A novel class of small molecule inhibitors targeting the chikungunya virus capping machinery with a high barrier to resistance

Abstract

Background: Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we identified a novel class of CHIKV inhibitors i.e. the CHVB series. Methods: CPE-reduction and virus yield assays were performed in Vero cells. Drug-resistant variants were selected using clonal resistance selection. The enzymatic assays for alphavirus capping were done using the non-structural protein 1 (nsP1) of Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV). Results: CHVB compounds inhibited the in vitro replication of CHIKV isolates with EC50 values in the low μM range. In virus yield assays, the most potent analogues reduced the viral load with 4-5 log10. CHVB-resistant variants were selected and found to carry (i) two mutations in the gene encoding nsP1 (responsible for viral RNA capping), (ii) one mutation in nsP2 and (iii) one mutation in nsP3. Reverse-engineering suggested that nsP1 is the target of CHVB, since both nsP1 mutations were needed to achieve 10-fold resistance. Interestingly, the CHVBres virus proved cross-resistant to the MADTP series, a class of CHIKV capping inhibitors that we described earlier, suggesting a similar mode of action. In enzymatic assays, CHVB proved a potent inhibitor of the methyltransferase and guanylyltransferase activities of nsP1 of SFV and VEEV. Conclusion: We identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity and the high barrier to resistance make this chemical scaffold a potential candidate for CHIKV drug development.