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

Abstract Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad‐spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS‐CoV, MERS‐CoV, hCoV‐229E, SARS‐CoV‐2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS‐CoV‐2. Thus, AT is an endogenous inhibitor of SARS‐CoV‐2 that may be involved in COVID‐19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti‐TMPRSS2 and anti‐SARS‐CoV‐2 activity of AT, suggesting that repurposing of native and activated AT for COVID‐19 treatment should be explored.


| INTRODUCTION
The emergence and pandemic spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major global health crisis.
Even though protective vaccines are available, there is still an urgent need for the development of specific antivirals to prevent SARS-CoV-2 spread into the lung or treat COVID-19. 1 SARS-CoV-2 infection is mediated by the viral Spike (S) glycoprotein, which comprises two subunits, S1 and S2. 2,3 The S1 subunit harbors the receptor binding domain (RBD) and binds to the cellular receptor ACE2, while the S2 subunit anchors the S protein in the viral membrane and harbors determinants required for virus-cell fusion. Following engagement of ACE2, proteolytic cleavage of the S2 subunit occurs by the cellular proteases transmembrane serine protease serine subtype 2 (TMPRSS2), cathepsins B or L or furin at the so-called S2′ site. 4,5 Cleavage is thought to prime the S protein for membrane fusion by triggering conformational changes allowing viral entry. 3,5,6 Inhibitors of TMPRSS2 enzymatic activity such as camostat or nafamostat prevent S protein priming and consequently viral infection, 5,[7][8][9][10][11] and are currently in the clinical evaluation as COVID -19 therapeutics.
Previous reports show that the serine protease inhibitor (serpin) α 1antitrypsin (α 1 AT, SERPINA1) is an endogenous inhibitor of TMPRSS2 activity and SARS-CoV-2 entry, with potential application for COVID-19 treatment. 12,13 Until today, 36 human serpins have been identified and linked to the regulation of physiological processes such as hemostasis, immunity, and inflammation. 14,15 However, it remains largely unclear which serpins can interfere with SARS-CoV-2 infection. The serpin antithrombin (SERPINC1) is a serum protein with a physiological concentration of~0.15 mg/ml, equivalent to~2.6 µM. 16 Its main function is the regulation of coagulation through inhibition of serine proteases including factor IXa, Xa, and thrombin. However, it also displays affinity to other proteases such as trypsin, papain, and cathepsin L. [17][18][19][20] The target protease recognizes and cleaves a distinct region of antithrombin (AT), the so-called reactive center loop (RCL). The subsequent formation of a covalently bound acyl-enzyme intermediate between serpin and protease triggers conformational changes in the serpin, which physically disrupts the protease reactive center and result in an irreversible inhibition. 21,22 Herein, we investigated whether AT inhibits TMPRSS2 and consequently SARS-CoV-2 infection.

| Computational modeling
For modeling the TMPRSS2-AT interaction, a TMPRSS2 homology model deposited in the SwissModel repository 23 (UniProtKB ID: O15393) was superimposed onto the crystal structure of the factor IXa-AT-pentasaccharide complex (PDB ID: 3KCG). 24 The initial docking model was refined with HADDOCK, 25 GalaxyRefineComplex, 26,27 and Rosetta. 28 The 10 best-scored solutions from each tool were selected and applied to Rosetta's 28 high-resolution refinement protocol. The best-scored solution was refined in the presence of explicit solvent and the system was neutralized with NaCl. To this end, NPT unrestrained simulated annealing MD simulations were performed (2 replicas) with the CHARMM36 force field 29 and the GROMACS simulation package. 30 The initial reference temperature was 300 K and the system was subjected to three 10 ns cycles of heating to 320 K and stepwise cooling to 275 K.

| Filtration and gel electrophoresis
AT preparations were subjected to a 10 kDa centrifugal filter (Amicon Ultra-0.5 ml Ultracel-10k) at 14,000g. Retentate and flowthrough were collected and adjusted to match the input volume. One microgram of protein per sample was then applied to reduce sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 4%-12% Bis-Tris protein gel (NuPAGE™) with MES running buffer. Before electrophoresis, samples were reduced with 50 mM TCEP and heated for 10 min at 70°C. The gel was stained with Coomassie G-250 (GelCode™ Blue Stain).

| Cytotoxicity assay
To assess the cytotoxicity of AT preparations, heparin, Fondaparinux  Virus stocks were aliquoted and stored at −80°C until use.

