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Fraser, Bryan

University of British Columbia

SARS-CoV-2 exploits the activity of surface-expressed human proteases to achieve maturation of its Spike (S) protein and undergo conformational changes that enable virus-host membrane fusion and efficient infection of the airways. Early in the COVID-19 pandemic, Transmembrane Protease, Serine-2 (TMPRSS2) was recognized as a key proteolytic driver of SARS-CoV-2 infection of the human aerodigestive tract, analogous to its role in SARS-CoV-1 cleavage activation, but no TMPRSS2-specific drugs had been developed. This lack of progress was in part due to challenges procuring a recombinant source of the active protease. This thesis describes the first production and purification of fully matured TMPRSS2 protease through protein engineering efforts targeting its zymogen activation motif, leading to its first crystal structures and biochemical interrogations of its interaction with recombinant SARS-CoV-2 S protein substrates. The closely related TMPRSS3 and -4 proteases, which belong to the same Type II Transmembrane Serine Protease (TTSP) family as TMPRSS2, were similarly produced and biochemical investigations revealed that matured TMPRSS3 efficiently cleaves the S protein, potentially explaining SARS-CoV-2 tropisms in cells expressing TMPRSS3 and not TMPRSS2. A platform to identify potent and selective TMPRSS-targeted antivirals was developed using the natural TMPRSS protease inhibitor protein, hepatocyte growth factor activator inhibitor-2 (HAI-2) as a novel scaffold. The protease engineering and inhibition strategies established here may enable future interrogations of TTSP pathobiology in vitro and in vivo and rationally select the most critical proteases for viral pathobiology to disable with potent and selective pharmacological inhibitors, improving our pandemic preparedness against respiratory viruses exploiting this entry pathway.

Text

2024-08-19

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88963

Interdisciplinary Oncology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/