A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

Abstract

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in 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 viral spike protein processing.

Potentin vitroanti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

“…Taken together, these data suggest that the antiviral activity of gallinamide A is not due to inhibition of viral (MPro and PLpro) protease targets but likely due to inhibition of host-derived CatL. The level of CatL differs substantially in these cell lines which supports published data that some cell lines are more sensitive to cysteine protease inhibition than others ( 23 ).…”

“…Given that CatB was detected in our proteomics studies using VeroE6 and A549 lysates ( Figure S2 ), and it has also been recently reported that a broad-spectrum activity-based probe for cysteine proteases was active against both proteases in cell lysates, ( 23 ) we also calculated the k inact /K i values for gallinamide A and the selected lead analogues against CatB. This revealed selectivity values ranging from 8-fold for compound 17 to 112-fold for gallinamide A ( Figure 4F, Figure S5 ) for CatL over CatB.…”

“…There are already a number of FDA-approved drugs that possess inhibitory activity against CatL ( 27 ). Furthermore, recent studies have shown that K777, a potent irreversible inhibitor of CatL consisting of a dipeptide-vinyl sulfone, was found to be well tolerated in rodents, dogs and nonhuman primates ( 56 ), and an IND has been opened with the FDA for its use as a therapeutic treatment of COVID-19 infection ( 23 ). Importantly, synthetic gallinamide A analogues have already been safely examined in vivo for other infectious indications ( 39 ).…”

“…Host cell entry requires priming of the S protein by cleavage at the S1/S2 and the S2ʹ site that enables fusion of viral and cellular membranes. This cleavage has been shown to be carried out primarily by the membrane-bound serine protease TMPRSS2, ( 19 ) but can also be performed by the cysteine protease, cathepsin L (CatL) ( 22, 23 ). Following entry of the virus into the cell via an endosomal pathway, CatL is responsible for S1 cleavage at acidic pH, conditions where TMPRSS2 is not catalytically functional.…”

“…This dual role of CatL in the establishment and progression of COVID-19 pathology has therefore reinforced the enzyme as a key drug target for SARS-CoV-2 ( 27 ). The importance of both TMPRSS2 and CatL for facilitating viral entry and replication is highlighted by the effectiveness of TMPRSS2 inhibitors, camostat mesylate ( 19 ) and nafamostat mesylate ( 28 ) and the pan-cysteine protease inhibitors E64d ( 19 ) and K777 ( 23 ) to reduce virus infection levels of SARS-CoV-2 (and SARS-CoV) in a range of human cell lines. Following uncoating and release of the viral RNA from the endosome, translation of the two large viral reading frames gives rise to the polyproteins pp1a and pp1ab that are subsequently processed by two viral proteases: the main protease (Mpro) and the papain-like protease (PLpro) ( 18 ).…”