Biology subjects

Fraser, B. J.

Publications and source records attributed to Fraser, B. J..

4 recordsLinked to original sources

bioRxiv2026

Hepatocyte growth factor activator inhibitor-2 rapidly inactivates airway-expressed human Type II Transmembrane Serine Proteases

Human Type II Transmembrane Serine Proteases (TTSPs) are essential entry factors for various influenza A and coronaviruses, and drive cancer metastasis when they are overexpressed by tumor cells. However, the natural inhibition mechanisms that regulate these proteases are not well understood. One natural transmembrane protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), has been shown to block TMPRSS2 activity and can prevent SARS-CoV-2 infection and reduce TMPRSS2-driven prostate cancer metastasis when overexpressed. In this study, we present biochemical and biophysical evidence showing that HAI-2 effectively inactivates TMPRSS2 and other TTSPs only after they have undergone zymogen activation. Through mutagenesis and ligand binding assays, we demonstrate that Kunitz Domain 1 (KD1) and KD2 can form stable ternary complexes with TMPRSS2 and other TTSPs, but do not employ the typical Laskowski inhibitor mechanism found for other macromolecular serine protease inhibitors. We also show that HAI-2 proteins do not inhibit the coagulation protease thrombin and that multivalent human IgG-tagged HAI-2 proteins are highly potent TMPRSS2 inhibitors. Our findings provide a mechanistic understanding of how TTSP activity is regulated in human airway cells and offer a foundation for developing engineered soluble HAI-2 proteins as anti-TTSP antivirals and anti-cancer therapeutics.

biochemistry↗

bioRxiv2025

Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 {+/-} 0.1 {micro}M). A 1.9 [A] crystal structure of the hCatK-Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.

biochemistry↗

bioRxiv2024

Structural basis of TMPRSS11D specificity and autocleavage activation

Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We demonstrate these proteases can spontaneously and efficiently cleave their own zymogen activation motifs, thereby activating their wider protease activity on other cellular substrates. We determined TMPRSS11D co-crystal structures in complexes with a native TMPRSS11D zymogen activation motif and with an engineered activation motif, providing insights into TMPRSS11D autocleavage activation and revealing unique regions of its substrate binding cleft. We further show that a protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. These insights into human protease viral tropism and into liabilities with existing human serine protease inhibition strategies will guide future drug discovery campaigns for these targets.

biochemistry↗

bioRxiv2021

Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation

Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-coronavirus-2 (SARS-CoV-2). Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) Protein, enabling virus-host membrane fusion and infection of the lungs. We present here an efficient recombinant production strategy for enzymatically active TMPRSS2 ectodomain enabling enzymatic characterization, and the 1.95 [A] X-ray crystal structure. To stabilize the enzyme for co-crystallization, we pre-treated TMPRSS2 with the synthetic protease inhibitor nafamosat to form a stable but slowly reversible (15 hour half-life) phenylguanidino acyl-enzyme complex. Our study provides a structural basis for the potent but non-specific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that will guide future generations of inhibitors to improve selectivity. TMPRSS2 cleaved recombinant SARS-CoV-2 S protein ectodomain at the canonical S1/S2 cleavage site and at least two additional minor sites previously uncharacterized. We established enzymatic activity and inhibition assays that enabled ranking of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.7 nM to 120 M and determination of inhibitor mechanisms of action. These results provide a body of data and reagents to support future drug development efforts to selectively inhibit TMPRSS2 and other type 2 transmembrane serine proteases involved in viral glycoprotein processing, in order to combat current and future viral threats. SUMMARY PARAGRAPHViruses hijack the biochemical activity of host proteins for viral invasion and replication. Transmembrane protease, serine-2 (TMPRSS2) is a surface-expressed protease implicated in the activation of influenza A, influenza B, and coronaviruses, including SARS-CoV-2, to drive efficient infection of the lungs1-5. TMPRSS2 is an attractive target for antiviral therapies, as inhibiting its proteolytic activity blocks efficient viral entry5,6. However, a structural and biochemical understanding of the protease has remained elusive and no selective inhibitors are available. We engineered on-demand activatable TMPRSS2 ectodomain and determined the 1.95 [A] X-ray crystal structure of the stabilized acyl-enzyme after treatment with nafamostat, a protease inhibitor under investigation as a COVID-19 therapeutic. The structure reveals unique features of the TMPRSS2 substrate recognition pocket and domain architecture, and explains the potent, but nonselective inhibition by nafamostat. TMPRSS2 efficiently cleaved the SARS-CoV-2 S protein at the canonical S1/S2 site as well as two minor sites previously uncharacterized. We further established a robust enzymatic assay system and characterized inhibition by two additional clinical protease inhibitors under study for COVID-19, camostat and bromhexine. Our results provide a body of data and reagents to enable ongoing drug development efforts to selectively inhibit TMPRSS2 and other TTSPs involved in viral glycoprotein processing, in order to combat current and future viral threats.

biochemistry↗