Discovery and characterization of novel chikungunya virus nsP2 inhibitors

Infectious diseases, including those caused by viral pathogens, remain a major cause of global morbidity and mortality. Chikungunya virus (CHIKV) is an emerging arbovirus responsible for explosive outbreaks of acute febrile illness that often gives rise to chronic, debilitating arthralgia. Despite its significant public health impact, there are currently no FDA-approved antivirals for CHIKV or other alphavirus infections. The viral non-structural protein 2 (nsP2) protease is an attractive target for therapeutics due to its essential role in viral replication and pathogenesis.Here, we report the development and optimization of a high-throughput screening platform for CHIKV nsP2 protease using an internally quenched fluorescent peptide substrate. Following assay optimization and validation, screening of diversity-oriented chemical libraries (>100,000 compounds) and a cysteine-reactive covalent fragment library identified the vinyl sulfone inhibitor RA-0002034 as a potent inhibitor of nsP2 protease enzymatic activity and alphavirus replication. Multipronged characterization efforts demonstrated selectivity of the inhibitor for the active site cysteine, as well as minimal off-target inhibition of human cysteine proteases. Subsequent medicinal chemistry approaches established the structure-activity relationship of the scaffold and yielded analogs with improved potency, covalent reactivity, and pharmacokinetic profiles. Additional discovery efforts utilizing display technologies and targeted protein degradation strategies, as well as efforts targeting the nsP2 helicase domain with small molecule inhibitors, are also presented, providing complementary approaches for the development of nsP2-targeted antivirals.