Biochemical characterization of Sapovirus 2C helicase and identification of pramipexole as a novel inhibitor in vitro.

Sapovirus (SaV) is a major causative agent of acute gastroenteritis in both humans and animals. The virally encoded 2C helicase, which shares low homology with host helicases, represents a promising target for the development of broad-spectrum antiviral drugs. However, the inherent instability of the 2C helicase has hindered its recombinant expression, biochemical characterization and the development of targeted therapies. In this work, the wild-type SaV-2C helicase and two motif mutants (SaV-2CGK10AA and SaV-2CDE47AA) were successfully expressed and purified firstly. Then nucleotide triphosphate (NTP) binding assays showed strong preference of SaV-2C for ATP, which was markedly reduced in the SaV-2CGK10AA mutant but unaffected in SaV-2CDE47AA mutant. Furthermore, NTP hydrolysis analysis demonstrated that SaV-2C can hydrolyze both ATP and GTP with the highest activity observed for ATP, and both mutations significantly impaired ATPase activity. SaV-2C also displayed high binding affinity for double-stranded RNA, which was compromised in the two motif mutants. Consistent with previous reports, guanidine hydrochloride was found to inhibit the ATPase activity of SaV-2C. In addition, several candidate compounds with low binding free energy to SaV-2C was identified through homology modeling and virtual screening. Among these, pramipexole (PPX) was validated as a lead inhibitor of SaV-2C ATPase function.These findings elucidate the crucial biochemical roles of SaV-2C and nominate pramipexole as a potent antiviral compound against SaV.