Role of Serine Proteases and Host Cell Receptors Involved in Proteolytic Activation, Entry of SARS-CoV-2 and Its Current Therapeutic Options

Abstract The current global pandemic of a novel severe acute respiratory syndrome coronavirus-2 continues with its public health disaster beginning from late December 2019 in Wuhan, Hubei province, China. The scientific community has tried to fight against this novel coronavirus through vaccine development and designing different candidate drugs. However, there is no well-defined therapy to prevent 2019-nCov infection, thus complete prevention of the virus remains difficult. Therefore, it is a critical factor for death of millions worldwide. Many clinical trials and insights are ongoing in the struggle with this pandemic of SARS-CoV-2. SARS-CoV-2 entry into the host cell requires host cell angiotensin-converting enzyme-2 (ACE2) and glucose regulated protein 78 (GRP78). On the other hand, proteolytic activation of the viral spike protein (S protein) needs the host cell serine proteases, including transmembrane serine protease 2 (TMPRSS2), cathepsins, trypsin and furin. This review focuses on the protein involved in the mechanism of entry, and proteolytic activation. In addition, it looks at current therapeutic options for SARS-CoV-2.


Introduction
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a global health pandemic, which originated and was disseminated from Wuhan, in the Chinese province of Hubei, and it causes millions of death worldwide. 1 The spike protein (S protein) of coronavirus is a structural surface glycoprotein, which is responsible for host-virus membrane fusion, binding of receptor and pathogenesis of virus. 2 It is composed of carboxyl intracellular terminus, transmembrane domain and N-terminal extracellular domain. 3 Spike glycoprotein consists of S1 and S2 subunits having a binding and hostviral membrane fusion activity, respectively. It facilitates viral entry into the host cell, and it is encoded by all types of coronavirus. However, the receptor binding domain (RBD) of S protein facilitates a viral-host receptor interaction specific to beta coronavirus subtypes. 4 The cleavage of S2 and S1 subunits at the boundary site causes a conformational change for membrane fusion of the virus and the host cells. 5 Host cell surface receptor, angiotensin-converting enzyme 2 (ACE2) is recognized by the SARS-CoV-2 S1 subunit of spike protein that contains RBD. 6 The activity of vital host-cell receptor, ACE2 is activated by androgen-dependent enzyme, including transmembrane protease serine 2 (TMPRSS2), thus a recent therapeutic strategy plan depends on blocking the activity of this cellular receptor. 7 Studies revealed that ACE2 is a determinant factor for the entry of SARS-CoV-2 rather than TMPRSS2 because the expression of TMPRSS2 is limited to a specific organ. 8 Cellular entry of SARS-CoV-2 also depends on the activity of serine proteases, such as TMPRSS2 at PH-independent state. 9 Fusion of SARS-CoV-2 requires proteolytic cleavage of spike glycoprotein at the boundary of S1 and S2 subunits. The priming of S protein depends on the activity of host cell proteases and it induces conformational changes in those subunits. 10 Studies showed that the activation of spike protein requires the activity of TMPRSS2 to cleave it. 11 On the other hand, the fusion of viral-host membrane requires furinbased priming at the S1/S2 site of S protein, hence proteolytic cleavage at this site induces the activity of TMPRSS2 to facilitate entry of SARS-CoV-2. 12 The S protein of SARS-CoV-2 consists of S1 and S2 cleavage sites required for the purpose of proteolytic activation induced by furin. 13 Studies state that effective priming of S protein of virus is activated by furin and it facilitates entry of the virus. 14 In addition to TMPRSS2 and furin, cathepsins induce endocytic-based fusion of host cell and viral membrane. 15 Although the activity of cathepsin has been well-studied in MERS-CoV and SARS-CoV-2, its potential role still needs further investigation. However, the investigation done on 293T cells having ACE2 showed that the entry of the virus was decreased by 76% in cathepsin L inhibitor treated pseudo virus. 1 Inhibition of the activity of cathepsin B/L by elevating endosomal pH leads to reduce the entry of SARS-CoV-2 in 293T cells. 16 In addition to this, the other cellular protease promotes the activation of S protein to facilitate viral entry into the host cell. 17 It has a similar effect with TMPRSS2 regarding cleavage of S protein of SARS-CoV-2. 9

