Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Antiviral Agents*/pharmacology ; Antiviral Agents*/chemistry ; SARS-CoV-2*/drug effects ; SARS-CoV-2*/enzymology ; Organoselenium Compounds*/pharmacology ; Organoselenium Compounds*/chemistry ; Isoindoles*/pharmacology ; Coronavirus 3C Proteases*/antagonists & inhibitors ; Coronavirus 3C Proteases*/metabolism ; Azoles*/pharmacology ; Azoles*/chemistry ; Proline*/analogs & derivatives ; Proline*/pharmacology ; Proline*/chemistry; Humans ; Molecular Docking Simulation ; Protease Inhibitors/pharmacology ; Protease Inhibitors/chemistry ; COVID-19 Drug Treatment ; COVID-19/virology ; HEK293 Cells ; Lactams ; Leucine/analogs & derivatives ; Sulfonic Acids

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S -transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

RSC Med Chem. 2020 Dec 21;12(3):370-379. (PMID: 34041486)
Commun Biol. 2023 Jul 5;6(1):694. (PMID: 37407698)
Commun Biol. 2022 Feb 25;5(1):169. (PMID: 35217718)
J Comput Chem. 2011 May;32(7):1456-65. (PMID: 21370243)
Science. 2021 Dec 24;374(6575):1586-1593. (PMID: 34726479)
J Biol Chem. 2024 Feb;300(2):105599. (PMID: 38159853)
Nucleic Acids Res. 2012 Jul;40(Web Server issue):W537-41. (PMID: 22570416)
J Virol. 2012 Nov;86(21):11754-62. (PMID: 22915796)
J Chem Phys. 2020 Apr 21;152(15):154102. (PMID: 32321259)
Signal Transduct Target Ther. 2020 Nov 17;5(1):269. (PMID: 33203855)
J Biol Chem. 2010 Jun 11;285(24):18134-8. (PMID: 20388714)
Molecules. 2023 Feb 02;28(3):. (PMID: 36771108)
J Chem Theory Comput. 2013 Jan 8;9(1):338-54. (PMID: 26589037)
Cell Res. 2020 Aug;30(8):678-692. (PMID: 32541865)
Biochem Biophys Res Commun. 2019 Sep 17;517(2):310-316. (PMID: 31353086)
Nutr Res Rev. 2020 Dec;33(2):190-217. (PMID: 32051057)
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W522-5. (PMID: 17488841)
Chemosphere. 2019 Aug;229:549-558. (PMID: 31100626)
Antiviral Res. 2021 Sep;193:105142. (PMID: 34303749)
Biol Trace Elem Res. 2013 Dec;156(1-3):36-44. (PMID: 24197605)
Sci Rep. 2023 Jun 6;13(1):9161. (PMID: 37280236)
J Chem Theory Comput. 2014 Apr 8;10(4):1518-1537. (PMID: 24803865)
J Mol Biol. 2007 Feb 23;366(3):965-75. (PMID: 17189639)
Protein Sci. 2018 Jan;27(1):112-128. (PMID: 28836357)
Phys Chem Chem Phys. 2016 Aug 10;18(32):22047-61. (PMID: 27215663)
Therap Adv Gastroenterol. 2012 May;5(3):179-88. (PMID: 22570678)
Nature. 2020 Jun;582(7811):289-293. (PMID: 32272481)
J Med Chem. 2022 May 12;65(9):6499-6512. (PMID: 35352927)
ACS Pharmacol Transl Sci. 2020 Oct 09;3(6):1265-1277. (PMID: 33330841)

19K07079 Ministry of Education, Culture, Sports, Science and Technology; 21H04920 Ministry of Education, Culture, Sports, Science and Technology; 19H05772 Ministry of Education, Culture, Sports, Science and Technology; 22K05345 Ministry of Education, Culture, Sports, Science and Technology; 24K09793 Ministry of Education, Culture, Sports, Science and Technology

Keywords: GC376; Mpro; SARS-CoV-2; binding energy calculation; boceprevir; carmofur; ebselen; selenoneine

0 (Antiviral Agents)
40X2P7DPGH (ebselen)
0 (Organoselenium Compounds)
0 (Isoindoles)
EC 3.4.22.28 (Coronavirus 3C Proteases)
0 (Azoles)
9DLQ4CIU6V (Proline)
89BT58KELH (N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide)
H1NMJ5XDG5 (GC376)
0 (Protease Inhibitors)
0 (Lactams)
GMW67QNF9C (Leucine)
0 (Sulfonic Acids)

Date Created: 20240619 Date Completed: 20240619 Latest Revision: 20240620

20240620

PMC11172239

10.3390/ijms25115767

38891954