Protein engineering of Serpins to alter protease specificity: Exploring the therapeutic potential of SerpinB3 and recombinant variants to inhibit SARS-CoV-2 cell entry and replication

The serpins (serine protease inhibitors), a diverse protein superfamily renowned for their roles as natural inhibitors of various plasma proteolytic processes, encompassing coagulation and fibrinolysis, while also exhibiting diverse functions such as tumour suppression and hormone transport. Serpins irreversibly bind to target serine or cysteine proteases, acting as suicide inhibitors. Some serpins have shown therapeutic potential, exemplified by recombinant alpha-1 antitrypsin (A1AT) administration for conditions like A1AT deficiency and COPD. A specific member of the human serpin family, SerpinB3, stands out for its ability to target cysteine proteases, particularly exhibiting potency against human cathepsin L (CatL) and moderate inhibition of cathepsins S, K, V. Cathepsins, cysteine proteases pivotal in lysosomal proteolysis, are implicated in various pathological processes such as cancer progression, osteoporosis, and parasitic infections. Cathepsin K (CatK), notably abundant in bone osteoclasts, plays a crucial role in bone resorption and remodelling, making it a prime target for diseases like osteoporosis, osteoarthritis, and metastatic bone disease. Despite extensive efforts, drugs targeting CatK have faced challenges, including off-target effects. A study utilizing combinatorial peptide libraries investigated cathepsin specificities, revealing distinct preferences, with CatK favouring proline in the P2 position. Following this, we hypothesized that altering the SerpinB3 reactive centre loop (RCL) might enhance specificity for CatK, potentially improving inhibition efficacy or generating a better substrate with reduced inhibition. Through site-directed mutagenesis, a P2 mutant (F352P) of SerpinB3 was generated, demonstrating improved inhibition of CatK, with preliminary data showing a 2.5-fold increase in inhibition while reducing CatL inhibition by 5-fold. Additionally, the designed SerpinB3 variant was assessed for glycosaminoglycan binding in the presence of heparin, revealing a significant 9-fold increase in inhibitory capacity when interacting with heparin. Amid the pandemic, the focus of the project shifted to COVID-19 in March 2020. COVID-19 infections, caused by the SARS-CoV-2 virus, are initiated when the virus binds to the ACE-2 receptor on host cells, leading to the cleavage of its spike (S) protein by host cell proteases such as TMPRSS2, furin, and CatL. While A1AT has shown efficacy in inhibiting TMPRSS2 and suppressing cell infection, its broad protease inhibition presents limitations. To address this, we engineered modified variants of SerpinB3, a natural CatL inhibitor, with a specific aim to target TMPRSS2 and furin. Mutagenesis of the RCL of SerpinB3 was carried out to alter amino acids to preferred residues for each protease. Recombinant proteins were expressed, purified, and their inhibitory profiles were successfully altered as anticipated. In vitro assays demonstrated their ability to block the cleavage of the SARS-CoV-2 spike protein by the relevant protease. Purified recombinant SerpinB3 and its variants were then tested in cell infection assays and shown to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and to restrict SARS-CoV-2 replication in VeroE6 cells expressing TMPRSS2. Notably, the B3-TMP construct designed to inhibit TMPRSS2 displayed greater potency and selectivity than A1AT, effectively inhibiting both pseudoparticle entry and viral replication. B3-TMP exhibited an 18-fold higher TMPRSS2 inhibition compared to A1AT and was more effective in blocking viral replication. Moreover, the B3-TMP construct demonstrated potent inhibition for both an Alpha clinical isolate (WT, D614G) and Omicron (BA.5) cell entry and replication, whereas the native SerpinB3 CatL inhibitor was only effective for Omicron (BA.5). These findings underscore the potential of serpins and designer serpins as promising biological anti-viral agents.