Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication

Abstract

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.

Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation

“…Indeed, α1AT is the most abundant inhibitor of NSPs in plasma and α1AT deficiency (AATD) is the most important genetic predisposition to emphysema and chronic obstructive pulmonary disease, where proteases drive the pathology. Moreover, α1AT binds to TMPRSS2 at high concentrations, impairing S processing by TMPRSS2 and preventing SARS-CoV-2 entry to some extent (48)(49)(50)(51). Our results, however, challenge the notion that reduction of JCI Insight 2024;9(7):e174133 https://doi.org/10.1172/jci.insight.174133 NSP activity is a critical target in severe COVID-19 patients and our data are supported by recent epidemiological and clinical trial results.…”

Bacterial Proteases as Potentially Exploitable Modulators of SARS-CoV-2 Infection: Logic from the Literature, Informatics, and Inspiration from the Dog

“…Given the exceptional proteomic diversity present within the pan-species protease/ antiprotease spectrum, the best actionable evidence might emerge from a confluence of corroborating evidence, such as exacerbatory or inhibitory roles of bacterial species that also encode proteases or antiproteases similar to COVID-19-related host proteins. As evidence of this, it should be noted that experimental mutagenetic manipulation of human SERPINb3 transformed an antiprotease with minimal capacity for TMPRSS2 inhibition into a TMPRSS2 blocker more potent than SERPINa3 [179]. Since evolutionary pressures can foster rapid bacterial mutations, it is possible that some forms of bacteria may have adapted their proteolytic capacity for the purpose of modifying human TMPRSS2 activity, as suggested in studies relating to the synergistic interplay among TMPRSS2, androgen control, and microbiome composition [180][181][182][183].…”

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions

“…A three-dimensional lung cell model using primary cells from multiple donors found that more resistant donors to an in vitro SARS-CoV-2 infection expressed higher transcript levels of SERPINA1 (AAT gene), SERPINE1, and SERPINE2 in the airway cells [70]. Variants of SerpinB3-through mutagenesis of its RCL-have been generated and found to be more potent in inhibiting TMPRSS2 than AAT [71]. We found that the AAT inhibition of TMPRSS2 activity in primary hAEcs was augmented significantly by enoxaparin [18].…”