Bose, M. R.; Sears, J. D.; Talbot, K. M.; Su, Y.-W. N.; Houliston, S.; Hossain, M. A.; Davis-Gilbert, Z. W.; Zhao, C.; Oh, H. J.; Brown, P. J.; Sanders, M. K.; Moorman, S. R.; Ojha, D.; Burdick, J. E.; Law, I.; Morales, N. L.; Martinez, S. A.; Loppnau, P.; Perez, J. G.; Drobish, A. M.; Morrison, T. E.; Streblow, Z. J.; Streblow, D. N.; Arrowsmith, C. H.; Vargason, A.; Counago, R. M.; Halabelian, L.; Arnold, J. J.; Cameron, C. E.; Moorman, N. J.; Heise, M. T.; Willson, T. M.

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum anti-alphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting anti-alphaviral drugs.