A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

Drake M. Mellott, Chien Te Tseng, Aleksandra Drelich, Pavla Fajtová, Bala C. Chenna, Demetrios H. Kostomiris, Jason Hsu, Jiyun Zhu, Zane W. Taylor, Klaudia I. Kocurek, Vivian Tat, Ardala Katzfuss, Linfeng Li, Miriam A. Giardini, Danielle Skinner, Ken Hirata, Michael C. Yoon, Sungjun Beck, Aaron F. Carlin, Alex E. ClarkLaura Beretta, Daniel Maneval, Vivian Hook, Felix Frueh, Brett L. Hurst, Hong Wang, Frank M. Raushel, Anthony J. O'Donoghue, Jair Lage De Siqueira-Neto, Thomas D. Meek, James H. McKerrow

Research output: Contribution to journalArticlepeer-review

62 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)642-650
Number of pages9
JournalACS Chemical Biology
Issue number4
StatePublished - Apr 16 2021

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine


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