Mast Cell Tryptase and Implications for SARS-CoV-2 Pathogenesis

Abstract

Mast cells (MCs) are heterogenous innate leukocytes producing many inflammatory mediators during viral infections that can be protective or damaging to the host, as is seen in the infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for the coronavirus disease that was first identified in 2019 (COVID-19). MCs can sense viruses by diverse mechanisms. They express angiotensin-converting enzyme 2 (ACE2), known as the principal entry receptor for SARS-CoV-2, and tryptase that can promote SARS-CoV-2 infection. Tryptase is one of the most abundant serine proteases released by MCs during degranulation and is reported to have both beneficial and detrimental roles in respiratory diseases. Reviewed here are the potential roles of MC-derived tryptase during COVID-19, the implications it has in the pathogenesis of SARS-CoV-2, and the possibility of treating COVID-19 by targeting tryptase.

1. Introduction

The roles of mast cells (MCs) in conditions outside of the context of allergies have been highlighted in recent decades. These cells mainly reside in areas exposed to external stimulators, such as the lungs, making them a part of the first line of a host’s defense against pathogens [1]. Cumulative data revealed the impact of bacteria and viruses on the activation of MCs [2] and release of their granule contents that are packed with various inflammatory mediators, including serine proteases tryptase and chymase [2]. It should be noted that MCs display heterogeneity, with mucosal MCs, predominantly expressing tryptase in their granules, and connective tissue-resident MCs having granules containing tryptase and chymase [3]. Viruses have the potential to stimulate MCs both directly and indirectly through viral and inflammatory products. A variety of viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), dengue virus (DENV), respiratory syncytial virus (RSV), herpes simplex virus (HSV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), and influenza viruses can trigger MC degranulation, protease release, and cytokine production [4,5,6]. It is generally accepted that proteases in MC granules, especially tryptase and chymase, biologically contribute to inflammatory responses. Moreover, evidence suggests that MC proteases may play a broad range of roles in regulating pathological processes, from a protective effect in HSV infection [5] to a detrimental function in DENV infection [7]. Concentrations of MC-derived proteases were shown to be raised in sera and lung tissues from patients diagnosed with COVID-19. Furthermore, the concentrations of these proteases were significantly correlated with disease severity [6]. However, a recent study [8] demonstrated that patients with COVID-19 that had MC activation disorders, such as MC activation syndrome, tended to mount immune responses driven by T cells with a T helper (Th)-1 phenotype. This contradicts the view that MC-mediated Th2-skewed immune responses are expected to put these patients at a high risk of severe COVID-19 due to the impaired Th1-dominant antiviral responses. We previously reviewed the functional roles of MCs in SARS-CoV-2 infections [9]. Here, the role of serine protease tryptase released by MCs in response to viruses will be discussed in the context of its effect on functional immunological outcomes and pathogenesis, with an emphasis on SARS-CoV-2 infections. Additionally, the current pharmacologic treatments that target MC tryptase signaling will be explored in the context of their potential to treat COVID-19.

2. Mast Cell Ontogeny and Heterogeneity

MCs typically begin as progenitor cells in the bone marrow and complete their development once they migrate to peripheral tissues [10], where they display distinct characteristics that serve unique roles depending on their anatomical location [11]. Separate populations of these granulocytes, including connective tissue MCs and mucosal MCs, display unique phenotypes. MCs with granules containing tryptase rather than chymase appear to have a phenotype like mucosal MCs, while those expressing both tryptase and chymase resemble serosal MCs. MCs are generally classified based on where they reside after differentiation and the types of cytokines or contents stored in their granules [3].

