key: cord-0953933-ykuke34v
authors: Korkmaz, Brice; Lesner, Adam; Marchand-Adam, Sylvain; Moss, Celia; Jenne, Dieter E.
title: Lung Protection by Cathepsin C Inhibition: A New Hope for COVID-19 and ARDS?: Miniperspective
date: 2020-07-21
journal: J Med Chem
DOI: 10.1021/acs.jmedchem.0c00776
sha: ed960124ecfbaa50d0c37f7c73435463735f482e
doc_id: 953933
cord_uid: ykuke34v

[Image: see text] Cathepsin C (CatC) is a cysteine dipeptidyl aminopeptidase that activates most of tissue-degrading elastase-related serine proteases. Thus, CatC appears as a potential therapeutic target to impair protease-driven tissue degradation in chronic inflammatory and autoimmune diseases. A depletion of proinflammatory elastase-related proteases in neutrophils is observed in patients with CatC deficiency (Papillon–Lefèvre syndrome). To address and counterbalance unwanted effects of elastase-related proteases, chemical inhibitors of CatC are being evaluated in preclinical and clinical trials. Neutrophils may contribute to the diffuse alveolar inflammation seen in acute respiratory distress syndrome (ARDS) which is currently a growing challenge for intensive care units due to the outbreak of the COVID-19 pandemic. Elimination of elastase-related neutrophil proteases may reduce the progression of lung injury in these patients. Pharmacological CatC inhibition could be a potential therapeutic strategy to prevent the irreversible pulmonary failure threatening the life of COVID-19 patients.

The infectious respiratory tract disease COVID-19 (coronavirus disease 2019) caused by a newly emergent coronavirus SARS-CoV-2 is a global pandemic, and it is urgent and vital for the medical scientific community to investigate new therapies. COVID-19 is the third emergence of a coronavirus in less than 20 years. Its clinical spectrum ranges from unapparent to very severe signs of a life-threatening disease presenting as acute respiratory distress syndrome (ARDS) due to a generalized viral pneumonia. The latter disease manifestation necessitates admission to a hospital in 20% and intensive care therapies in 5% of all infected persons. 1 ARDS, the major cause of morbidity and mortality of COVID-19 patients, is a type of respiratory failure characterized by acute lung injury and edema (Figure 1 ). While the mechanism that causes the most severe forms of COVID- 19 is not yet fully understood, accumulating evidence points to an inappropriate exaggerated response of the innate immune system leading to severe and potentially irreversible lung injury and death from respiratory failure.

The development of viral hyperinflammation resulting in increased influx of neutrophils and monocyte-macrophages was observed in severe cases of COVID-19 2 as well as in previous coronavirus infections (SARS, severe acute respiratory syndrome, or MERS, Middle East respiratory syndrome). 3 Every minute, 30 billion neutrophils (assuming a cardiac output of 5 L/min and 6000 neutrophils/μL blood) with a large arsenal of mature, ready to use proteases are squeezed through lung capillaries and are at the forefront of sensing subtle changes in the lung tissue and local cytokine production. In addition to the freely circulating neutrophils, a large fraction of neutrophils are tethered to the lining of the lung vasculature, and this so-called marginated pool represents the most prominent reservoir and almost 40% of total body neutrophils. 4 As shown by pulmonary intravital microscopy, neutrophils firmly associated with lung endothelial cells form an efficient vascular antibacterial filter to remove circulating bacteria and endotoxin. 5 Neutrophil activation and neutrophilinitiated local proteolysis often at very low but sometimes at a very fast pace are a common theme in chronic inflammatory and autoimmune diseases of the lung. 6−8 On the basis of the accumulated data from preclinical and clinical studies, neutrophils indeed play a crucial role in acute lung injury by releasing elastase-related serine proteases and reactive oxygen species under rapidly evolving deteriorating health conditions. 6, 7, 9 Decondensation of nuclear chromatin is promoted by neutrophil elastase released from primary granules and leads to neutrophil extracellular trap formation 10 which very recently has been inferred as a driver of severe COVID-19 pneumonia. 11−14 Neutrophil elastase-related serine proteases recognized as pharmacological targets in neutrophilic inflammatory diseases thus appear as promising targets of therapeutic intervention in COVID-19.

