Dual Inhibition of Cathepsin L and 3CL-Pro by GC-376 Constrains SARS Cov2 Infection Including Omicron Variant
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (ScreenIT)
Abstract
Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 were capable of inhibiting prototypic (USA-WA1/2020, Lineage A) SARS CoV2 induced CPE in highly susceptible cell lines. Moreover, these drugs inhibited delta (Lineage-B.1.617.2) and omicron (Lineage-B.1.1.529) infection with equal potency showing that the newer mutations harbored in these variants did not affect the mechanism of action of these drugs such as cathepsin-L or 3CL-Pro inhibition. Moreover, our early evidence that 3CL-Pro inhibition can effectively inhibit omicron-induced CPE in highly susceptible cell lines suggests that the recently FDA-approved oral drug, a 3CL-Pro inhibitor which is a combination of nirmatrelvir/ritonavir (Paxlovid) could be effective against omicron variant which shows reduced sensitivity to vaccines and mAb.
Importance
We report that cathepsin-L and 3CL-Pro as major targets for designing antivirals against SARS CoV2. Dual inhibition of cathepsin-L and 3CL-Pro by GC-376 renders it effective in inhibiting SARS CoV2-induced cytopathic effect in highly susceptible cell lines. Moreover, this candidate drug is equally effective against prototypic SARS CoV2 lineage A and emerging variants such as delta and omicron which show reduced sensitivity to vaccines and monoclonal antibodies. Given the recent wave of SARS CoV2 omicron variant infection around the world, and 3CL-Pro inhibitor nirmatrelvir is one of the components of the FDA-approved Paxlovid, our findings are timely, important and should be of broad interest.
Article activity feed
-
SciScore for 10.1101/2022.02.09.479835: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experiments were approved and performed per the guidelines set forth by the Institutional Biosafety Committee (IBC) and the Environmental Health and Safety Office (EHSO) at the University of Illinois at Chicago. 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 Cleavage of the spike protein was detected using anti-SARS-CoV Spike Protein (SDelta3) monoclonal antibody (7G12, Thermo Fisher Scientific, CA, USA)) raised against immunogen aa168-461 of the S1 domain. anti-SARS-CoV Spike Protein (SDelta3suggested: (Thermo Fisher Scientific Cat# …SciScore for 10.1101/2022.02.09.479835: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experiments were approved and performed per the guidelines set forth by the Institutional Biosafety Committee (IBC) and the Environmental Health and Safety Office (EHSO) at the University of Illinois at Chicago. 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 Cleavage of the spike protein was detected using anti-SARS-CoV Spike Protein (SDelta3) monoclonal antibody (7G12, Thermo Fisher Scientific, CA, USA)) raised against immunogen aa168-461 of the S1 domain. anti-SARS-CoV Spike Protein (SDelta3suggested: (Thermo Fisher Scientific Cat# MA5-35945, RRID:AB_2866557)Experimental Models: Cell Lines Sentences Resources Briefly, 1 x 10^6 293T ACE2 cells were nucleofected with sgRNA:TracrRNA: Cas9 complex using Amaxa Cell Line Nucleofector Kit V and cultured for 72h. ACE2suggested: NonePseudovirus entry inhibition assay: SARS CoV2 pseudotyped virus particles were produced by transfecting HEK293T cells with the following plasmids 1) lentiviral backbone plasmid-pHAGE-CMV-Luc2-IRES-ZsGreen that expresses luciferase and ZsGreen reporters. HEK293Tsuggested: None25 x 103 human ACE2 expressing 293T cells were seeded in 96 well plates and infected with bald pseudovirus without spike glycoprotein (negative control) and SARS CoV2 spike protein-expressing pseudovirus and treated with 1 μM concentration (50μl Infection medium 2% FBS + 150 μl pseudovirus) of the candidate inhibitory small molecules. 293Tsuggested: NoneCPE assay using infectious SARS CoV2: 25 x 103 hACE2-293T (BEI resources) and human TMPRSS2 expressing Vero E6 cells (Ordered from Japanese Collection of Research Bioresources Cell Bank (JCRB) and distributed by Sekisui XenoTech, LLC, KS) or Vero E6 cells (ATCC), Vero E6 Cells Expressing TMPRSS2 and Human ACE2 (Vero E6-TMPRSS2-T2A-ACE2) (NR-54970, BEI) were seeded in each well of 96 well plates in 100 μl of 5% DMEM medium. Vero E6suggested: NoneRecombinant DNA Sentences Resources 2) pHDM-Hgpm2-lentiviral helper plasmid expressing HIV Gag-Pol. pHDM-Hgpm2-lentiviralsuggested: None3) pHDM-tat1b: lentiviral helper plasmid expressing HIV Tat. pHDM-tat1bsuggested: RRID:Addgene_164442)4) pRC-CMV-Rev1b: lentiviral helper plasmid expressing HIV Rev, and pHDM expressing the SARS CoV2 Wuhan-Hu-1 Spike envelope Glycoprotein (Procured from BEI resources). pRC-CMV-Rev1bsuggested: RRID:Addgene_164443)Software and Algorithms Sentences Resources Statistical analysis: Statistical analyses were performed using Prism GraphPad (V9.0). GraphPadsuggested: (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:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- 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.
-
