Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

Sears, J. D.; Ojha, D.; Taft-Benz, S.; Sylvester, P. A.; Streblow, Z.; Gramm, A. J.; Drobish, A. M.; Ramalingam, B. M.; Hossain, M. A.; Talbot, K. M.; Sanders, W.; Chang, C.-K.; Law, I.; Martinez, S. A.; Burdick, J. E.; Wiltshire, R.; Saba, N. A.; Poplarski, J. H.; Counago, R. M.; Brown, P. J.; Anand, G. S.; Morrison, T. E.; Arnold, J. J.; Cameron, C. E.; Streblow, D. N.; Willson, T. M.; Heise, M. T.; Moorman, N. J.

doi:10.1101/2025.06.03.657755

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bioRxiv · 10.1101/2025.06.03.657755

Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

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Abstract

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence SummaryThis study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity.

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Sears, J. D., Ojha, D., Taft-Benz, S., Sylvester, P. A., Streblow, Z., Gramm, A. J., Drobish, A. M., Ramalingam, B. M., Hossain, M. A., Talbot, K. M., Sanders, W., Chang, C.-K., Law, I., Martinez, S. A., Burdick, J. E., Wiltshire, R., Saba, N. A., Poplarski, J. H., Counago, R. M., Brown, P. J., Anand, G. S., Morrison, T. E., Arnold, J. J., Cameron, C. E., Streblow, D. N., Willson, T. M., Heise, M. T., Moorman, N. J.. 2025-06-04. Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase. https://doi.org/10.1101/2025.06.03.657755

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