Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

Abstract

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin–TMPRSS2–AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2–AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

“…Mechanistically, this infers that the expulsion of the AT RCL, rather than the formation of the ternary complex between protease, antiprotease, and its activator, is responsible for this effect. A recent study described a reduction of electrostatic repulsions between the serpin α 1 AT and the low molecular weight heparin (LMWH) enoxaparin, 85 which might also contribute to the activation of AT. Heparin or LMWH are administered as the standard of care in the majority of COVID‐19 patients at daily intravenous doses of several thousand international units (IU), while FPX is administered at 2.5 mg, respectively 74,80,86 .…”

Alpha-1-antitrypsin antagonizes COVID-19: a review of the epidemiology, molecular mechanisms, and clinical evidence

“…Because AAT is a potent serpin, it has the potential to inhibit both TMPRSS2 and SARS-CoV-2 infection. Indeed, in HEK293T cells engineered to overexpress TMPRSS2, physiologic concentrations of AAT potently inhibited TMPRSS2 activity [ 86 , 87 ]. Ritzmann et al [ 88 ] showed in both two-dimensional submerged airway epithelial cells and three-dimensional airway epithelial organoid cultures that physiologic concentrations of AAT (1 and 5 mg/ml) significantly inhibited TMPRSS2 activity and decreased intracellular burden of SARS-CoV-2 [ 88 ].…”

Indirect effect of alpha-1-antitrypsin on endotoxin-induced IL-1β secretion from human PBMCs

“…It is important to point out that AAT interacts with cytokines, free heme, heparins, lipoproteins, and other substances (Bergin et al, 2010;Karnaukhova et al, 2012). For example, AAT shows better anti-elastase activity in complex with high-density lipoprotein or heparin than by itself (Houghton et al, 2010;Lechowicz et al, 2020;Bai et al, 2022). AAT also interacts with chemoattractants, such as IL-8 and leukotriene B4 (LTB4), and thus modulates neutrophil adhesion and chemotaxis (Mcelvaney et al, 2020).…”