{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T07:00:53Z","timestamp":1775890853187,"version":"3.50.1"},"posted":{"date-parts":[[2025,2,2]]},"group-title":"Biochemistry","reference-count":55,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,2,2]],"date-time":"2025-02-02T00:00:00Z","timestamp":1738454400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,2,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Chikungunya virus (CHIKV) is an emerging pathogen with pandemic potential. CHIKV infection in humans is transmitted by mosquitoes and induces common symptoms of high fever, arthralgia and myalgia. Because no specific antiviral drugs for treatment of CHIKV infection are available, drug development remains a central goal. The chikungunya virus protease nsP2 (CHIKVP) has emerged as a key drug target due to its indispensable role in viral replication via cleavage of the viral polyprotein. To date, effective tools for screening for CHIKVP inhibitors that reflect the most critical polyprotein cleavage sites have been lacking, hampering drug-development efforts. We found that the recognition ability of CHIKVP is sensitive to the length of peptide substrates. In this study, we report a robust fluorogenic substrate comprising a 15-mer peptide derived from the nsP3\/4 junction from the CHIKV polyprotein. This peptide is flanked by an ACC-Lys(dnp) donor-quencher pair. Our new substrate acc-CHIK\n                  <jats:sub>15<\/jats:sub>\n                  -dnp shows a 20-fold improved signal-to-noise ratio as compared to the previously reported edab\n                  <jats:sub>8<\/jats:sub>\n                  substrate, which is also based on the nsP3\/4 junction. We found acc-CHIK\n                  <jats:sub>15<\/jats:sub>\n                  -dnp is recognized only by CHIKVP but not by other alphavirus proteases. This is surprising due to the high level of sequence conservation in the alpha virus polyprotein junctions and indicates that the P-side residues are more important than the P\u2019-side sequence for effective CHIKVP cleavage. The robust signal-to-noise ratio obtained using acc-CHIK\n                  <jats:sub>15<\/jats:sub>\n                  -dnp derived from the nsP3\/4 cleavage site enabled much improved small molecule HTS on CHIKV relative to other fluorogenic reporters.\n                <\/jats:p>","DOI":"10.1101\/2025.02.01.636040","type":"posted-content","created":{"date-parts":[[2025,2,2]],"date-time":"2025-02-02T22:40:23Z","timestamp":1738536023000},"source":"Crossref","is-referenced-by-count":0,"title":["A Robust Fluorogenic Substrate for Chikungunya Virus Protease (nsP2) Activity"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7622-1502","authenticated-orcid":false,"given":"Sparsh","family":"Makhaik","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States"},{"name":"Center for Bioactive Delivery in the Institute for Applied Life Sciences, University of Massachusetts, Amherst, 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