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

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ACS Chem Biol


. 2021 Mar 31.
doi: 10.1021/acschembio.0c00875. Online ahead of print.
A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells


Drake M Mellott, Chien-Te Tseng ¹ , Aleksandra Drelich ¹ , Pavla Fajtov? ^{2 3} , Bala C Chenna, Demetrios H Kostomiris, Jason Hsu ¹ , Jiyun Zhu, Zane W Taylor, Klaudia I Kocurek, Vivian Tat ¹ , Ardala Katzfuss, Linfeng Li, Miriam A Giardini ² , Danielle Skinner ² , Ken Hirata ² , Michael C Yoon ² , Sungjun Beck ² , Aaron F Carlin ⁴ , Alex E Clark ² , Laura Beretta ² , Daniel Maneval, Vivian Hook ² , Felix Frueh, Brett L Hurst, Hong Wang, Frank M Raushel, Anthony J O'Donoghue ² , Jair Lage de Siqueira-Neto ² , Thomas D Meek, James H McKerrow ²



Affiliations

• PMID: 33787221
• DOI: 10.1021/acschembio.0c00875

Abstract

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 ?M), 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 ?M. There was no toxicity to any of the host cell lines at 10-100 ?M 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.