Identification and characterization of novel chikungunya virus polymerase inhibitors

Chikungunya virus (CHIKV) and other alphaviruses in the Togaviridae family are positive-sense RNA viruses and major human pathogens, causing millions of infections worldwide. In humans, alphaviruses, such as CHIKV, Mayaro, and Ross River viruses, typically cause arthritogenic disease characterized by debilitating arthralgia, joint inflammation, fever, and rash. Although a vaccine was recently approved for use against CHIKV, no vaccines are licensed against other alphaviruses. No antiviral treatments are available to prevent or treat infections by any alphavirus. To address this unmet need, we used a CHIKV nanoluciferase reporter virus to develop a high-throughput screening assay for novel small-molecule inhibitors. From this campaign, we identified several unique inhibitors of CHIKV replication. Mechanistic characterization of two inhibitors suggests that both target the nsP4 RNA-dependent RNA polymerase. Susceptibility profiling pinpointed unique nsP4 mutations that specifically conferred resistance but also decreased viral fitness in competition studies. In silico docking and resistance profiling analyses indicated potential binding sites of the inhibitors near the polymerase active site. Collectively, these results define multiple chemotypes for further development and highlight novel molecular targets within nsP4 for CHIKV inhibition.IMPORTANCEChikungunya virus is a mosquito-borne pathogen that has caused millions of human infections worldwide, producing severe fever, rash, and long-lasting joint pain that can persist for months. Related viruses, such as Mayaro and Ross River viruses, also cause debilitating disease, yet no antiviral drugs are available to treat any infection caused by this family of viruses. In this study, we developed a high-throughput assay that allowed us to rapidly identify compounds capable of blocking chikungunya virus replication. We discovered new hit compounds that inhibit virus growth. Our results suggest that the two most promising hit candidates target the viral nsP4 polymerase. By identifying these novel inhibitors and characterizing both their mechanisms of action and resistance profiles, we have established the groundwork for future efforts to develop much-needed therapies against chikungunya virus and related pathogens.