Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1ß from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1ß, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1ß and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.

Severity of SARS-CoV-2 infection is associated with high numbers of alveolar mast cells and their degranulation.

Background: The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective: To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods: MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe; severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FcεR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry. Results: The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion: In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme.

Biochemical and biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro) from the novel coronavirus SARS-CoV 2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here may improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the discovery of drugs and design of antiviral therapeutics against COVID-19.

Kinins and chymase: the forgotten components of the renin-angiotensin system and their implications in COVID-19 disease.

The unique clinical features of COVID-19 disease present a formidable challenge in the understanding of its pathogenesis. Within a very short time, our knowledge regarding basic physiological pathways that participate in SARS-CoV-2 invasion and subsequent organ damage have been dramatically expanded. In particular, we now better understand the complexity of the renin-angiotensin-aldosterone system (RAAS) and the important role of angiotensin converting enzyme (ACE)-2 in viral binding. Furthermore, the critical role of its major product, angiotensin (Ang)-(1-7), in maintaining microcirculatory balance and in the control of activated proinflammatory and procoagulant pathways, generated in this disease, have been largely clarified. The kallikrein-bradykinin (BK) system and chymase are intensively interwoven with RAAS through many pathways with complex reciprocal interactions. Yet, so far, very little attention has been paid to a possible role of these physiological pathways in the pathogenesis of COVID-19 disease, even though BK and chymase exert many physiological changes characteristic to this disorder. Herein, we outline the current knowledge regarding the reciprocal interactions of RAAS, BK, and chymase that are probably turned-on in COVID-19 disease and participate in its clinical features. Interventions affecting these systems, such as the inhibition of chymase or blocking BKB1R/BKB2R, might be explored as potential novel therapeutic strategies in this devastating disorder.

Potential anti-SARS-CoV-2 drug candidates identified through virtual screening of the ChEMBL database for compounds that target the main coronavirus protease.

A novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or 2019 novel coronavirus] has been identified as the pathogen of coronavirus disease 2019. The main protease (Mpro , also called 3-chymotrypsin-like protease) of SARS-CoV-2 is a potential target for treatment of COVID-19. A Mpro homodimer structure suitable for docking simulations was prepared using a crystal structure (PDB ID: 6Y2G; resolution 2.20 Å). Structural refinement was performed in the presence of peptidomimetic α-ketoamide inhibitors, which were previously disconnected from each Cys145 of the Mpro homodimer, and energy calculations were performed. Structure-based virtual screenings were performed using the ChEMBL database. Through a total of 1 485 144 screenings, 64 potential drugs (11 approved, 14 clinical, and 39 preclinical drugs) were predicted to show high binding affinity with Mpro . Additional docking simulations for predicted compounds with high binding affinity with Mpro suggested that 28 bioactive compounds may have potential as effective anti-SARS-CoV-2 drug candidates. The procedure used in this study is a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that may significantly shorten the clinical development period with regard to drug repositioning.