NEUTROPHIL ELASTASE-RELATED PROTEASES Direct Inhibition. As proteases are easily understood as major actors in the degradation of tissue, their targeting by therapeutic inhibitors appears to represent a straightforward, easy to achieve goal. 7, 15, 16 Unexpectedly, direct inhibition of neutrophil elastase-related serine proteases has faced a lot of unresolved difficulties regarding the selection of the most relevant protease targets and their respective inhibitors with an appropriate physicochemical profile, prompting proposals for alternative approaches. 7 Many studies have been conducted with an elastase-selective competitor inhibitor, called sivelestat (N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulfonylamino]-aminoacetic acid, research name ONO-5046 marketed as Elaspol, IC 50 -elastase = 0.044 μM, K i = 0.2 μM). 17, 18 While approved in Japan for the treatment of acute respiratory failure, Eli Lilly stopped the development of this drug in the U.S. in view of disappointing results. 19 Destructive proteolytic processes with organ damage are mediated by multiple proteases, and hence inhibition of a single entity is not appropriate and unlikely to be effective in neutrophil-mediated lung injury. Nonetheless, monospecific highly selective inhibitors developed by the pharmaceutical industry were extraordinarily successful with small compounds inhibiting nonredundant proteases of proteolytic cascades like thrombin or the clotting factor Xa.

Indirect Inhibition. An original and innovative recent strategy was dedicated to the development of an efficient antiproteolytic therapy upstream of elastase-related serine proteases by blocking their maturing enzyme, cathepsin C (CatC, EC 3.4.14.1), 7 during the promyelocytic stage of neutrophil maturation in the bone marrow. CatC, also known as dipeptidyl peptidase I, is a lysosomal tetrameric ( Figure 2 ) amino peptidase belonging to the papain family of cysteine peptidases (family C1, clan CA). 20 CatC is initially synthesized as a ∼60 kDa single chain glycosylated monomer (Asp 1 -Leu 439 ) that associates to form the inactive proCatC homodimer. 21 ProCatC dimer is processed and converted to its proteolytically active, mature form by proteolytic activities of CatL-like cysteine proteases in two steps. 21, 22 This maturation is initiated by an almost complete excision of the internal propeptide (Thr 120 -His 206 ) (step 1) and then continued by the processing of the catalytic papain-like structure (Asp 207 -Leu 439 ) (step 2). The three generated chains of mature CatC, the exclusion domain (Asp 1 -Gly 119 ), the heavy (Asp 207 -Arg 370 ) and the light (Asp 370 -Leu 439 ) chains, are tightly associated by noncovalent interactions. The processing of papain-like structure in the second step is essential for achievement of active tetrameric CatC in which the N-terminal exclusion domain responsible for the diaminopeptidase activity is localized and stabilized 22 beyond the S2 pocket 20 according to the nomenclature of Schechter and Berger. 23 CatC, which is ubiquitously expressed in mammals, is recognized as a major intracellular processing protease. 20 CatC catalyzes the dipeptide removal of two residues from the free N-termini of peptides and proteins. The best known function of CatC is the activation of immune cell-associated serine proteases such as proinflammatory neutrophil elastase-related serine proteases (elastase, proteinase 3, cathepsin G, and NSP4) by the removal of their N-terminal dipropeptide. 24, 25 Loss of function mutations in the CatC gene (gene symbol CTSC) in humans cause Papillon−Lefevre syndrome (PLS, OMIM 245000) characterized by a severe prepubertal periodontitis and palmoplantar keratoderma without marked immunodeficiency. 26, 27 The lack of CatC activity results in an almost total elimination of elastase-related serine proteases in neutrophils from PLS patients 25, 28, 29 ( Figure 3A ). Moreover, PLS cells are incapable of producing neutrophil extracellular traps (NETs), networks of fibers, primarily composed of DNA. 30, 31 In spite of their deficiency in CatC, PLS patients do not present marked immunodeficiency or recurrent viral infections which means that a transitory pharmacological inhibition of CatC activity in the precursor cells of the bone marrow could well be an attractive therapeutic strategy to regulate activity of elastase-related serine proteases in inflammatory and immune disorders. 6,7 Small molecule Computed tomography (CT) images from two patients showing bilateral multifocal ground-glass opacities (GGO) (patient 1) and consolidation lesions (patient 2). Chest CT of patient 1 was performed 10 days after initial onset of symptoms. The survival and functional outcome of the patient 1 were favorable after 20 days in the intensive care unit. Chest CT of patient 2 was performed 16 days after initial onset of symptoms. Patient 2 died on day 31 despite thorough treatment in the intensive care unit. GGO is a nonspecific finding on CT scans consisting of a hazy opacity that does not obscure the underlying bronchial structures or pulmonary vessels and indicates a partial filling of air spaces in the lung by exudate, transudate, fibrosis, or malignancy. Pulmonary consolidation is a region of normally compressible lung tissue that has filled with liquid or cells instead of air.

