Species-Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with In Vivo Antialphaviral Activity.

RA-0002034 (1) is a potent covalent inhibitor targeting the 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 chikungunya (CHIKV) viral loads in multiple tissues. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a potential antialphaviral therapeutic to further define the contribution of hepatic and non-hepatic GST metabolism to its clearance in humans.