Development of High Throughput Screening to Discover SARS-CoV-2 Main Protease Inhibitors with NanoBit Complementation: OAJBS Publishers

Development of High Throughput Screening to Discover SARS-CoV-2 Main Protease Inhibitors with NanoBit Complementation by Jianxi Liu* in Open Access Journal of Biomedical Science (OAJBS)

SARS-CoV-2 has become a serious global public health problem and there is lack of effective drugs to fight it. Main protease (Mpro) of SARS-CoV-2 is an important antiviral drug target. We aim to establish a high-throughput screening (HTS) assay to identify inhibitors of SARS- CoV-2 Mpro. A Mpro NanoBiT assay for HTS was developed by using NanoBiT complementary strategy. Mpro peptide substrate was flanked by subunits of Nano luciferase (NanoLuc), SmBiT and LgBiT respectively at the amino and carboxyl termini to obtain the structure Mpro cleavage site-NanoBiT. Mpro cleavage site-NanoBiT showed strong NanoLuc activity, which was modulated with Mpro peptide digestion, indicating the inhibitory effect of compounds on Mpro could be read out with NanoLuc activity. We optimized the experiment conditions including the quantity of Mpro and Mpro cleavage site-NanoBiT, DMSO tolerance, enzyme digestion time, and plate readout time after adding NanoLuc substrate. We further evaluated with two Mpro reference inhibitors, GC376 and Mpro inhibitor 11a, and found the assay was suitable for HTS of Mpro inhibitors. The miniaturized 384-well plate format assay was further used to screen 6,400 compounds from FDA-approved Drug Library and Natural Bioactive Compound Library. Control experiments showed a significant separation of relative activity of the negative and positive controls leading to an acceptable HTS Z’ value of 0.67, indicating satisfactory assay performance. Our results demonstrated that our established Mpro NanoBiT assay by inserting protease substrate peptides between SmBiT and LgBiT of NanoBiT could be applied for discovery of inhibitors of different proteases.
https://biomedscis.com/fulltext/development-of-high-throughput-screening-to-discover-sars-cov-2-main-protease-inhibitors-with-nanobit-complementation.ID.000360.php

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