Synthesis, screening and validation of cysteine-reactive fragments as chikungunya virus protease inhibitors

Alphaviruses like the Chikungunya virus cause severe outbreaks; however, no specific treatments are available. Their viral replication depends on the nsP2 cysteine protease, a promising but underexplored target for drug discovery. In this study, we report a covalent fragment screening against Chikungunya virus nsP2 protease, resulting in the identification of three inhibitors that can serve as starting points for future drug development. Careful validation proved indispensable in eliminating false-positive hits from a Fo<spacing diaeresis>rster resonance energy transfer (FRET)-based inhibition assay, wherein interference was caused by the inner filter effect between the fluorescent substrate and coloured compounds. Jump-dilution experiments accompanied by reactivity studies with cysteine and the recombinant protein indicate covalent inhibition via thia-Michael addition. We further demonstrate cross-inhibition of the related alphavirus nsP2 protease from Sindbis virus. The study provides early insights into nsP2 inhibition by electrophilic fragments featuring non-promiscuous N-arylacrylamides, thus advancing the search for antivirals targeting Chikungunya and other alphaviruses of concern.