Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Abstract

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (M^pro), its moderate activity in M^pro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Fig. 1. Cell entry pathways of SARS-CoV-2.

After initial virus binding with its S protein to the ACE2 receptor, cell entry proceeds via two pathways. In case of cell surface entry, the S-protein is cleaved by TMPRSS2, inducing membrane fusion and viral genome release. In case of the endosomal entry, the viral particle is internalized by clathrin-mediated endocytosis and subsequently the S-protein is activated by CatL in the endosome, followed by membrane fusion and genome release.

Fig. 2. Inhibitor binding to the active site of CatL.

a Domain structure of CatL with Calpeptin derived from prodrug S-Calpeptin bound to the active site. b Close-up view of the active site of CatL with bound S-Calpeptin. Residues involved in the catalytic mechanism are highlighted in stick representation. c Surface representation of the empty active site of CatL with the catalytic cysteine highlighted in yellow. Calpeptin derived from S-Calpeptin (d) and the GC-376 aldehyde (e) bound to the CatL active site with highlighted subsites S1–S3.

Fig. 3. Calpeptin and S-Calpeptin inhibit SARS-CoV-2 infection.

The viral titer (●) and the cell viability (♦) of VERO-CCL81 (a, b) and human LC-HK2 (c, d) cells were determined by RT-qPCR and CellTiter-Glo luminescence method, respectively. EC₅₀ values for viral titers are shown. Individual data points and the mean (―) of three replicates of two biological experiments are displayed as line. EC₅₀ was calculated by fitting the data using GraphPad Prism version 8.0.

Fig. 4. Calpeptin and S-Calpeptin inhibit SARS-CoV-2 mediated CPE.

Calpeptin or S-Calpeptin were added to VERO-CCL81 at 2-fold serial dilutions (a, b) and to human LC-HK2 at 10-fold serial dilutions (c, d). CPE inhibition was determined via CellTiter-Glo® Luminescent Assay. EC₅₀-values are shown. Individual data points (●) and the mean (―) of three replicates of two biological experiments for VERO-CCL81 cells (a, b) and LC-HK2 cells (c, d) are displayed. Compounds concentrations are presented in log scale for interpolation. EC₅₀ was calculated by fitting the data using GraphPad Prism version 8.0.

Fig. 5. S-Calpeptin treatment reduces viral load in trachea in a hamster SARS-CoV-2 infection model.

Golden Syrian hamsters were intranasally inoculated with SARS-CoV-2 on day 0. Daily treatment started on day 1 with 1 mg/kg S-Calpeptin subcutaneously (s.c.) and 1:100 DMSO (dimethyl sulfoxide) as control. Viral load was measured at day 3, 5, and 7 post infection in lungs and trachea. a SARS-CoV-2 RNA level expressed as viral RNA copies/copies of β-actin/g of tissue. b Infectious viral particles expressed as 50% tissue culture infective dose/g of tissue. n = 5 animals per group. Values are expressed in median, interquartiles and range. Statistically significant differences (p ≤ 0.05) were determined by two-sided Mann–Whitney U-test.