Integrated in silico and in vitro approaches reveal novel coumarin hybrids targeting influenza A virus RNA-dependent RNA polymerase.

Abstract

Influenza A virus (IAV) remains a significant public health threat due to its high mutation rate and increasing resistance to existing antiviral drugs. The viral RNA-dependent RNA polymerase (RdRp) is essential for viral replication and represents a promising target for new therapeutics. We screened our in-house chemical library of 103 compounds against two domains of the IAV RdRp complex using molecular docking. Based on computational findings, we selected eleven coumarin-phenolic acid hybrids and one thiazolidinone compounds for in vitro evaluation against H1N1 and H5N1. LMed 93 and LMed 99 exhibited potent antiviral activity, with EC50 values of 5.66 and 1.21 μM against H1N1 respectively, and high selectivity indices. Mechanistic studies showed that LMed 99 acts throughout early to late stages of infection, while LMed 93 impacts early and intermediate stages. Both compounds significantly inhibited viral RNA synthesis, with LMed 99 achieving 82% inhibition at 1- and 3-h post-infection (hpi) and 80% at 6 hpi, and LMed 93 achieving 82% (1 hpi), 78% (3 hpi), and 60% (6 hpi). LMed 99 reduced viral proteins and mRNA in a time-dependent manner, indicating interference with viral transcription. A mini-replicon assay showed that LMed 93, at 70 and 35 μM, reduces polymerase activity by 53.7% and 38.7%, while LMed 99 at 9 and 4.5 μM increased expression by 108.97% and 61.37%, respectively. Structure-activity relationship analysis highlighted that catechol groups and halogenation enhance antiviral potency. These findings suggest that LMed 93 and LMed 99 are promising coumarin-based scaffolds for developing new anti-Influenza A agents, also offering potential strategies for combating other RNA viruses.