chemical inhibitors of CatC resulted in similar protection of knockout mice against neutrophil-mediated tissue damage. 32

Several chemical inhibitors of CatC have been synthesized, 33−36 some of which are now being tested in preclinical/clinical trials. 6 Most are based on particular dipeptide substrates and carry electrophilic warheads that form reversible or irreversible covalent bonds with the enzyme's active site Cys234. 33, 35 The main issue in developing CatC inhibitors is metabolic stability which comes at the cost of inhibitory activity. Different cell assays were used including rat liver microsomes in an attempt to optimize stability, and a small number of compounds progressed to in vivo studies of pharmacokinetics or biological effects. 6 Design and Synthesis of Brensocatib and IcatC XPZ-01 . Nitrile-based inhibitors of cysteine cathepsins, which react reversibly with the Cys active site to form a thioimidate adduct, have been studied the most ( Figure 4A ). Investigators have focused on three chemical classes of nitrile (cyanamides, aryl or heteroaryl nitriles, and amino-or amidoacetonitriles), which differ in their electrophilic effects. 6, 34 Nitrile-based inhibitors of CatC are mostly dipeptidyl nitriles with a P2 side chain which determines inhibitory potency. 33, 37, 38 The S2 subsite of CatC presents as a deep pocket ( Figure 4B ) containing a chloride ion at the bottom 20, 39 and an Asp1 with a carboxylic side chain which interacts with the free N-terminal amino group on the inhibitor. The S1 subsite at the surface of CatC is exposed to solvent 39 so it can accommodate P1 residues bearing aliphatic, hydrophobic, polar, basic, or acidic natural amino acid side chains 40 but might not tolerate long aliphatic side chains directed at proline 3 at the P1 position 32 ( Figure 4B ).

The first dipeptidyl nitrile compounds, derived from Abu-Bip-CN ( Figure 5A ) with an aminobutyric acid (Abu) residue at P2 and biphenyl (Bip) at P1, were not stable in plasma because the amide bond was rapidly hydrolyzed. 33, 34, 37 Stabilization of the peptide bond can be achieved by substituting the N-terminal amino acid with one displaying a piperidine or cyclohexyl ring as side chain 33−36 or by inserting a 1,1-cyclopropylaminonitrile moiety in P1. 41, 42 By use of these strategies, brensocatib (formerly INS1007/AZD7986, (S)-N- Figure 5B ) and cyclopropyl inhibitor IcatC XPZ-01 ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4′-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide), IC 50 -CatC = 15 nM) 32 ( Figure 5C ) were developed by AstraZeneca and Neuprozyme Therapeutics, respectively. These appear to be potent CatC inhibitors with good species crossover for rodent CatC, suitable metabolic stability, and resistance to hydrolytic degradation.

