Study explores structure, activity, and inhibition of TMPRSS2 implicated in SARS-CoV-2 activation

Viruses inhibit the biochemical activity of host proteins and use them for invasion and replication inside the host. Transmembrane protease serine-2 (TMPRSS2) is an endothelial cell surface protein implicated in the activation of influenza A, B, and coronaviruses, including the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to drive infection of the lungs. It is a key host cell factor that helps viral entry and pathogenesis of the novel coronavirus, SARS-CoV-2.

TMPRSS2 proteolytically processes the spike (S) protein of SARS-CoV-2 and enables virus-host membrane fusion and infection of the lung tissue. This makes TMPRSS2 an attractive target for antiviral therapies, as inhibition of the proteolytic activity of TMPRSS2 blocks viral entry. However, a sound structural and biochemical understanding of the protease is lacking, and selective inhibitors of TMPRSS2 are not available.

Production and structure of TMPRSS2 ectodomain

TMPRSS2 is a transmembrane serine protease (TTSP) with a single-pass transmembrane domain, an intracellular domain, and a biologically active ectodomain with 3 subdomains: a Class A scavenger receptor cysteine-rich (SRCR) domain, a low-density lipoprotein receptor type-A (LDLR-A) domain, and a C-terminal trypsin-like serine peptidase (SP) domain with a canonical Ser441-His296-Asp345 catalytic triad.

TMPRSS2 is produced as a single-chain proenzyme, or zymogen, and requires cleavage at a conserved Arg255- Ile256 peptide bond within its SRQSR255↓IVGGE activation motif to achieve full maturation of its enzymatic activity.

Researchers from Canada recently presented an efficient strategy for recombinant production of enzymatically active TMPRSS2 ectodomain, enabling enzymatic characterization and the 1.95 Å X-ray crystal structure. Their work is published on the bioRxiv* preprint server while awaiting peer-review.

The researcher synthesized the on-demand activatable TMPRSS2 ectodomain and illustrated the 1.95 Å X-ray crystal structure of the stable acyl-enzyme after treatment with nafamostat. This synthetic protease inhibitor is being investigated for use as a COVID-19 therapeutic.

The researchers pre-treated TMPRSS2 with nafamostat to stabilize the enzyme for co-crystallization. The pretreatment forms a stable but slowly reversible phenylguanidino acyl-enzyme complex with a 15-hour half-life.

“We have produced and characterized a source of TMPRSS2 enzyme that will enable rapid inhibitor development as antivirals and thorough molecular interrogation of coronavirus and influenza virus activation.”

Findings offer valuable data to help future drug development efforts to selectively inhibit transmembrane serine proteases

The study offers a structural basis for the potent and non-specific inhibition of TMPRSS2 by nafamostat. It also identifies the distinct features of the TMPRSS2 substrate-binding pocket that guides future generations of inhibitors and improves selectivity.

Engineered activation and structural characterization of stabilized TMPRSS2 ectodomain. a Full-length, membrane bound TMPRSS2 zymogen undergoes autocleavage activation at the Arg255-Ile256 peptide bond and the matured enzyme proteolytically processes SARS-CoV-2 Spike protein docked to the ACE2 receptor to drive viral membrane fusion. b Engineered recombinant TMPRSS2 ectodomain containing the low-density lipoprotein receptor type-A (LDLR) domain, a Class A Scavenger Receptor Cysteine-Rich (SRCR) domain and a C-terminal trypsin-like serine peptidase (SP) domain, features an enteropeptidase-cleavable DDDDK255 substitution to facilitate controlled zymogen activation. The non-catalytic (LDLR+SRCR) and catalytic (SP) chains are tethered by a disulfide bond and the activation status can be interrogated by SDS-PAGE under non-reducing and reducing (5% β-mercaptoethanol) conditions. c X-ray crystal structure of activated TMPRSS2 ectodomain pre-treated with nafamostat (yellow sticks). d The interdomain disulfide pair (Cys244-Cys365) maintains covalent attachment of the SRCR and SP domains. e Close-up view of the SP catalytic triad residues (His296, Asp345 and Ser441) and the post-activation Asp440:Ile256 salt bridge showing complete maturation of the protease. Nafamostat treatment results in phenylguanidino acylation of Ser441. Polar contacts are shown as yellow dashed lines.
Engineered activation and structural characterization of stabilized TMPRSS2 ectodomain. a Full-length, membrane bound TMPRSS2 zymogen undergoes autocleavage activation at the Arg255-Ile256 peptide bond and the matured enzyme proteolytically processes SARS-CoV-2 Spike protein docked to the ACE2 receptor to drive viral membrane fusion. b Engineered recombinant TMPRSS2 ectodomain containing the low-density lipoprotein receptor type-A (LDLR) domain, a Class A Scavenger Receptor Cysteine-Rich (SRCR) domain and a C-terminal trypsin-like serine peptidase (SP) domain, features an enteropeptidase-cleavable DDDDK255 substitution to facilitate controlled zymogen activation. The non-catalytic (LDLR+SRCR) and catalytic (SP) chains are tethered by a disulfide bond and the activation status can be interrogated by SDS-PAGE under non-reducing and reducing (5% β-mercaptoethanol) conditions. c X-ray crystal structure of activated TMPRSS2 ectodomain pre-treated with nafamostat (yellow sticks). d The interdomain disulfide pair (Cys244-Cys365) maintains covalent attachment of the SRCR and SP domains. e Close-up view of the SP catalytic triad residues (His296, Asp345 and Ser441) and the post-activation Asp440:Ile256 salt bridge showing complete maturation of the protease. Nafamostat treatment results in phenylguanidino acylation of Ser441. Polar contacts are shown as yellow dashed lines.

TMPRSS2 cleaved recombinant viral spike protein ectodomain at the canonical S1/S2 cleavage site and at least 2 additional previously uncharacterized minor sites. The researchers established enzymatic activity and inhibition assays enabling ranking of clinical protease inhibitors with half-maximal inhibitory concentrations from 1.7 nM to 120 μM and determination of mechanisms of actions of inhibitors.

The researchers further established a powerful enzymatic assay system and characterized inhibition by two other clinical protease inhibitors under investigations for use in the treatment of COVID-19 - camostat and bromhexine.

According to the authors, biochemical characterization of secreted enzymes is required to interpret their activation status and subunit organization, as an active form of TMPRSS2 in the extracellular environment could have significant implications in pathobiology and therapeutic targeting.

The findings of this study offer valuable data and reagents to support drug development efforts in the future to selectively inhibit TMPRSS2 and other types of 2 transmembrane serine proteases that have a role in viral glycoprotein processing in order to fight against the current and future viral pandemic threats.

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.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Susha Cheriyedath

Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.

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