Alphavirus capsid protease inhibitors as potential antiviral agents for Chikungunya infection

Chikungunya virus (CHIKV) is an arthritogenic alphavirus and currently, no antiviral drug is available to combat it. Capsid protein (CP) of alphaviruses present at the N-terminus of the structural polyprotein possesses auto-proteolytic activity which is essential for initiating the structural polyprotein processing. We are reporting for the first time antiviral molecules targeting capsid proteolytic activity. Structure-assisted drug-repositioning identified three molecules: P1,P4-Di(adenosine-5') tetraphosphate (AP4), Eptifibatide acetate (EAC) and Paromomycin sulphate (PSU) as potential capsid protease inhibitors. A FRET-based proteolytic assay confirmed anti-proteolytic activity of these molecules. Additionally, in vitro cell-based antiviral studies showed that EAC, AP4, and PSU drastically stifled CHIKV at the post-entry step with a half-maximal effective concentration (EC₅₀) of 4.01 μM, 10.66 μM and 22.91 μM; respectively. Interestingly, the inhibitors had no adverse effect on viral RNA synthesis and treatment of cells with inhibitors diminished levels of CP in virus-infected cells, which confirmed inhibition of capsid auto-proteolytic activity. In conclusion, the discovery of antiviral molecules targeting capsid protease demystifies the alphavirus capsid protease as a potential target for antiviral drug discovery.