Evaluation of IcatC XPZ-01 and Brensocatib in Cell-Based Assays. IcatC XPZ-01 . In studies using human immature myeloid PLB-985 cells and HL-60 promyelocytic leukemia cells as a model for neutrophilic precursors at different stages of maturation, IcatC XPZ-01 almost completely inhibited the activation of elastase-related proteases. 44 However, the protease levels achieved by pharmacological CatC inhibition using IcatC XPZ-01 were not as low as those observed in neutrophils from PLS patients. 42, 44 This may be explained by a difference in protease content of immortalized cell lines compared with bone marrow precursor cells. To test this, we pulse-chased neutrophil progenitors from human bone marrow up to 5 days in the presence of IcatC XPZ-01 . 45 There was no disturbance of neutrophil differentiation and almost total disappearance of proteolytic degradation as seen in PLS patients. Treating human CD34 + hematopoietic stem cells from umbilical cord blood with IcatC XPZ-01 for 10 days during neutrophil differentiation gave similar results.

Brensocatib. In a study using human primary bone marrowderived CD34 + neutrophil progenitor cells, brensocatib almost completely inhibited the activation of elastase-related proteases in a concentration-dependent manner. 43 Evaluation of IcatC XPZ-01 and Brensocatib in Vivo. IcatC XPZ-01 . In mice, IcatC XPZ-01 reached high enough levels in bone marrow to inhibit CatC. In a murine model of rheumatoid arthritis induced by anti-collagen antibodies, subcutaneous administration of IcatC XPZ-01 (1.2 or 4.8 mg/kg twice daily) resulted in sustained antiarthritic activity measured as reduced mean total and rear paw arthritis scores and mean rear paw thickness. 32 This demonstrates that incomplete CatC inhibition with 60−80% reduction of elastase-related protease activity can have a therapeutic effect and that total elimination of the proteases may not be necessary.

Neutrophil elastase-related proteases are implicated in vascular compromise and inflammation following lung transplantation, the so-called ischemia−reperfusion response with primary graft dysfunction. 9 We hypothesized that IcatC XPZ-01 , by blocking elastase maturation in the bone marrow, might minimize this damage. We tested this in an orthotopic mouse lung transplantation (LTx) model after 18 h of cold storage of the graft. Recipient mice treated with subcutaneous IcatC XPZ-01 for 10 days (1.2 mg/kg; twice daily) prior to LTx showed reduced proteolytic activity in bone marrow neutrophils, improved early graft function, and disappearance of active elastase-related proteases in the transplanted lung. Pretreatment with a CatC inhibitor to reduce elastase-related proteases might be a therapeutically useful strategy to minimize the immediate ischemia−reperfusion response to LTx.

Inhibition of elastase-related proteases by IcatC XPZ-01 has also been demonstrated in a non-human primate model of acute lung inflammation. Subcutaneous administration of IcatC XPZ-01 (4.5 mg/kg; twice daily; 12 days) resulted in almost complete elimination of elastase-related proteases in white blood cells ( Figure 3B ) which could still be recruited to the lung in response to lipopolysachharide-induced airway inflammation. 45 These preclinical results confirm that a reduction in elastase-like proteases comparable to that seen in PLS patients is possible using pharmacological inhibitors of bone marrow CatC. Temporary inhibition of CatC to rebalance the protease load during chronic inflammatory diseases might offer new therapeutic possibilities in humans.

Brensocatib. The effect of CatC inhibition by brensocatib on downstream elastase-related protease activation was studied in vivo in naive rats: brensocatib administered twice daily for 8 days resulted in a dose-dependent decrease in protease activity in bone-marrow cell lysates.