Angiotensin-Converting Enzyme 2 Mediated SARS-CoV-2 Entry
Tissues express exopeptidase angiotensin-converting enzyme 2 (ACE2) to activate the binding of SARS-CoV-2 spike glycoprotein to host cell receptors. 18 The expression of ACE2 is not only associated with the lung, but also with extra-pulmonary tissues, which indicates the possible dissemination of virus to other tissues. 19 Dissemination of the virus to the lungs, gastrointestinal tract, kidneys, and heart indicates the expression of ACE2 in those vital organs. 20 A recent investigation done on Chinese pigs, civets, and bats showed that HeLa cells having ACE2 surface receptors were infected with SARS-CoV-2. 21 Studies had correlated and confirmed the binding interaction of ACE2 with former SARS-CoV and current SARS-CoV-2 S protein. Therefore, they notified the presence of the same model interaction. 22 Thus, SARS-CoV-2 entry to the host cell depends on the presence of ACE2 on the host cell membrane. 23 The investigation done on animal models showed that the severity of SARS-CoV infection is associated with elevated expression of this cellular surface receptor. 24 Inhibition of binding interaction of ACE2 and S protein requires down-regulation of ACE2 host cell receptor. However, ACE2 undergoes endocytosis through angiotensin II type 1 receptor (AT1R) to facilitate its physiological and biological action within the cell. Thus, downregulating the expression of ACE2 has its own complications in lungs. 25 The role of ACE2 in the entry mechanism of SARS-CoV-2 has similar activity to the former SARS-CoV, thus RBD of spike glycoprotein binds to ACE2. 26 The receptor binding domain found on S1 subunit of S protein is vital for the binding of ACE2 to the virus. 27 Binding interaction of ACE2 to RBD region of S protein is a determinant factor for entry of the virus to the host cell 28 ( Figure 1). The receptor binding domain consists of 318-510 amino acid residues that are responsible for binding with ACE2. 29 In vivo experimental studies done in China revealed that ACE2 is recognized by A475 and F486 amino acid residues found in RBD of S protein. 28 In addition to viral entry, the transmission of the virus among humans is also associated with tight binding of S protein with ACE2. 5

Glucose Regulated Protein 78 Mediated SARS-CoV-2 Entry
Cellular stress condition is a factor for the release of glucose regulated protein 78 (GRP78) from endoplasmic reticulum and its exportation to cell membrane, which in turn leads to entry mechanism of SARS-CoV-2. 30 Recent data predicted that the binding and entry activity of the virus is related to the interaction of S protein with membrane bounded protein, including GRP78. 31 Binding of SARS-CoV-2 to GRP78 is expected to be facilitated by a molecular docking mechanism that occurs through the interaction between GRP78 substrate binding domain region and region IV of this novel coronavirus. 32 The molecular docking model (HADDOCK software) utilized by researchers revealed that SARS-CoV-2 S protein residue at C480-C488 was responsible for binding with GRP78, and they identified more than four interaction sites between GRP78 and viral S protein. 33  researchers confirmed that S protein of virus may interact with GRP78 in a head-to-head fashion in the III and IV regions of substrate binding domain of GRP78. 30 In addition to GRP78, multi-walled carbon nanotube mechanistic studies attempted to identify the binding activity of SARS-CoV-2 of RBD-ACE2 complex (6LZG) and main viral proteases (6LU7) and they confirmed the presence of strong binding activity between ACE2 and viral S protein RBD. 34 Endoplasmic reticulum (ER) stress increases in SARS-CoV-2 infected cells, which in turn activates an unfolded protein response. Storage of unfolded protein within ER leads to the release of GRP78 to activate the enzymes such as inositol requiring enzyme 1 (IRE1), protein kinase RNAlike endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) to refold and synthesize protein. 35 The level of GRP78 becomes elevated as a result of unfolded protein response activity within ER. 36 Law and his coworkers in 2020 revealed that the genetic expression of GRP78 increased because ER stress is caused by the influence of SARS-CoV-2 S protein. 37 Elevated levels of GRP78 and S protein within endoplasmic reticulum (ER) in turn affect unfolded protein response. 38 Thus, the cellular stress condition leads to the exportation of GRP78 to the plasma membrane to activate virus-host binding interaction and activate the viral entry mechanism 39 ( Figure 1). Studies notified that inhibition of GRP78 ATPase activity using celecoxib derivative, AR12 (OSU-03012) may act as a possible therapeutic option to diminish its protein renaturation capability and decrease S protein synthesis. 40

