Identification of C270 as a novel site for allosteric modulators of SARS-CoV-2 papain-like protease
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Abstract
The papain-like protease (PL pro ) in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins, and plays an important role in dysregulation of host immune response. PL pro is a promising therapeutic target with a major challenge in inhibitor design due to the restricted S1/S2 sites for two consecutive glycine of substrates. Here we reported the discovery of two activators of the SARS-CoV-2 PL pro from a biochemical screening, and the identification of the unique residue, C270, as an allosteric and covalent regulation site for the activators. This site was also specifically modified by glutathione oxidized, resulting in the S-glutathionylation and activation of the protease. Furthermore, one compound was found to allosterically inhibit the protease by covalent binding to this crucial site. Together, these results elucidated an unrevealed molecular mechanism for allosteric modulation of the protease’s activity, and provided a new strategy for discovery of allosteric inhibitors of the SARS-CoV-2 PL pro .
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SciScore for 10.1101/2022.03.30.486313: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Membranes were first incubated with the primary antibody (Anti-Glutathione antibody [D8], ab19534, Anti-Glutathionesuggested: (Abcam Cat# ab19534, RRID:AB_880243); Mouse anti DDDDK-Tag (FLAG-tag) mAb, AE005, ABclonal) in 5% BSA TBST for 16 h at 4 °C, washed with TBST three times, then incubated with the secondary antibody (HRP-labeled Goat Anti-Mouse IgG(H+L), A0216, Beyotime) in 5% BSA TBST (1 h, 25 °C), and washed with TBST three times. anti DDDDK-Tag (FLAG-tagsuggested: NoneAnti-Mouse…SciScore for 10.1101/2022.03.30.486313: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Membranes were first incubated with the primary antibody (Anti-Glutathione antibody [D8], ab19534, Anti-Glutathionesuggested: (Abcam Cat# ab19534, RRID:AB_880243); Mouse anti DDDDK-Tag (FLAG-tag) mAb, AE005, ABclonal) in 5% BSA TBST for 16 h at 4 °C, washed with TBST three times, then incubated with the secondary antibody (HRP-labeled Goat Anti-Mouse IgG(H+L), A0216, Beyotime) in 5% BSA TBST (1 h, 25 °C), and washed with TBST three times. anti DDDDK-Tag (FLAG-tagsuggested: NoneAnti-Mouse IgG(H+L)suggested: NoneExperimental Models: Cell Lines Sentences Resources In the present study, HEK293T cells were seeded in 12-well plates overnight. HEK293Tsuggested: NoneRecombinant DNA Sentences Resources For bacterial expression, the cDNA encoded the SARS-CoV-2 PLpro with E. coli codon optimization was ordered from GenScript and cloned into the pET15b expression vector with an N-terminal 6 × His-SUMO2 fusion tag. pET15bsuggested: RRID:Addgene_129689)For transfection of mammalian cell, the cDNA encoded the SARS-CoV-2 PLpro with mammalian codon optimization was also ordered from GenScript and cloned into the pcDNA 3.1 with an C-terminal FLAG tag. pcDNA 3.1suggested: RRID:Addgene_20407)The sequence of pcDNA3-PL-flipGFP-T2A-mCherry was designed based on plasmid pcDNA3-TEV-flipGFP-T2A-mCherry (Addgene catalog NO.124429) where TEV cleave site was replaced by SARS-CoV-2 PLpro cleavage site (LRGGAPTK), and ordered from GenScript. pcDNA3-PL-flipGFP-T2A-mCherrysuggested: NonepcDNA3-TEV-flipGFP-T2A-mCherrysuggested: NoneSoftware and Algorithms Sentences Resources The resulting kobs values were then plotted versus compound concentrations ([C]), then kinact and Ki or Ka values were calculated according to the equation: kobs = kinact × ([C]/([C] + Ki or Ka)) using GraphPad Prism. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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