Brensocatib was the first nitrile CatC inhibitor to reach clinical trials 43 with randomized, placebo controlled human phase 1 studies commencing in 2014. The safety, tolerability, and pharmacokinetics/pharmacodynamics of single and multiple oral doses were assessed in 81 healthy subjects treated for 28 days with brensocatib or placebo. 46 Daily doses of 10, 25, and 40 mg of brensocatib resulted in 30%, 49%, and 59% reduction in whole blood neutrophil elastase activity. 46 Several dose-dependent, nonserious skin manifestations were observed, including peeling and hyperkeratosis. These symptoms seem unrelated to elastase activity and were not considered sufficiently significant to prohibit further clinical development. 46 In 2016, the biotechnology company Insmed Incorporated announced a licensing agreement with the pharmaceutical company AstraZeneca for global exclusive rights to brensocatib. In the WILLOW phase 2 study evaluating safety, efficacy, and pharmacokinetics, 256 adults with noncystic fibrosis bronchiectasis were treated once daily for 24 weeks with 10 mg or 25 mg of brensocatib or placebo. Results from this international, randomized, double-blind placebo-controlled trial were announced recently. As well as achieving the primary and a key secondary end point, there was a significant reduction in sputum neutrophil elastase which is an important biomarker for CatC inhibition (www.insmed.com). The Immunoblot of white blood cell (WBC) lysates using an antielastase Ab: blood samples were collected from two healthy controls (Ct) and two PLS patients (P). The cells from PLS patients were lacking elastase zymogens which were degraded during neutrophil differentiation in the bone marrow. The blood samples and pictures were taken after informed consent was obtained, and the study was conducted according to Declaration of Helsinki principles. 29 (B) Pharmacological inactivation of CatC by prolonged administration of IcatC XPZ-01 in a Macaca fascicularis experimental model of acute lung inflammation. The image shows broncho-alveolar lavage of an anesthetized macaque. After 11 days administration of IcatC XPZ-01 no dental or dermatological manifestations were observed. For the immunoblot of white blood cell (WBC) lysates using an antielastase Ab, blood samples were collected at days 1, 10, 12 from the macaque treated with IcatC XPZ-01 . CatC inhibition resulted in elimination of elastase zymogens as observed in PLS. All primate experiments and procedures were approved by the local animal experimentation ethic committee (Committee d'Ethique de Val de Loire (CEFA VdL, no. 2013-01-2). 45 The Western-blot results shown in the figure are partially modified and reproduced with permission from Biochemical Pharmacology (https://www. sciencedirect.com/journal/biochemical-pharmacology), 45 Copyright 2017 Elsevier, and from Pharmacology & Therapeutics (https:// www.sciencedirect.com/journal/pharmacology-and-therapeutics), 6 Copyright 2018 Elsevier. company plans to advance brensocatib to phase 3 trials for the treatment of noncystic fibrosis bronchiectasis. Moreover, the company announced very recently that brensocatib will be evaluated in the STOP-COVID19 (Superiority Trial of Protease Inhibition in COVID-19, EudraCT no. 2020-001643-13) trial in up to 300 hospitalized patients with COVID-19.

These preclinical and clinical results provide a powerful argument for testing CatC inhibitors in other neutrophil-driven inflammatory conditions.

CatC, which is ubiquitously expressed in mammals, is considered to be a major intracellularly located, tetrameric dipeptidyl exopeptidase processing a small restricted subset of substrates at the amino terminus. 20 This unique property distinguishes it from other lysosomal and endosomal cathepsins with a broader substrate profile. After cellular uptake of viral particles, endosomal cathepsins, in particular cathepsin L, can cleave the coronavirus surface spike glycoprotein, which is required for membrane fusion and entry of the corona virus mRNA into the cytosol of host cells. 47 The spike protein of SARS-COV-2, however, is susceptible to structurally diverse proteases and to furin-like and trypsin-like TMPRSS2 proteases. 48, 49 However, these proteases cannot be inhibited collectively to prevent the virus from spreading. In many cases, the initiation and progression of pulmonary disease are the result of an excessive and uncontrolled inflammatory response. The molecular and cellular mechanisms involved can culminate in complete respiratory failure. The pathology of viral pneumonia COVID-19 is typical of inflammatory deregulation in the lungs often culminating in ARDS and sometimes death. ARDS is characterized by diffuse alveolar damage with severe hypoxia requiring hospitalization for oxygen therapy administered if necessary by artificial ventilation on an intensive care unit. It appears that COVID-19 can lead to a deadly cytokine release ("cytokine storm"). Neutrophils seem to play a central role in this hyperinflammation which results in acute lung injury and sometimes irreversible degradation of lung tissue. Their recruitment in the respiratory tract is associated with a poor prognosis. Thus, the major threat is not the SARS-CoV-2 virus per se but the inappropriately exaggerated response of the innate immune system. Neutrophils produce proinflammatory cytokines, NETs and proteases, in particular elastase-related serine proteases whose proteolytic activities contribute to acute lung injury and escalate inflammation. Inhibiting elastaserelated serine proteases represents a particularly promising approach to combating inflammatory processes in lung diseases characterized by neutrophilic inflammation. CatC increasingly attracts the attention of both scientists and clinicians because of its role in the activation of proinflammatory neutrophil elastase-related serine proteases im- (2) Lagunas-Rangel, F. A. Neutrophil-to-lymphocyte ratio and lymphocyte-to-C-reactive protein ratio in patients with severe

