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Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays
Chunlong Ma; Haozhou Tan; Juliana Choza; Yuying Wang; Jun Wang.
Affiliation
  • Chunlong Ma; University of Arizona
  • Haozhou Tan; University of Arizona
  • Juliana Choza; University of Arizona
  • Yuying Wang; University of Arizona
  • Jun Wang; University of Arizona
Preprint in English | bioRxiv | ID: ppbiorxiv-458041
Journal article
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ABSTRACT
SARS-CoV-2 main protease (Mpro) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based Flip-GFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/458041v1_ufig1.gif" ALT="Figure 1"> View larger version (41K) org.highwire.dtl.DTLVardef@b0c310org.highwire.dtl.DTLVardef@d652deorg.highwire.dtl.DTLVardef@da8d0corg.highwire.dtl.DTLVardef@62449b_HPS_FORMAT_FIGEXP M_FIG C_FIG Flip-GFP and Protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate the reported SARS-CoV-2 Mpro inhibitors.
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Full text: Available Collection: Preprints Database: bioRxiv Type of study: Prognostic study Language: English Year: 2021 Document type: Preprint
Full text: Available Collection: Preprints Database: bioRxiv Type of study: Prognostic study Language: English Year: 2021 Document type: Preprint
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