Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

被引：0
|
作者：
Voget, Rabea [1 ]
Breidenbach, Julian [1 ]
Claff, Tobias [1 ]
Hingst, Alexandra [1 ]
Sylvester, Katharina [1 ]
Steinebach, Christian [1 ]
Vu, Lan Phuong [1 ]
Weisse, Renato H. [2 ]
Bartz, Ulrike [3 ]
Straeter, Norbert [2 ]
Mueller, Christa E. [1 ]
Guetschow, Michael [1 ]
机构：
[1] Univ Bonn, Pharmaceut Inst, Pharmaceut & Med Chem, D-53121 Bonn, Germany
[2] Univ Leipzig, Inst Bioanalyt Chem, Ctr Biotechnol & Biomed, D-04103 Leipzig, Germany
[3] Univ Appl Sci Bonn Rhein Sieg, Dept Nat Sci, D-53359 Rheinbach, Germany
来源：
ACTA PHARMACEUTICA SINICA B | 2024年 / 14卷 / 05期
关键词：
COVID-19; SARS-CoV-2; Main protease; Peptide nitriles; Fluorogenic substrates; Active-site titration; X-ray crystallography; Inner filter effect; SUBSTRATE-INHIBITION; FLUORESCENCE;
D O I：
10.1016/j.apsb.2024.03.0012211-3835
中图分类号：
R9 [药学];
学科分类号：
1007 ;
摘要：
A titrant for the SARS-CoV-2 main protease (M-pro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active M-pro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of M-pro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the M-pro-catalyzed cleavage of these substrates were determined.
引用
收藏
页码：2349 / 2357
页数：9
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