Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

Ma, Chunlong; Tan, Haozhou; Choza, Juliana; Wang, Yuying; Wang, Jun

doi:10.1016/j.apsb.2021.10.026

Cited by 59 publications

(104 citation statements)

References 68 publications

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“…The mechanism of action of EACC, KY-226, and tropifexor in inhibiting SARS-CoV-2 PL pro was further characterized in the thermal shift assay and the cell-based FlipGFP PL pro assay. 15,[19][20]23 Similar to the positive control GRL0617, all three hits displayed dose-dependent binding to PL pro as revealed by the enhanced melting temperatures with increasing drug concentration (Figure 2). Next, we tested the three hits in the FlipGFP PL pro assay.…”

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 97%

“…The hits were further counter screened against the SARS-CoV-2 M pro to rule out promiscuous cysteine protease inhibitors. [19][20][21][22] It was found that EACC and KY-226 were not active (IC 50 ≥ 60 µM), while tropifexor had weak inhibition with an IC 50 of 43.65 µM, which corresponds to a selectivity index (SI) of 8.5 (Figure 1E). These results suggest the inhibition of SARS-CoV-2 PL pro by tropifexor is specific.…”

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

“…Next, we tested the three hits in the FlipGFP PL pro assay. 15,[19][20] The FlipGFP PL pro was recently developed by us as a surrogate assay to quantify the cellular activity of PL pro inhibitors in the biological safety level 2 facility, and we have shown that there is positive correlation between the FlipGFP IC 50 values with the SARS-CoV-2 antiviral EC 50 values. 15 The FlipGFP assay is a virus free cell-based protease assay in which the 293T cells were transfected with PL pro and the GFP reporter.…”

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

“…The advantages of FlipGFP assay compared to the FRET assay is that it can rule out compounds that are cytotoxic, membrane impermeable, and having off-target effects that prevent cellular on-target engagement. [19][20] In the FlipGFP assay, the positive control GRL0617 showed dose-dependent inhibition with an IC 50 of 14.67 µM, while the negative control GC376 was not active (IC 50 > 60 µM) (Figure 3A, B). The results from EACC and KY-226 were not conclusive due to the cytotoxicity of the compounds.…”

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

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Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

Wang

Choza

et al. 2021

Preprint

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The global COVID-19 pandemic underscores the dire need of effective antivirals. Encouraging progress has been made in developing small molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 µM. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 µM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC50 of 4.03 µM, a 7.8-fold increase compared to GRL0617 (EC50 = 31.4 µM). Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.

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Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 97%

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

Section: High-throughput Screening Of Sars-cov-2 Pl Pro Inhibitorsmentioning

confidence: 99%

See 2 more Smart Citations

Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

Wang

Choza

et al. 2021

Preprint

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“…(Cao et al, 2020 ; Chu et al, 2004 ; Li & De Clercq, 2020 ; Ma et al, 2021 ; Sheahan, Sims, Leist, et al, 2020 ; Zhang, Lin, et al, 2020 )…”

Section: Types Of Agents In Clinical Trailsunclassified

Races of small molecule clinical trials for the treatment of COVID‐19: An up‐to‐date comprehensive review

Jiang

Xiang

et al. 2021

Drug Development Research

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The coronavirus disease‐19 (COVID‐19) pandemic has become a global threat since its first outbreak at the end of 2019. Several review articles have been published recently, focusing on the aspects of target biology, drug repurposing, and mechanisms of action (MOAs) for potential treatment. This review gathers all small molecules currently in active clinical trials, categorizes them into six sub‐classes, and summarizes their clinical progress. The aim is to provide the researchers from both pharmaceutical industries and academic institutes with the handful information and dataset to accelerate their research programs in searching effective small molecule therapy for treatment of COVID‐19.

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SARS‐CoV‐2 Papain‐Like Protease: Structure, Function and Inhibition

Ullrich

Nitsche

2022

ChemBioChem

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Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PL pro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.

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Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

Cited by 59 publications

References 68 publications

Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

Races of small molecule clinical trials for the treatment of COVID‐19: An up‐to‐date comprehensive review

SARS‐CoV‐2 Papain‐Like Protease: Structure, Function and Inhibition

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