A new mechanism of respiratory syncytial virus entry inhibition by small-molecule to overcome K394R-associated resistance.

Infection with respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in young children and older people. Despite intensive efforts over the past few decades, no direct-acting small-molecule agents against RSV are available. Most small-molecule candidates targeting the RSV fusion (F) protein pose a considerable risk of inducing drug-resistant mutations. Here, we explored the in vitro and in vivo virological properties of the K394R variant, a cross-resistant mutant capable of evading multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in vitro and more pathogenic than the parental strain in vivo. The small molecule (2E,2'E)-N,N'-((1R,2S,3S)-3-hydroxycyclohexane-1,2-diyl)bis(3-(2-bromo-4-fluorophenyl) acrylamide) (CL-A3-7), a structurally optimized compound derived from a natural caffeoylquinic acid derivative, substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant. Mechanistically, CL-A3-7 significantly inhibited virus-cell fusion during RSV entry by blocking the interaction between the viral F protein and the cellular insulin-like growth factor 1 receptor (IGF1R). Collectively, these results indicate severe disease risks caused by the K394R variant and reveal a new anti-RSV mechanism to overcome K394R-associated resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance.

Discovery of cyperenoic acid as a potent and novel entry inhibitor of influenza A virus.

Influenza therapeutics with new targets and modes of action are urgently needed due to the frequent emergence of mutants resistant to currently available anti-influenza drugs. Here we report the in vitro and in vivo anti-influenza A virus activities of cyperenoic acid, a natural compound, which was isolated from a Chinese medicine Croton crassifolius Geise. Cyperenoic acid could potently suppress H1N1, H3N2 and H9N2 virus replication with IC50 values ranging from 0.12 to 15.13 µM, and showed a low cytotoxicity against MDCK cells (CC50 = 939.2 ± 60.0 µM), with selectivity index (SI) values ranging from 62 to 7823. Oral or intraperitoneal treatment of cyperenoic acid effectively protected mice against a lethal influenza virus challenge, comparable to the efficacy of Tamiflu. Additionally, cyperenoic acid also significantly reduced lung virus titers and alleviated influenza-induced acute lung injury in infected mice. Mechanism-of-action studies revealed that cyperenoic acid exhibited its anti-influenza activity during the entry stage of viral replication by inhibiting HA-mediated viral fusion. Simulation docking analyses of cyperenoic acid with the HA structures implied that cyperenoic acid binds to the stalk domain of HA in a cavity near the fusion peptide. Collectively, these results demonstrate that cyperenoic acid is a promising lead compound for the anti-influenza drug development and this research provides a useful small-molecule probe for studying the HA-mediated viral entry process.