The heterogeneity of MCs is evident in that they rely on signals from their microenvironment, allowing each unique subset of MCs to execute distinct functions in their specific locations [3]. As a result of their heterogeneity, MCs in the same location can produce different responses to the same stimulus [12]. In addition to their microenvironment, differentiation of the subgroups of MCs depends on the species, gene expression, and the origin of development. For instance, previous studies have shown the higher expression of Mas-related G protein-coupled receptor X2 (MRGPRX2) receptor in connective tissue MCs of the skin and adipose tissues and a lower expression in those residing in the lungs or colon [13]. Sex may also affect MC heterogeneity as it has been observed in mouse models that females have elevated concentrations of released mediators, including tryptase, compared to males [13]. Therefore, depending on the situation, heterogeneity allows MCs to play contrasting roles in immunity by prolonging or dampening immune responses during infections [3]. Moreover, accumulated evidence revealed that the immunoregulatory function of MCs is not restricted to viral infections. Indeed, on some occasions, MCs can release granule contents and cytokines that suppress chronic inflammation and allergic reactions [14].

The location of MCs has a significant role in their functions [15]. For instance, MCs of the skin are quite mobile and may traffic to other tissues when stimulated. Furthermore, the properties of MCs in the skin may differ in terms of the mediators and proteases in their granules [16]. For example, MCs in the foreskin have more histamine and chymase than those found in other areas of the skin. MCs in the skin also produce a unique response to pathogens that is different from other MCs in the body due to their numerous membrane receptors, including various types of Toll-like receptors (TLRs). Similarly, MCs along the entire gastrointestinal tract have functional differences as demonstrated by the MCs of the colon expressing more TLRs because of the numerous bacterial colonies in the area, compared to the MCs of the small intestine, which express fewer TLRs. In the respiratory system, there are very few MCs expressing both chymase and tryptase in the submucosal layer of the lungs. These variations are all examples of the heterogeneity of MCs and how it contributes to a vast range of functions [3]. Increases in the numbers of degranulated MCs that lead to the release of granule contents and augment the concentrations of proteases have been demonstrated in several illnesses and diseases, including renal complications, pancreatitis, and COVID-19 [3,6]. It should also be noted that different growth factors regulate distinct subsets of tissue MCs. For example, murine mucosal MCs are susceptible to regulation by transforming growth factor-beta (TGF-β), while connective tissue MCs are not [13].

3. MC-Derived Tryptase and Chymase in Inflammation

MC-derived serine proteases are potent chemoattractants for inflammatory cells [17,18]. Angiotensin-converting enzyme activity [19,20] and proteolytic cleavage of type I procollagen [21] increase the migration of leukocytes to the inflamed regions and lead to tissue remodeling. Eosinophilia is a hallmark of severity and prognosis of allergic reactions [22]. Terakawa et al. [23] showed that in addition to their anti-parasitic functions, eosinophils can be stimulated by MC-derived chymase to release and upregulate the neutrophil chemoattractant interleukin (IL)-8 and macrophage inflammatory protein-2 (MIP-2) at allergic inflamed sites. The secretion of IL-8 from neutrophils is supposed to be mediated by G protein-coupled receptor and mitogen-activated protein kinases. Indeed, Tani et al. [24] indicated that the development of inflammatory responses following neutrophil accumulation may also directly result from the chemotactic activity of chymase. Furthermore, Temkin et al. [25] reported that tryptase-related secretion of IL-8 and IL-6 from human eosinophils can be triggered by both connective tissue- and mucosa-resident MCs. This process is driven by the mitogen-activated protein kinase/activating protein-1 pathway, which is mediated by the cleaving of protease-activated receptor-2. MCs contain different types of tryptases, including α, β, and γ. However, tetrameric β tryptase is considered the dominant form of mature tryptase [26]. In this regard, it has been observed that the recruitment of neutrophils into affected sites is significantly abrogated in tetramer tryptase-knockout mouse models [27]. Tryptase elevates the secretion of IL-8 by up-regulating intracellular adhesion molecule-1 in bronchial epithelial cells [28]. Endothelial cells, as key participants in inflammatory reactions, are also influenced by tryptase proinflammatory activity. MC-derived tryptase induces the expression of inflammatory cytokines, such as IL-1β and IL-8 from endothelial cells in a dose-dependent manner [29].

