Biochemical screening for SARS-CoV-2 main protease inhibitors

Abstract

The Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) pandemic represents a global challenge. SARS-CoV-2's ability to replicate in host cells relies on the action of its non-structural proteins, like its main protease (M pro). This cysteine protease acts by processing the viruses' precursor polyproteins. As proteases, together with polymerases, are main targets of antiviral drug design, we here have performed biochemical high throughput screening (HTS) with recombinantly expressed SARS-CoV-2 M pro. A fluorescent assay was used to identify inhibitors in a compound library containing known drugs, bioactive molecules and natural products. These screens led to the identification of 13 inhibitors with IC 50 values ranging from 0.2 μM to 23 μM. The screens confirmed several known SARS-CoV M pro inhibitors as inhibitors of SARS-CoV-2 M pro , such as the organo-mercuric compounds thimerosal and phenylmercuric acetate. Benzophenone derivatives could also be identified among the most potent screening hits. Additionally, Evans blue, a sulfonic acid-containing dye, could be identified as an M pro inhibitor. The obtained compounds could be of interest as lead compounds for the development of future SARS-CoV-2 drugs.

The Inhibitory Effects of Plant Derivate Polyphenols on the Main Protease of SARS Coronavirus 2 and Their Structure–Activity Relationship

“…The COVID-19 pandemic started in December 2019 and has extended until now; there are no approved therapeutics for this disease. Although numerous researchers performed computational chemistry [ 45 ] and virtual screening of FDA-approved drugs [ 46 ] and flavonoid compounds [ 47 , 48 , 49 , 50 ] with M pro , their efficiencies against M pro need to be confirmed by in vitro and in vivo experiments. To the best of our knowledge, among our tested polyphenols, EGCG and tannic acid have been reported to have inhibitory effects on M pro with IC 50 values of 7.6 μg/mL and 2.1 μM, respectively [ 23 , 45 ].…”

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

“…First, the influence of different buffers was investigated. Most SARS-CoV-2 M pro biochemical assays were conducted using Tris buffer 9, 12–15, 19–21 or FIEPES buffer. 11, 22–23 We found no significant difference between Tris and phosphate buffer, whereas the fluorescence increase over time was lower when using a HEPES buffer (Figure 1A).…”

Biochemical features and mutations of key proteins in SARS-CoV-2 and their impacts on RNA therapeutics

“…In another study, with a recombinant M pro , Coelho et al conducted HTS using a fluorescent assay to identify potential inhibitors from drugs, small molecules, and natural products, leading to the discovery of 13 M pro inhibitors (e.g. thimerosal, phenylmercuric acetate, and benzophenone derivatives) with a low IC50 values (0.2–23 μM) [28] .…”