Author:

Abstract:

The last decade, chikungunya virus (CHIKV) re-emerged in many parts of Africa and Asia, and recently also for the first time in Central and South America. The global re-emergence of this virus and its high morbidity rate emphasize the need for potent antivirals for treatment. We recently identified MADTP_314, a [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one, as a potent inhibitor of CHIKV replication with no toxicity to the host cells. The antiviral activity of this molecule was confirmed against several clinical isolates of CHIKV, including the Caribbean St-Martin strain. To identify the viral protein(s) involved in the mechanism of action of this molecule, drug-resistant CHIKV variants were isolated. In all independently-selected drug-resistant isolates, a single mutation was identified in the nsP1 coding sequence that results in a P34S amino acid substitution. Reverse-engineering corroborated the link between this mutation and the compound-resistant phenotype. nsP1 is the central enzyme for the viral mRNA capping and interestingly, the proline at position 34 is located near the conserved histidine (H38) in a region involved in methyltransferase/guanylyltransferase functions. Enzymatic assays using the nsP1 of Venezuelan equine encephalitis virus (VEEV) showed that MADTP_314 did not, or poorly, interfered with the N7 methylation of GTP performed by nsP1 but rather inhibited the covalent binding of N7-GMP on H38 of nsP1. Interestingly, analogues of MADTP_314 with more potent anti-CHIKV activity did not only inhibit the guanylyltransferase activity but also the methyltransferase activity of nsP1. Moreover, the corresponding resistance mutation on VEEV nsP1 (D34S) abrogated the inhibitory effect of MADTP compounds on nsP1 capping functions. In conclusion, a novel class of CHIKV inhibitors targeting the viral capping machinery was discovered.