MC-derived serine proteases also have important roles in all phases of wound repair, including hemostasis, inflammation, proliferation, and remodeling [30]. Anti-coagulant activity, the recruitment and activation of inflammatory cells, angiogenesis, epithelialization, and fibroblast proliferation are among the numerous central functions of these mediators. It seems that tetramer-forming MC tryptases have a specific role in tissue remodeling and the proliferation phase of wound healing [31]. They can activate pro-stromelysin/MMP3 and degrade extracellular matrices (ECMs). Indeed, tryptase-β⁺/heparin⁺ complexes released by MCs extend bleeding time in order to prevent excessive life-threatening clotting during the wound repair process [32].

The long-lasting presence of MCs and excessive degranulation of serine proteases can result in the formation of keloid and hypertrophic scars. Keloid [33] and hypertrophic scars are common complications of the wound healing process resulting from abnormal connective tissue responses [16]. These benign tumor-like scars are accompanied by extra collagen accumulation and impairment of the remodeling process in inflamed sites. Individuals with these scars complain of itching and erythema. However, these symptoms are resolved by corticosteroid therapy, confirming the main role of MCs in this process.

4. MC Tryptase and Chymase in Viral Infections

4.1. Flaviviruses

Flaviviruses, such as West Nile virus (WNV), DENV, JEV, and ZIKV are known as arthropod-borne viruses (arboviruses) [34]. They help demonstrate how MCs play either beneficial or detrimental roles in response to viral infections. Following the bite of infected mosquitoes with DENV, MCs at the infected site act as a major sensory arm of the innate immune system and will recognize DENV viral components via RNA sensors, such as protein kinase R (PKR), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5). These interactions lead to the production of type I interferons and different chemokines, including CCL4, CCL5, and CXCL10 [35,36,37,38,39]. The anti-viral responses by type I IFNs suppress viral replication and protect other cells from infection [40] and the MC-derived chemokines recruit T cells and natural killer cells to the site of infection, resulting in viral clearance [38,41,42]. However, severe forms of DENV disease, known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) can cause more serious pathophysiological conditions. The presence of pre-existing antibodies against the virus in the blood of individuals with secondary DENV infection or infants born to DENV-immune mothers results in antibody-dependent enhancement (ADE) and increases the critical symptoms of the disease. The poor prognosis in the acute form of the disease is associated with systemic vascular permeability. Although there is no strong evidence linking the direct attack of DENV to endothelial cells, immune-mediated pathology may explain the mechanism of vascular leakage during DHF and DSS [43]. MCs express FcγRII on their surface enabling them to capture soluble IgG or antibody-antigen complexes [44]. Consequently, MCs are activated and several chemokines, vasoactive cytokines, and proteases are released leading to exaggerated inflammatory reactions and the loss of vascular integrity [45,46]. However, MCs can also degranulate in response to complement components [47] or directly without antibody sensitization [7]. While some MC-dependent mediators, such as tumor necrosis factor can enhance vascular complications, they are not assumed to be associated with DENV severity [48]. It seems that MCs with the tryptase-chymase phenotype (MCTC) is related to the aberrant release of tryptase and chymase in submucosal and connective tissues of patients with DHF and DSS. These mediators promote inflammation and tissue remodeling and are prognostic biomarkers associated with dengue disease severity. Intriguingly, the concentration of chymase may help to differentiate the broad clinical spectrum of the disease, while other early clinical signs are not able to predict the prognosis of the disease in patients infected with DENV [49,50]. Chymase is released from the same granules as histamine and tryptase and can induce microvascular leakage for a longer duration [51]. Secretion of chymase in the form of a macromolecular complex [52] along with a high affinity for the extracellular components of basement membranes [53] localizes the actions of chymase. While both chymase and tryptase are structurally stable proteins, tryptase seems to be a more potent inducer of vascular perturbation than chymase due to its longer half-life in circulation [54]. Moreover, chymase can downregulate the expression of the coagulation factor FXIIIA and subsequently raise the bleeding time in individuals infected by DENV [55]. Tryptase exerts its effect by cleaving protease-activated receptors (PARs) at the inter-endothelial junctions [56]. In this regard, disseminated intravascular coagulation and low concentration of circulating fibrinogen in patients with DHF were reported in the Philippines [57] and Thailand [58], which would be the consequence of tryptase activity.

