Assessing Chikungunya Virus Protease Activity and Pharmacological Inhibition Using the acc-CHIK15-dnp Fluorogenic Peptide Substrate.

Chikungunya virus, a mosquito-borne alphavirus, poses a significant global health threat due to its rapid spread and lack of approved antiviral therapies. The viral nonstructural protein 2 protease (nsP2p) plays a critical role in processing the viral polyprotein to produce key viral enzymes, making it an attractive target for drug development. In this study, we describe the expression and purification of recombinant CHIKV nsP2 protease and establish a robust fluorogenic assay to measure its enzymatic activity using the acc-CHIK15-dnp peptide substrate. The assay enables real-time monitoring of proteolytic cleavage and provides a platform for evaluating small-molecule inhibitors. We present optimized conditions for protein expression in E. coli, affinity purification, and tag removal, achieving high-purity nsP2 protease suitable for biochemical characterization. Using this system, we also provide a robust protocol for determining IC₅₀ values for candidate inhibitors. This work provides a reproducible methodology for high-throughput screening of CHIKV protease inhibitors, facilitating the discovery of potential antiviral compounds against chikungunya virus.