A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, N.I.H., Extramural
Grant support
N01 AI030048
NIAID NIH HHS - United States
R24 AI120942
NIAID NIH HHS - United States
- MeSH
- Antiviral Agents pharmacology MeSH
- Chlorocebus aethiops MeSH
- Phenylalanine pharmacology MeSH
- Spike Glycoprotein, Coronavirus chemistry metabolism MeSH
- Cysteine Proteinase Inhibitors pharmacology MeSH
- Virus Internalization drug effects MeSH
- Cathepsin L antagonists & inhibitors metabolism MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Cell Line, Tumor MeSH
- Piperazines pharmacology MeSH
- Protein Domains MeSH
- Proteolysis MeSH
- SARS-CoV-2 drug effects MeSH
- Tosyl Compounds pharmacology MeSH
- Vero Cells MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, N.I.H., Extramural MeSH
- Names of Substances
- Antiviral Agents MeSH
- Phenylalanine MeSH
- Spike Glycoprotein, Coronavirus MeSH
- Cysteine Proteinase Inhibitors MeSH
- Cathepsin L MeSH
- N-pip-phenylalanine-homophenylalanine-vinyl sulfone phenyl MeSH Browser
- Piperazines MeSH
- spike protein, SARS-CoV-2 MeSH Browser
- Tosyl Compounds MeSH
Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.
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