In the case of JEV, chymase can also be released primarily from MCs near the blood-brain barrier (BBB) in response to infection [59]. The release of granule-associated proteases during infections with JEV breaks the tight junctions between endothelial cells in the brain and augments the BBB permeability. This process promotes virus entry into the central nervous system and leads to neurologic impairments. Moreover, chymase can activate some matrix metalloproteinases (MMPs), such as MMP2 and MMP9 into their enzymatically active forms, further exacerbating the breach of the BBB by perturbing the vasculature [59]. MMP9 has also been shown to facilitate access of WNV to the brain by degrading ECM molecules in the basement membranes of the BBB [60].

4.2. Respiratory Viruses

Viruses are known to have the potential to trigger allergic sensitization, and overwhelming evidence supports the notion that respiratory viruses play a major role in the exacerbation of asthma [61]. Patients with asthma have significantly larger numbers of MCs in the airway submucosa [62] and bronchial epithelial layer [63]. These MCs play a cardinal role in initiating and maintaining allergic inflammation. After IgE-antigen-mediated crosslinking of surface FcεRI in patients with asthma, MCs are activated and release large quantities of chymase [64] and tryptase [65]. These proteases are crucial contributors to the pathophysiological inflammatory responses, including bronchial hyperresponsiveness and bronchoconstriction. Synergism between allergic airway inflammation and respiratory virus infections could be involved in the development of asthma [66]. RSV is a well-known risk factor for the development of asthma in children and it has been suggested that [67] human lung fibroblasts (HLFs) infected by RSV can establish a hyaluronan-enriched ECM that would enhance MC retention and MC protease production at the sites of inflammation.

Tryptase and chymase elicit the secretion of the alarmin adenosine triphosphate (ATP) and pro-inflammatory mediators IL-8 and IL-6 and contribute to airway epithelial barrier dysfunctions [68,69]. MC-derived chymase promotes airway remodeling by either the secretion of plasminogen activator inhibitor-1 (PAI-1) or activation of the TGF-β1 pathways [70] and impairs wound healing by activating pro-MMP-2 [69]. Tryptase also decreases the expression of viral-induced type-I and -III interferons and pattern recognition receptors (PRRs). These all promote increased epithelial permeability and lead to exacerbation of asthma. There is evidence to suggest that the concentration of tryptase in the lungs rises with age. This is unfortunate because high concentrations of tryptase are a risk factor for severe asthma in elderly people [71].

4.3. Other Viruses

MC-mediated injuries are reported for various viral infections, namely hepatitis C virus (HCV) [72], cytomegalovirus (CMV) [73], hantavirus [74], and human immunodeficiency virus-1 (HIV-1) [75] infections. Hu et al. [76] suggested that MC activation and tryptase secretion were associated with lung lesions and apoptosis in mice infected with the H5N1 influenza virus. Viral bronchiolitis and airway hyperresponsiveness during infection with Sendai virus were related to proliferation and the recruitment of MCs to the airways [77]. Accordingly, MC tryptase has been detected in pig lungs after being challenged with the Sendai virus [78]. A positive correlation between MC tryptase activity and the presence of infectious bursal disease virus (IBDV)-derived antigens in chickens has been documented [79]. Furthermore, similar to IBDV, the inhibition of MC mediators, such as tryptase in chickens infected with Newcastle disease virus alleviated virus-induced tissue injury [80].

Using a murine model of heart failure, Kitaura-Inenaga et al. [81] showed that tryptase and chymase were involved in the progression of heart failure and viral cardiac remodeling. They indicated that the expression of high concentrations of MC chymase, tryptase, and MMP-9 correlated with the development of myocardial necrosis and fibrosis during infection with encephalomyocarditis virus (EMCV). In this regard, chymase and tryptase incited cardiac remodeling through the activation of specific molecules, including procollagen or IL-1β. In addition, they activated MMP-3 resulting in MMP-9 activation and ECM degradation.

