[PEPTIDE INSTITUTE] Fluorescent Substrates of SARS-CoV/SARS-CoV-2 Main Proteases
Once severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells, large precursor proteins are translated from the viral RNA. The precursor proteins are cleaved by various proteases to form functional proteins required for viral replication, such as RNA-dependent RNA polymerase. SARS-CoV-2 Mpro, one of the cleavage products, plays a major role in the cleavage of precursor proteins and thus is a protease essential for the replication of the virus. Therefore, inhibitors of this "main protease" have the potential to become antiviral drugs and are being developed for practical use worldwide.
Peptide Institute, Inc. provides three types of substrate for SARS-CoV main protease (SARS-CoV Mpro or 3CLpro) and SARS-CoV-2 main protease (SARS-CoV-2 Mpro). Protease activity can be determined by observing increased fluorescence due to the cleavage of substrates by the main protease. Please utilize these substrates for exploration of inhibitors or other research.
This product is a FRET-based, quenching fluorescent substrate of SARS-CoV/SARS-CoV-2 Mpro1),2),3). A fluorescent Edans group and a quenching Dabcyl group are bound to the precursor protein sequence Thr-Ser-Ala-Val-Leu-Gln↓Ser-Gly-Phe-Arg-Lys-Met [↓: cleavage site]. Fluorescence increases when SARS-CoV Mpro cleaves the Gln-Ser sequence between the fluorescent and quenching groups. Protease activity is determined by observing this fluorescence.。
[KM=17 μM、kcat=1.9 s-1 (kinetic parameters for SARS-CoV Mpro]1)
[KM=33 μM (kinetic parameter for SARS-CoV-2 Mpro)]3)
Figure. Method for measuring protease activity of SARS-CoV/SARS-CoV-2 Mpro using a FRET-based, quenching fluorescent substrate
These products are 4-methylcoumarin-7-amide (MCA)-type fluorescent substrates; aminomethylcoumarin (AMC) is bound to two sequences with high affinity for SARS-CoV-2 Mpro, as reported by Drag et al.4). When the nonfluorescent MCA substrate is cleaved by the SARS-CoV-2 Mpro, highly fluorescent AMC is released. Protease activity is determined by observing this fluorescence. (Ex: 380 nm / Em: 460 nm)
- C.-J. Kuo, Y.-H. Chi, J.T.-A. Hsu, and P.-H. Liang, Biochem. Biophys. Res. Commun., 318, 862 (2004).
- L. Zhang, D. Lin, X. Sun, U. Curth, C. Drosten, L. Sauerhering, S. Becker, K. Rox, and R. Hilgenfeld, Science, 368, 409 (2020).
- C. Ma, M. D. Sacco, B. Hurst, J. A. Townsend, Y. Hu, T. Szeto, X. Zhang, B. Tarbet, M. T. Marty, Y. Chen, and J. Wang, bioRxiv2020.04.20.051581.
- W. Rut, K. Groborz, L. Zhang, X. Sun, M. Zmudzinski, R. Hilgenfeld, and M. Drag, bioRxiv2020.03.07.981928.
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