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

Abstract

of High Throughput Screening to Discover SARS-CoV-2 Main Protease Inhibitors with NanoBit ABSTRACT SARS-CoV-2 has become a serious global public health problem and there is lack of effective drugs to fight it. Main protease (M pro ) 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 M pro . A M pro NanoBiT assay for HTS was developed by using NanoBiT complementary strategy. M pro peptide substrate was flanked by subunits of Nano luciferase (NanoLuc), SmBiT and LgBiT respectively at the amino and carboxyl termini to obtain the structure M pro cleavage site-NanoBiT. M pro cleavage site-NanoBiT showed strong NanoLuc activity, which was modulated with M pro peptide digestion, indicating the inhibitory effect of compounds on M pro could be read out with NanoLuc activity. We optimized the experiment conditions including the quantity of M pro and M pro cleavage site-NanoBiT, DMSO tolerance, enzyme digestion time, and plate readout time after adding NanoLuc substrate. We further evaluated with two M pro reference inhibitors, GC376 and M pro inhibitor 11a, and found the assay was suitable for HTS of M pro 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 M pro NanoBiT assay by inserting protease substrate peptides between SmBiT and LgBiT of NanoBiT could be applied for discovery of inhibitors of different proteases.