Discovery of Novel Andrographolide Derivatives as Antiviral Inhibitors against Human Enterovirus A71.

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

Discovery of 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide as EV-A71 infection inhibitor.

Human enterovirus A71 (EV-A71) is a major etiological agent of hand-foot-and-mouth disease (HFMD) and there is presently no internationally approved antiviral against EV-A71. In this study, it is disclosed that 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide (2) was discovered to be an effective inhibitor against EV-A71 infection showing significant reduction of viral titre. In addition to EV-A71, compound 2 exerts broad-spectrum antiviral effects against other enteroviruses. It is revealed that compound 2 inhibits the post-entry stages of EV-A71 viral replication cycle and significantly reduces viral protein expression of structural proteins such as VP0 and VP2 via inhibiting EV-A71 RNA replication. Moreover, the inhibitory property of compound 2 is specific to viral RNA replication. Furthermore, compound 2 is more likely to target a host factor in EV-A71 RNA replication. As a result, introduction of epoxide at positions 8 and 17 of andrographolide is effective for anti-EV-A71 infection and is a potential anti-EV-A71 strategy. Further work to discover more potent andrographolide derivatives and elucidate comprehensive SAR is under way.