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bioRxiv · 10.1101/2025.01.13.632788

Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity

Hossain, M. A.·Mayo, A. K.·Ghoshal, A.·Taft-Benz, S. A.·Anderson, E. J.·Morales, N. L.·Pressey, K. D.·Vargason, A. M.·Brouwer, K. L. R.·Moorman, N. J.·Heise, M. T.·Willson, T. M.
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Abstract

RA-0002034 (1) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/632788v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8c1558org.highwire.dtl.DTLVardef@cd7e21org.highwire.dtl.DTLVardef@113e88aorg.highwire.dtl.DTLVardef@7c7dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Hossain, M. A., Mayo, A. K., Ghoshal, A., Taft-Benz, S. A., Anderson, E. J., Morales, N. L., Pressey, K. D., Vargason, A. M., Brouwer, K. L. R., Moorman, N. J., Heise, M. T., Willson, T. M.. 2025-01-16. Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity. https://doi.org/10.1101/2025.01.13.632788

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