5. MC Tryptase in COVID-19

Cytokine storms are a potential driver of immunopathology in cases of severe COVID-19 [82]. MC activation syndrome (MCAS) caused by SARS-CoV-2 plays an important part in initiating and maintaining the heightened immune responses [83]. According to Gebremeskel et al. [6], systemic MC activation and elevated MC-specific proteases in the sera and lung tissues of patients with SARS-CoV-2 could be related to inflammatory cytokine secretions contributing to COVID-19 complications. In support of this, the infiltration of c-kit-positive MC progenitors and mature MCs in perivascular spaces and alveolar septa were observed in the post-mortem lung biopsies of patients with COVID-19 [84]. A cytokine expression profile towards a type 2 response in severe COVID-19 was another indication that there is uncontrolled and excessive MC activation occurring in these patients [85]. The concentration of tryptase was a helpful clinical predictor for COVID-19 severity due to the associated specificity and strong effect on vascular leakage [86]. Although ACE2 is a major cell entry receptor for SARS-CoV-2 [87], it is unlikely that MCs get infected directly by the virus. There is no significant association between cytokine storms and ACE2 expression on leukocytes, such as MCs. It is speculated that the secondary activation of MCs via the engagement of PPRs, TLR3, TLR7, and TLR8, by viral RNA intermediates may be linked with augmented inflammatory responses [6]. MC degranulation can also be associated with FcεRI-mediated binding to IgE (e.g., post-vaccination anaphylactic reactions), activation of complement (C1q, C3a C4, C5a, and Factor B)-associated anaphylatoxins, or direct activation of MRGPRX2. The level of tryptase secretion might vary based on the MC activation mechanism. Although tryptase concentration may remain in the normal range in the direct activation of MCs via MRGPRX2, it increases dramatically when MCs are activated via complement and IgE-dependent pathways [88].

It is assumed that the “COVID-19 brain fog” symptom partially results from the activation of MC-derived vasoactive mediators, namely tryptase, that can augment the permeability of the BBB [89,90]. Furthermore, post-COVID-19 pulmonary fibrosis may also be due to tryptase secretion from MCs [91]. Tryptase activates PAR2 expressed on fibroblasts and triggers the migration and proliferation of lung fibroblasts resulting in airway remodeling. It also upregulates fibroblast collagen synthesis and consequently promotes the progression of lung fibrosis [92,93]. The role of MCs in the pathogenesis of COVID-19 has been further supported by a recent study in France [8] demonstrating that patients with COVID-19 with concomitant MC activation disorders elicit Th1 phenotype immune responses, which is considered ideal for antiviral responses.

6. Drug Therapies

Considering the roles of tryptase in viral infections and respiratory complications, MC stabilizers that reduce tryptase release from MC granules, and tryptase inhibitors could be regarded as potential pharmaceutical agents in the management of COVID-19 (Figure 1).

6.1. MC Stabilizers

This group of medications restricts the degranulation of MCs through diverse mechanisms leading to the inhibition of tryptase release from these cells. Cromolyn sodium is considered the leading medication in this group acting through inhibiting protein kinase C, limiting chloride transport into MCs, and blocking the activity of macrophages. Some other examples of these agents include ketotifen, flavonoids, coumarins, terpenoids, alkaloids, and antihistamines. We thoroughly reviewed this category of medications and their mechanisms of action in a previous paper discussing the role of MCs in cytokine storms [9]. As such, they will not be described here.