| RESULTS
Proper interaction between the serpin and the protease, as well as the presence of a compatible scissile bond in the serpins RCL are prerequisites for serpin-induced protease inhibition. 33 To gain insight into the putative interactions of AT with TMPRSS2, we performed a computational docking analysis using a hepsin-based TMPRSS2 homology model and the crystal structure of AT ( Figure 1A). Subsequent structural refinement (Supporting Information:  This suggests that, in the AT-TMPRSS2 complex, the scissile bond in the RCL is located between residues S392 and L393, and implies an AT-mediated inhibition of TMPRSS2 ( Figure 1A, inset).
To corroborate this finding, we used commercially available AT formulations, Anbinex and Kybernin, which are derived from human serum and are in clinical use for treatment of AT deficiency. 34 Interestingly, only FPX significantly increased inhibition of cellassociated TMPRRS2 activity by AT ( Figure 5B). In contrast to previously published data, activated AT had no effect on furin activity 43 (Supporting Information: Figure 9). Of note, heparin or FPX alone neither affected cell-free (Supporting Information: Figure 10A) nor cell-associated (Supporting Information: Figure 10B) TMPRSS2 activity. F I G U R E 4 Antithrombin reduces SARS-CoV-2 infection. Caco2 cells were treated with AT (Anbinex) or small molecule inhibitor CM for 1 h before infection with SARS-CoV-2 isolates Wuhan/Hu-1 (Spike mutation D614G) or VoC Alpha and Delta at an MOI of 0.005. Infection rates were assessed by flow cytometric analysis of SARS-CoV-2 nucleocapsid (N) protein expression at 1 dpi. Signal from PBS-treated, infected cells served as control and was used for normalization. Data are shown as means± SEM derived from n = 3 independent experiments. AT, antithrombin; CM, camostat mesylate; MOI, multiplicity of infection; PBS, phosphate-buffered saline; SEM, standard error of the mean; VoC, variants of concern.
We then studied whether heparin and FPX may confer antiviral activity to an Anbinex concentration otherwise insufficient to reduce authentic SARS-CoV-2 infection ( Figure 5). Flow cytometric quantification of N-positive cells revealed~45% reduced infection rates for the maximum heparin concentration applied (22.2 µM). In comparison, activation with ≥9.3 µM of FPX inhibited genuine SARS-CoV-2 infection almost entirely ( Figure 5C). Heparin or FPX alone did not reduce SARS-CoV-2 infection (Supporting Information: Figure 10C). Similarly, activation of a non-antiviral Kybernin concentration by FPX but not heparin resulted in almost complete inhibition of SARS-CoV-2 infection (Supporting Information: Figure 11). As shown in Figure 5D, FPX also increased the antiviral activity of AT against SARS-CoV-2 VOCs Alpha and Delta. These data show that activated AT is a highly potent inhibitor of SARS-CoV-2 infection. Thus, FPX-mediated activation of AT boosts the inhibitory effect on SARS-CoV-2 infection.
To determine whether AT also inhibits SARS-CoV-2 replication (i.e., multiple rounds of infection) in human lung cells, Calu-3 cells were treated with AT (13.8 µM), FPX-activated AT (13.8 µM), FPX alone, or CM (100 µM) and then infected with a low MOI of SARS-CoV-2 (day 0, inoculum). The entire supernatants were collected on Days 1, 2, and 3 and replaced by fresh medium containing the respective compounds. Viral RNA copies were determined by qPCR and revealed an exponential growth in the mock control from 10 5 RNA copies (inoculum) to more than 10 9 RNA copies on Day 3 postinfection ( Figure 6A). AT (Anbinex) and CM markedly reduced SARS-CoV-2 replication ( Figure 6A). Notably, FPX-activated AT prevented viral replication almost entirely, while FPX alone displayed no antiviral activity ( Figure 6A). Analysis of cumulative viral RNA copy production revealed a more than 340-(>99.7%) and 2400-fold (>99.99%) reduction by AT or activated AT, respectively. In  AT is a clinically approved anticoagulant in patients with hereditary and acquired antithrombin deficiency 70  increased AT levels will also exert beneficial effects by direct inhibition of SARS-CoV-2 replication.
Our data further suggest that activation of AT with the synthetic pentasaccharide FPX and-to a lesser extent-heparin increases its anti-TMPRSS2 and antiviral activity. 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