A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

Resnick, Samuel J; Iketani, Sho; Hong, Seo Jung; Zask, Arie; Liu, Hengrui; Kim, Sung‐Soo; Melore, Schuyler; Nair, Manoj S.; Huang, Yaoxing; Tay, Nicholas E. S.; Rovis, Tomislav; Yang, Hee Won; Stockwell, Brent R.; Ho, David D.; Chavez, Alejandro

doi:10.1101/2020.08.29.272864

Cited by 11 publications

(14 citation statements)

References 45 publications

(56 reference statements)

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“…GRL-0496 also inhibited SARS-CoV-2 3CL pro in cells, but its activity and cytotoxicity were poorly separated, such that luciferase reporter activity actually decreased from 25 to 100 µM ( Figure 5 , Table 1 ). GRL-0496 was originally developed as an inhibitor of SARS-CoV 3CL pro [ 44 ] but has also been found to be active against SARS-CoV-2 [ 45 ]. None of the remaining compounds had significant activity in our assay ( Figure S1 , Table 1 ), despite reported inhibition against SARS-CoV-2 3CL pro in vitro and/or virus replication in cell culture.…”

Section: Resultsmentioning

confidence: 99%

“…Two additional cell-based reporter assays have been described in pre-print manuscripts. Resnick and co-workers have developed a system in which inhibitors are identified by their ability to prevent 3CL pro -mediated cytotoxicity, as measured by crystal violet staining [ 45 ]. While simple in principle, approaches based on measuring cytotoxicity could potentially identify compounds that inhibit 3CL pro -mediated toxicity through indirect effects, such as effects on gene expression or protein stability.…”

Section: Discussionmentioning

confidence: 99%

“…None of these compounds was able to inhibit SARS-CoV-2 3CL pro to the same extent as GC376, however. All four compounds have been previously reported to block SARS-CoV-2 replication in cell culture [ 18 , 20 , 24 , 45 ].…”

Section: Discussionmentioning

confidence: 99%

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Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

Rawson

Duchon

Nikolaitchik

et al. 2021

Viruses

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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

Rawson

Duchon

Nikolaitchik

et al. 2021

Viruses

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“…To confirm that baicalein and the crude extract inhibit viral replication by directly targeting SARS-CoV-2 3CL pro in cells, we tested their anti-SARS-CoV-2 3CL pro activity under complex cell environment. As the expression of SARS-CoV-2 3CL pro is toxic to mammalian cells 36 , we used the mixture of HEK293T cell lysate and purified SARS-CoV-2 3CL pro for the test. Both baicalein and the crude extract showed inhibition activity under this condition (Figure S2).…”

Section: Baicalein Binds Sars-cov-2 3cl Pro To Inhibit Its Activitymentioning

confidence: 99%

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

Liu

Sun

et al. 2021

Journal of Enzyme Inhibition and Medicinal Chemistry

222

151

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COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL pro ) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL pro activity in vitro with IC 50 's of 8.52 mg/ml and 0.39 mM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC 50 's of 0.74 mg/ml and 2.9 mM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL pro activity at mM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL pro , demonstrating their potential as broad-spectrum anti-coronavirus drugs.

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“…To provide physiologically meaningful testing of molecules without the use of live SARS-CoV-2 virus at biosafety level 3 (BSL3), specific assays namely “surrogate assays” were investigated [ 127 ]. Resnick S.J.…”

Section: Enzymatic Assaysmentioning

confidence: 99%

SARS-CoV-2 Mpro: A Potential Target for Peptidomimetics and Small-Molecule Inhibitors

et al. 2021

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The uncontrolled spread of the COVID-19 pandemic caused by the new coronavirus SARS-CoV-2 during 2020–2021 is one of the most devastating events in the history, with remarkable impacts on the health, economic systems, and habits of the entire world population. While some effective vaccines are nowadays approved and extensively administered, the long-term efficacy and safety of this line of intervention is constantly under debate as coronaviruses rapidly mutate and several SARS-CoV-2 variants have been already identified worldwide. Then, the WHO’s main recommendations to prevent severe clinical complications by COVID-19 are still essentially based on social distancing and limitation of human interactions, therefore the identification of new target-based drugs became a priority. Several strategies have been proposed to counteract such viral infection, including the repurposing of FDA already approved for the treatment of HIV, HCV, and EBOLA, inter alia. Among the evaluated compounds, inhibitors of the main protease of the coronavirus (Mpro) are becoming more and more promising candidates. Mpro holds a pivotal role during the onset of the infection and its function is intimately related with the beginning of viral replication. The interruption of its catalytic activity could represent a relevant strategy for the development of anti-coronavirus drugs. SARS-CoV-2 Mpro is a peculiar cysteine protease of the coronavirus family, responsible for the replication and infectivity of the parasite. This review offers a detailed analysis of the repurposed drugs and the newly synthesized molecules developed to date for the treatment of COVID-19 which share the common feature of targeting SARS-CoV-2 Mpro, as well as a brief overview of the main enzymatic and cell-based assays to efficaciously screen such compounds.

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A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

Cited by 11 publications

References 45 publications

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

SARS-CoV-2 Mpro: A Potential Target for Peptidomimetics and Small-Molecule Inhibitors

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