6.2. Tryptase Inhibitors

6.2.1. Heparin Antagonists

Although most tryptase inhibitors target heparin and its active site, antagonists of heparin could also be regarded as potential agents against tryptase function. Tryptase tetramers bind to heparin proteoglycan cofactors and form macromolecular complexes, which are necessary for the effective enzymatic activity of tryptase. Therefore, polycationic compounds may destabilize tryptase through binding competitively to proteoglycans [94,95]. Several proteins with heparin-binding capacity, namely myeloperoxidase (MPO) [96], lactoferrin [97], and antithrombin III [98] have been noted in the literature to inhibit tryptase. Lactoferrin, a natural tryptase inhibitor is the functional mammalian homolog of leech-derived tryptase inhibitor (LDTI) [99] that is generated by the epithelium of mucosa and neutrophils in inflammation and reduces heparin’s capacity for stabilizing tryptase [100,101]. MPO, another antimicrobial product of activated neutrophils, breaks down the tryptase-bound heparin through enzymatic reactions [96]. In addition, Hallgren et al. revealed the competitive inhibition of human lung tryptase and a recombinant mouse tryptase by protamine, as a heparin antagonist. In the latter investigation, polybrene also limited the enzymatic activity of active tryptase non-competitively by dissociating the tetramer into monomers [95]. In another study, poly-Arginine and poly-Lysine of diverse molecular weights could strongly inhibit tryptase. The reversal of tryptase inhibition following heparin addition also confirmed the inhibitory role of these polycationic peptides by affecting the heparin complex [102]. However, Sommerhoff et al. reported that targeting heparin might be challenging, especially in vivo, with tryptase concentrations, affinity, and binding specificity as the influential factors in the success rate [94].

6.2.2. Natural Tryptase Inhibitors

Some natural compounds have been reported to exert inhibitory effects on tryptase, the most well-known being LDTI, which is a Kazal-type protein obtained from Hirudo medicinalis leeches [94]. The two N-terminal lysine residues of this agent can have electrostatic interactions with the Asp132 and Asp136 of the tryptase monomer leading to reduced tryptase activity [103]. LDTI inhibited tryptase in vitro, thereby reducing the tryptase-associated proliferation of human fibroblasts and keratinocytes [104]. Another natural inhibitor is a tick-derived protease inhibitor (TdPI), which is a Kunitz-related inhibitor of human tryptase [105] obtained from the salivary gland of the Rhipicephalus appendiculatus tick. It has been suggested to block three of the four catalytic sites of tryptase [106]. Peptide leucine arginine (pLR) is a small cyclic peptide, which can be isolated from frog skin. It was demonstrated to alleviate the symptoms of asthma and chronic airway inflammation in mice by inhibiting tryptase [107]. Lactoferrin and MPO, mentioned in the previous section, are the other members of the natural group of tryptase inhibitors. Recently, cathelicidin-MH (cath-MH), which is an antimicrobial peptide derived from the skin of Microhyla heymonsivogt, was revealed to reduce tryptase enzymatic activity in mice [108].

Cysteine knot miniproteins could also be classified as natural tryptase inhibitors isolated from a variety of sources, including spiders, snails, and various plant species. However, they have also been expanded in recent years by protein engineering and novel technologies [109]. An example of cystine knot peptides with an herbal origin is the inhibitors of trypsin-like serine proteases [110]. MCoTI-II is a cyclic cystine knot miniprotein, which is derived from the seeds of Momordica cochinchinensis. It has been demonstrated to have the potential to inhibit all tryptase monomers making it one of the most potent existing tryptase inhibitors [111]. Furthermore, engineered MCoTI-II analogs can act against tryptase and human leukocyte elastase as two therapeutically important serine proteases [112,113]. Although the oral administration of peptides is challenging due to the gastrointestinal enzymes that can hydrolyze peptides, these miniproteins have been shown to be resistant to these enzymes [109]. This feature along with the high anti-tryptase potential of these agents might make them promising pharmaceutical options in disease complications caused by MCs and tryptase.

6.2.3. Synthetic Tryptase Inhibitors

According to the literature, synthetic anti-tryptase agents can be classified into three classes based on their binding mechanism, namely canonical inhibitors, zinc-mediated inhibitors, and dibasic and bivalent inhibitors [94].

Canonical Inhibitors

Canonical inhibitors target the substrate-binding site (S) and S’ sites in the active site cleft. N-(1-hydroxy-2-napthoyl)-L-arginyl-L-prolinamide (APC-366) is among the popular medicines of this group. Four APC-366 molecules irreversibly bind the subunits of a tryptase tetramer with their arginine residue. As a result, airway hyperreactivity of tryptase is reduced [65,114]. APC-2059 has also been developed as a subsequent compound to APC-366 for treating several conditions, such as asthma [115]. Other examples of this group include RWJ-56423 [116]. The latter agent was shown by X-ray to interact with the serine and histidine residues of human tryptase [117].

