Fig 2: SerpinB3 variants designed with modified RCL sequences to inhibit TMPRSS2 and furin. (A) Schematic representation of coronavirus spike protein with cleavage sites. (B) The structure of native serpin archetype with RCL highlighted in magenta, containing a protease cleavage site (P1-P1’). Amino acid substitutions are highlighted in blue in the table, with the SerpinB3-WT sequence shown in black. The image was made in PyMol using the PDB file code 1QLP.

Fig 3: SerpinB3 variants inhibited cell entry of lentiviral S-pseudoparticles and SARS-CoV-2 infection and replication. (A) A549-ACE2-TMPRSS2 cells were incubated for 1 h with media (control) or with increasing concentrations of SerpinB3 variants or CM, followed by infection with SARS-CoV-2 S pseudoparticles (in 1:1 ratio). Luciferase activities (representing pseudoparticle entry) were analysed in cell lysates at 48 h post-pseudoparticle infection. (B) VeroE6/TMPRSS2 cells were treated with increasing concentrations of SerpinB3 variants 1 h before, simultaneously with, and 18 h post-infection with SARS-CoV-2, with the percentage of SARS-CoV-2 infected cells determined using flow cytometry to detect virus nucleocapsid staining. The mean ± SEM from n = 2 (A) or n = 3 (B) independent experiments performed in triplicates. Two-way ANOVA with Tukey’s comparison test: * p < 0.05, ** p < 0.002, *** p < 0.0005. CM, Camostat Mesylate.

Fig 4: Structures of rationally designed covalent reversible kbt inhibitors (4 through 7) of TMPRSS2.

Fig 5: Combined PS-SCL for substrate specificity of TMPRSS2, matriptase, hepsin, and HGFA. Using protease substrate terminology, the cleavage (hydrolysis) site for protease protein substrates is between the N-terminal P1 and P1' positions of the peptide (P) substrate, while S1 and S1' refer to the subsite (S) 1 and 1' of the protease where the P1 and P1' amino acid side chain binds (46). Red color designates overlap of TMPRSS2 amino acid specificity with other proteases. Single letter designations for amino acids are shown.