Aprotinin Inhibits SARS-CoV-2 Replication

Abstract

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.

Keywords: 2019-nCoV; COVID-19; antiviral; aprotinin; drug discovery; severe acute respiratory syndrome coronavirus; severe acute respiratory syndrome coronavirus 2.

Figure 1

Anti-severe acute respiratory syndrome virus 2 (SARS-CoV-2) effects of aprotinin and SERPINA1/alpha-1 antitrypsin. (A) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. (B) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1. (C) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.

Figure 2

Effects of aprotinin on SARS-CoV-2-induced caspase 3/7 activation. Caspase 3/7 activity was determined in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) 48 h post-infection.

Figure 3

Anti-SARS-CoV-2 effects of aprotinin when administered post-infection. For post-infection experiments, the cells were incubated with the virus for a one-hour adsorption period. Then, the cells were washed three times in PBS prior to the addition of the drug. (A) The effects of aprotinin and the RNA polymerase inhibitor remdesivir (a positive control drug that interferes with virus replication after virus entry) on virus replication as determined by qPCR in SARS-CoV-2/FFM1 (MOI of 0.1)-infected Caco2 cells 8 h post-infection (after approximately one round of replication). * p < 0.05 as determined by one-way ANOVA and Dunnett’s multiple comparison test. (B) The effects of aprotinin on cytopathogenic effect (CPE) formation in SARS-CoV-2/FFM1 (MOI of 0.01)-infected Caco2 cells were determined 48 h post-infection.

Figure 4

Regulation of host cell protease inhibitors in SARS-CoV-2-infected cells. (A) Total protein levels based on a publicly available proteomics dataset [35], indicating cellular levels of endogenous protease inhibitors in SARS-CoV-2 (MOI of 1)-infected Caco2 cells 2 h and 24 h post-infection. Data were normalized using summed intensity normalization for sample loading, followed by internal reference scaling and trimmed mean of M normalization. * p-values as determined using a two-sided Student’s t-test. (B) Mean protein translation of endogenous protease inhibitors in arbitrary units (AU) (normalized and corrected summed peptide spectrum matches (PSMs) were averaged) in SARS-CoV-2 (MOI of 1)-infected Caco2 cells 2 h and 24 h post-infection based on a publicly available translatome dataset [35]. * p-values as determined using a two-sided Student’s t-test.

Figure 5

Antiviral effects of aprotinin in SARS-CoV-2-infected air–liquid interface (ALI) cultures from primary bronchial epithelial cells. (A) Abundance of the SARS-CoV-2 proteins N (nucleocapsid) and M (membrane) in primary bronchial epithelial cell ALI cultures infected with SARS-CoV-2/FFM1 (MOI of 1) in the presence or absence of aprotinin (20 µM) as determined 5 days post-infection by multiplexed mass spectrometry analysis using acquisition targeting of previously identified viral peptides modified with TMTpro. The detailed data are presented in Table S1. (B) Western blots indicating cellular SARS-CoV-2 N and TMPRSS2 levels in primary bronchial epithelial cell ALI cultures infected with SARS-CoV-2/7/Human/2020/Frankfurt (FFM7) (MOI of 1) in the presence or absence of aprotinin as detected 5 days post infection. GAPDH was served as the loading control. Uncropped Western blots are shown in Figure S4.