A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Chen, K.Y.; Krischuns, Tim; Varga, L.; Harigua‐Souiai, Emna; Paisant, Sylvain; Zettor, Agnès; Chiaravalli, Jeanne; Delpal, Adrien; Courtney, David; O’Brien, Amornrat; Baker, Susan C.; Decroly, Étienne; Isel, Catherine; Agou, Fabrice; Jacob, Yves; Blondel, Arnaud; Naffakh, Nadia

doi:10.1016/j.antiviral.2022.105272

Cited by 14 publications

(9 citation statements)

References 75 publications

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“…9 b Encouraged by the importance of this target and guided by the results obtained from the computational analysis through docking and molecular dynamics simulations, we tested the two hits for their ability to inhibit M pro in a highly sensitive cell-based luciferase assay that we developed to monitor SARS-CoV-2 main protease activity. 36 This gain-of-function assay is based on a reverse-nanoluciferase (Rev-Nluc) reporter in which two nanoluciferase domains are permuted and linked together by a cleavage site recognized by M pro . Co-expression with the wild-type SARS-CoV-2 M pro results in cleavage of the reporter and thereby a significant reduction in luciferase activity.…”

Section: Resultsmentioning

confidence: 99%

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors

et al. 2023

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Section: Resultsmentioning

confidence: 99%

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors

et al. 2023

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“…This is especially valuable, since rescue systems for CoVs are challenging to develop due to their large genome size and the risk of altering the virus fitness due to genomic manipulation. Several cell-based assays were previously developed to measure 3CL pro intracellular activity, and techniques such as Flip-GFP, Flip-Firefly luciferase, reverse nano-luciferase were shown to be useful to conduct chemical screen and identify potential new inhibitors. , The ability to measure 3CL pro activity in cells using a reporter with high sensitivity and a wide dynamic range is a powerful tool for chemical compound screens. Moreover, other factors such as cell integrity, permeability, and compound toxicity can be simultaneously evaluated, providing a more comprehensive and accurate analysis of potential candidate compounds.…”

Section: Resultsmentioning

confidence: 99%

Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path

Bram

Duan

Nilsson-Payant

et al. 2022

ACS Bio Med Chem Au

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The SARS-CoV-2 pandemic is an ongoing threat to global health, and the continuing emergence of contagious variants highlights the urgent need for additional antiviral therapy to attenuate COVID-19 disease. The SARS-CoV-2 main protease (3CLpro) presents an attractive target for such therapy due to its high sequence conservation and key role in the viral life cycle. In this study, we designed a fluorescent–luminescent cell-based reporter for the detection and quantification of 3CLpro intracellular activity. Employing this platform, we examined the efficiency of known protease inhibitors against 3CLpro and further identified potent inhibitors through high-throughput chemical screening. Computational analysis confirmed a direct interaction of the lead compounds with the protease catalytic site and identified a prototype for efficient allosteric inhibition. These developments address a pressing need for a convenient sensor and specific targets for both virus detection and rapid discovery of potential inhibitors.

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“…T A B L E 3 Overview of active compounds and their labels used in the manuscript. , respectively [85,86] (see Supporting Information Section 4). A first brute-force screening at 100 μM showed a single compound for each of the three proteases (see red bars in Figure 4a-c).…”

Section: Experimental Testing Of Candidatesmentioning

confidence: 99%

“…The increase in fluorescence intensity over time is proportional to the rate constant of the protease, and by adding compounds at different concentrations, inhibitors can be identified. As positive controls, GC376 (IC 50 = 9.4 � 2.5 nM) and GRL0617 (IC 50 = 2.8 � 0.4 μM) were used for Nsp5 and Nsp3, respectively[85,86] (see Supporting Information Section 4). A first brute-force screening at 100 μM showed a single compound for each of the three proteases (see red bars in Figure…”

mentioning

confidence: 99%

A community effort in SARS‐CoV‐2 drug discovery

Schimunek,

Seidl,

Elez

et al. 2023

Molecular Informatics

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The COVID‐19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small‐molecule drugs that are widely available, including in low‐ and middle‐income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the “Billion molecules against COVID‐19 challenge”, to identify small‐molecule inhibitors against SARS‐CoV‐2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find ‘consensus compounds’. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding‐, cleavage‐, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS‐CoV‐2 treatments.

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A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Cited by 14 publications

References 75 publications

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors

Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path

A community effort in SARS‐CoV‐2 drug discovery

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