COVID-19): a meta-analysis

Immunopathogenesis of coronavirus infections: implications for SARS

Innate immunity and pulmonary host defense

The lung is a host defense niche for immediate neutrophil-mediated vascular protection

Therapeutic targeting of cathepsin C: from pathophysiology to treatment

Neutrophil elastase, proteinase 3 and cathepsin G as therapeutic targets in human diseases

Serine protease cathepsin G regulates adhesiondependent neutrophil effector functions by modulating integrin clustering

Premedication with a cathepsin C inhibitor alleviates early primary graft dysfunction in mouse recipients after lung transplantation

Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

Targeting potential drivers of COVID-19: neutrophil extracellular traps

Neutrophil extracellular traps (NETs) are increased in the alveolar spaces of patients with ventilator-associated pneumonia

The role of Neutrophil Extracellular Traps in Covid-19: Only an hypothesis or a potential new field of research?

Selective inhibitors of human neutrophil proteinase 3

Inhibitors and antibody fragments as potential anti-inflammatory therapeutics targeting neutrophil proteinase 3 in human disease

Pharmacological profile of a specific neutrophil elastase inhibitor, Sivelestat sodium hydrate

ONO-5046, a novel inhibitor of human neutrophil elastase

Neutrophil elastase inhibition in acute lung injury: results of the STRIVE study

Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases

Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing

Processing and maturation of cathepsin C zymogen: a biochemical and molecular modeling analysis

On the size of the active site in proteases. I. Papain

Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

Papillon-Lefevre syndrome: correlating the molecular, cellular, and clinical consequences of cathepsin C/dipeptidyl peptidase I deficiency in humans

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis

Haim-Munk syndrome and Papillon-Lefevre syndrome are allelic mutations in cathepsin C

NSP4 is stored in azurophil granules and released by activated neutrophils as active endoprotease with restricted specificity

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

Papillon-Lefevre syndrome patient reveals species-dependent requirements for neutrophil defenses

Characterization of neutrophil function in Papillon-Lefevre syndrome

Structure-based design and in vivo anti-arthritic activity evaluation of a potent dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C

Design and synthesis of dipeptidyl nitriles as potent, selective, and reversible inhibitors of cathepsin C

Cathepsin C inhibitors: property optimization and identification of a clinical candidate

Inhibitors of cathepsin C (dipeptidyl peptidase I)

Discovery of novel cyanamidebased inhibitors of cathepsin C

Dipeptidyl nitriles as human dipeptidyl peptidase I inhibitors

DPP1 inhibitors: exploring the role of water in the S2 pocket of DPP1 with substituted pyrrolidines

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity

In vivo inhibition of serine protease processing requires a high fractional inhibition of cathepsin C

Inhibition of the activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing

Discovery of second generation reversible covalent DPP1 inhibitors leading to an oxazepane amidoacetonitrile based clinical candidate (AZD7986)

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

Dipeptidyl peptidase 1 inhibitor AZD7986 induces a sustained, exposure-dependent reduction in neutrophil elastase activity in healthy subjects

Cathepsin L-selective inhibitors: a potentially promising treatment for COVID-19 patients

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 Site