YFV, WNV, hPIV3, and HIV, eliminating the possibility of a broad spectrum of action. Time-of-drug-addition assays, viral protein expression, and virus growth kinetics experiments characterized our compounds as early inhibitors of virus replication. The lack of inhibitory response against virus neuraminidases helped eliminating that enzyme as their target. Finally, serial passages of the eliminating that enzyme as their target. Finally, serial passages of the virus in the presence of the lead compounds were done to generate drug resistant variants that could pinpoint more accurately their target.;Although the exact mechanism by which compounds QMV-13, QMV-13B, QMV-15, QMV-15A, and QMV-15B exert their anti-influenza activity requires further investigation, we have repeatedly characterized them as early inhibitors of viral infection, finding that has significantly reduced the number of possible targets and may increase their value as novel therapeutics due to their potential applicability as prophylactic agents.