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[Preprint]. 2020 Aug 11:2020.07.17.207019.
doi: 10.1101/2020.07.17.207019.

Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

Wei Zhu  1 , Miao Xu  1 , Catherine Z Chen  1 , Hui Guo  1 , Min Shen  1 , Xin Hu  1 , Paul Shinn  1 , Carleen Klumpp-Thomas  1 , Samuel G Michael  1 , Wei Zheng  1
Affiliations

Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

Wei Zhu et al. bioRxiv. .

Update in

Abstract

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emphasized the urgency to develop effective therapeutics. Drug repurposing screening is regarded as one of the most practical and rapid approaches for the discovery of such therapeutics. The 3C like protease (3CL pro ), or main protease (M pro ) of SARS-CoV-2 is a valid drug target as it is a specific viral enzyme and plays an essential role in viral replication. We performed a quantitative high throughput screening (qHTS) of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds using a SARS-CoV-2 3CL pro assay. Twenty-three small molecule inhibitors of SARS-CoV-2 3CL pro have been identified with IC50s ranging from 0.26 to 28.85 μM. Walrycin B (IC 50 = 0.26 µM), Hydroxocobalamin (IC 50 = 3.29 µM), Suramin sodium (IC 50 = 6.5 µM), Z-DEVD-FMK (IC 50 = 6.81 µM), LLL-12 (IC 50 = 9.84 µM), and Z-FA-FMK (IC 50 = 11.39 µM) are the most potent 3CL pro inhibitors. The activities of anti-SARS-CoV-2 viral infection was confirmed in 7 of 23 compounds using a SARS-CoV-2 cytopathic effect assay. The results demonstrated a set of SARS-CoV-2 3CL pro inhibitors that may have potential for further clinical evaluation as part of drug combination therapies to treating COVID-19 patients, and as starting points for chemistry optimization for new drug development.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Schematic representation of the fluorogenic SARS-CoV-2 protease enzymatic assay. The peptide substrate exhibits low fluorescent because the fluorescence intensity of Edans in the C-terminal is quenched by the Dabcyl in the N-terminal of the substrate. The protease cleaves the substrate which breaks the proximity of the quencher molecule Dabcyl with the fluorophore Edans, resulting in an increase in fluorescence signal. This increase in fluorescence signal is proportional to the protease activity.
Figure 2.
Figure 2.
SARS-CoV-2 3CLpro enzyme assay optimization. (a) Concentration-response curve of enzyme titration. With a fixed concertation of substrate (20 μM), the fluorescent intensity increased with enzyme concentrations. The linear response was observed at low enzyme concentrations. Measurement was conducted 2 h after initiating the reaction at RT. (b) The signal-to-basal (S/B) ratios of three enzyme concentrations within the linear range, at various incubation times. Dotted line represents the S/B = 1. (c) Enzyme kinetics. Michealis-Menton plot exhibited a Km of 75.41 μM and Vmax of 1392 RFU/min for SARS-CoV-2 3CLpro. (d) The S/B ratios determined at RT and 37 °C. No difference was observed in 1 h incubation between the two temperatures.
Figure 3.
Figure 3.
(a) Concentration response of the known 3CLpro inhibitor, GC376. An IC50 of 0. 17 μM was determined for the inhibition of SARS-CoV-2 3CLpro. The substrate concentration was 20 μM and enzyme concentration was 50 nM in this experiment. (b) Scatter plot of the results from a DMSO plate in the 3CLpro enzymatic assay in a 1536-well plate, where columns 1 and 2 in the plate contain substrate only, column 3 includes GC376 titration (1:3 dilution series from 57.5 μM), and columns 5–48 contain DMSO (23 nL DMSO in 4 μL reaction solution).
Figure 4.
Figure 4.
Concentration-response curves of the 6 most potent compounds with IC50 values < 15 μM and maximal inhibition > 80% determined in the SARS-CoV-2 3CLpro enzyme assay. Enzyme assay (blue) and counter screen (red) curves correspond to left y-axis showing inhibitory results, CPE (black) and cytotoxicity (green) curves correspond to right y-axis showing cell viability. (a) Walrycin B, IC50 = 0.26 μM. (b) Hydroxocobalamin, IC50 = 3.29 μM. (c) Z-DEVD-FMK, IC50 = 6.81 μM. (d) Suramin sodium, IC50 = 6.5 μM. (e) LLL-12, IC50 = 9.84 μM. (f) Z-FA-FMK, IC50 = 11.39 μM. Primary CPE and cytotoxicity screens were conducted in 4 concentrations, only the hits were further confirmed with 8 concentrations with dilution ratio of 1:3.
Figure 5.
Figure 5.
Concentration-response curves of the 9 compounds with partial quenching effect and CPE activity. (a) Anacardic Acid, CPE efficacy = 89.62%. (b) Adomeglivant, CPE efficacy = 49.2%. (c) Eltrombopag olamine, efficacy = 74.39%. (d) GSK-3965, efficacy = 22.51. (e) GW574, CPE efficacy = 63.3%. (f) Hexachlorophene, CPE efficacy = 47.57%. (g) MK-886, CPE efficacy = 69.18%. (h) AMG-837, CPE efficacy = 106.31%. (i) MG-149, CPE efficacy = 70%.
Figure 6.
Figure 6.
Predicted binding models of (a) Z-DEVD-FMK and (b) Z-FA-FMK bound to the active site of 3CLpro. The protein 3CLpro (grey) is represented in ribbons and the active site is shown with the hydrophobic protein surface. Small molecule inhibitors are shown in sticks. The catalytic residue Cys145 in the binding pocket is highlighted.
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