A group of canonical inhibitors is the non-peptidic ones, such as basic ketoacid 4-amidinophenylpyruvic acid (APPA) [94]. Beta-lactam-based inhibitors, namely BMS-262084 and BMS-363131 could also be classified in this group [94]. They consist of a central beta-lactam, which plays a role in binding to Ser 95 in the bioactive site of tryptase [118]. A popular non-peptidic inhibitor is nafamostat mesylate, which has a high potency. It has been shown to block vascular leakage caused by DENV in vivo [54]. Moreover, significant clinical improvement was reported following nafamostat therapy in a patient with COVID-19 pneumonia and disseminated intravascular coagulation [119].

Zinc-Mediated Inhibitors

BABIM [bis(5-amidino-2benzimidazolyl) methane] inhibitors utilize zinc as a cofactor for inhibiting tryptase [94]. The zinc ion was found to be tetrahedrally coupled with two BABIM benzimidazole nitrogens and two tryptase active site residues [100]. BABIM has a low selectivity for tryptase, while APD 10, a follow-up compound, is more selective. In vivo investigation of these agents in sheep models revealed reduced airway hyperresponsiveness [115].

Dibasic and Bivalent Inhibitors

These inhibitors are composed of three structural components, including two basic head groups as ligands for the S1 pockets, a core orienting the head groups toward the adjacent S1 pockets, and spacer elements that adjust the optimal distance [94]. MOL-6131 is an example of this group, which diminished the number of eosinophils in both bronchoalveolar lavage fluid and airway tissue, goblet cells hyperplasia, peribronchial edema, as well as IL-4 and IL-13 production in a murine model of asthma [120]. Furthermore, it has been demonstrated to inhibit human lung tryptase with good selectivity [115].

6.2.4. Tryptase-Specific Antibodies: Next Generation Tryptase Inhibitors

Tryptase-specific antibodies are being assessed and developed in recent years as adequately specific anti-tryptase agents. B12, as a monoclonal IgG1 isotype, is an anti-tryptase that interferes non-competitively with the tetrameric structure of heparin-stabilized tryptase resulting in the generation of monomers, which are inactive in the neutral pH of most bodily fluids [121]. Moreover, Maun et al. developed 31A.v11 IgG4, as an allosteric noncompetitive tryptase-specific antibody, that disrupts active tetramers into inactive monomers. The anti-tryptase inhibited the activity of endogenous tryptase, which was obtained from the supernatant of degranulated human MCs in vitro. Furthermore, in vivo studies by the same group, showed that the antibody was efficient in humanized mouse and cynomolgus monkey models. The researchers recommended the antibody as a suitable clinical candidate for the treatment of severe asthma [122].

Recently, the first published human study on tryptase-specific antibodies investigated MTPS9579A (RG6173), which is a full-length humanized IgG4 with the ability to bind to human and cynomolgus monkey tryptase. It inhibits tryptase by disrupting the active tryptase tetramer into inactive monomers in an irreversible manner. This phase I randomized observer-blinded placebo-controlled single and multiple ascending-dose study was performed on 106 healthy adult participants. They reported the inhibition of tryptase in the upper airway with no severe adverse effects [123]. Currently, a phase II multicenter randomized placebo-controlled double-blind study is being conducted to assess the efficacy, safety, and pharmacokinetics of MTPS9579A in patients with asthma, who require inhaled corticosteroids and a second controller. The investigation is estimated to be completed in 2022 [NCT04092582].

7. Conclusions

The heterogeneity of MCs is defined by their protease contents, which are under the regulation of the tissue they reside in. MC-derived proteases during viral infections can be protective or damaging to the host, as is seen in cases of infection with the SARS-CoV-2. Evidence presented in this review suggests that MCs are involved in the regulation of infection and inflammation through the release of proteases. Thus, they are a target for treating viral diseases. Contributing to tissue homeostasis and repair, MC-derived tryptase could be specifically targeted and manipulated as a therapeutic agent for human diseases, including the treatment of COVID-19.