Department of NanoEngineering, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Angew Chem Int Ed Engl. 2022 Feb 21;61(9):e202113617. doi: 10.1002/anie.202113617. Epub 2022 Jan 14.
The main protease (M ) and papain-like protease (PL ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of M and PL is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of M and PL by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by M and PL . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 M and PL . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.
主蛋白酶（M）和木瓜蛋白酶样蛋白酶（PL）在 SARS-CoV-2 复制中发挥着关键作用，是抗病毒抑制剂的有前途的靶标。同时可视化 M 和 PL 对于 SARS-CoV-2 的检测和快速抑制剂筛选非常有价值。然而，由于缺乏合适的探针，这样的重要研究仍然具有挑战性。我们现在已经开发了一种双色探针（3MBP5），用于通过荧光（或Förster）共振能量转移（FRET）同时检测 M 和 PL。该探针通过 M 和 PL 切割的 Cy3 和 Cy5 荧光团产生荧光。用重组蛋白、抑制剂、转染质粒的 HEK 293T 细胞和 SARS-CoV-2 感染的 TMPRSS2-Vero 细胞证明了 3MBP5 的激活特异性。双色探针的结果首先验证了 SARS-CoV-2 M 和 PL 的同时检测和细胞内分布。这是一种用于同时检测不同蛋白酶的强大工具，对于快速筛选抑制剂具有重要价值。
