Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro.
| Autor: | Kovar P; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Richardson PL; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Korepanova A; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Afanador GA; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Stojkovic V; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Li T; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Schrimpf MR; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Ng TI; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Degoey DA; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Gopalakrishnan SM; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA., Chen J; SMTPT, AbbVie Discovery, AbbVie, 1 N Waukegan Rd., North Chicago, IL 60065, USA. Electronic address: jun.chen@abbvie.com. |
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| Jazyk: | angličtina |
| Zdroj: | SLAS discovery : advancing life sciences R & D [SLAS Discov] 2024 Sep; Vol. 29 (6), pp. 100179. Date of Electronic Publication: 2024 Aug 14. |
| DOI: | 10.1016/j.slasd.2024.100179 |
| Abstrakt: | The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2024. Published by Elsevier Inc.) |
| Databáze: | MEDLINE |
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