Transmembrane Serine Protease 2 Mediated SARS-CoV-2 Entry
Transmembrane serine protease 2 (TMPRSS2) is a host cellgenerated protease capable of priming S protein to activate the binding of the SARS-CoV-2 to ACE2 because it consists of the extracellular domain utilized for virus-receptor interaction. 23

DovePress
In addition to other host cell proteases, TMPRSS2 is a major protease for SARS-CoV-2 infection, and invasion of human lung cells. 41 ACE2 is the main cellular surface receptor involved in the SARS-CoV-2 entry mechanism. 42 After the virus binds to ACE2 of the host cell, TMPRSS2 activates priming and fusion of virus-host cell membrane, which also leads to the entry of the virus to the host cell 43 (Figure 1). The S protein of coronavirus consists of S1 and S2 subunits having RBD and membrane fusion functional elements, respectively. Transmembrane domain, hepatid and fusion peptides are the main components of S2 subunit of CoV S protein. 44 Although the multi-basic amino acid residues of S1/S2 of SARS-CoV-2 are undefined, it is found in other human coronaviruses, including MERS-COV, HKU1 and OC43. 41 After binding of S protein of virus to ACE2, it undergoes conformational changes to be proteolytically degraded in the midpoint of S1 and S2 subunits by host cell proteases, including TMPRSS2. 45 In addition to cathepsin B/L mediated endocytosis, cleavage of S protein by TMPRSS2 also activates the entry of SARS-CoV into host cells. 46 Although the mechanism of TMPRSS2 induced activation of S protein is not well-defined, studies confirmed that TMPRSS2 mediates host cells and viral membrane fusion. 47 Researchers revealed that fusion of plasma and viral membrane during the entry mechanism provokes TMPRSS2 to induce extracellular priming of S protein as the virus binds to ACE2. 43 Transmembrane serine protease 2 activates and cleaves S protein to induce infectivity and spread of the virus. 18,43,44 It has a potential therapeutic insight after the identification of its impact on the pathogenesis of H7N9, H3N2 and H1N1 influenza A virus. 1 Proteins Involved During Proteolytic Activation of SARS-CoV-2 Spike Protein Cathepsins Cathepsins are members of various proteases that are involved in the antigen presenting process, and proteolytic activation. 48 Proteolytic activation of coronavirus S protein activates the generation of a better conformational state for the purpose of receptor binding activity and virus-host cell membrane fusion. 49 Recent investigations tried to assess proteases regarding cleavage of S1/S2 site of S protein of SARS-CoV, and they showed that cathepsin B is capable of actively degrading S1/S2 site of S protein. 48 As TMPRSS2 acts on host-viral membrane fusion, cathepsin B/L undergoes their proteolytic activation after endocytosis of the virus. 50 Cathepsins play a major role in the fusion of endosomal and viral membrane because they cause priming of S protein following endocytosis of the virus. 43 The entry of CoV to the target cell depends on the cathepsin levels of these cells during the endocytosis process. 51 The cathepsin found in the lysosome facilitates endocytic dependent entry of SARS-CoV-2 to the host cell. 52 Studies assessed the role of cathepsins for the entry mechanism of the virus, thus cathepsin L inhibitor (calpain (E64D)) and cathepsin L, B, H inhibitors were introduced to HEK 293/hACE2 cells. In this investigation, the introduction of cathepsin L to these cells induces a marked decline in the SARS-CoV-2 entry mechanism compared with cathepsin B. 51 In this regard, cathepsin L induces splitting of S protein to increase viral entry and infection. 53 An investigation done on animals indicates that SARS-CoV-1 utilizes cathepsin-L for infection. 54 Although inhibition of cathepsin L by calpain inhibitor XII is not clearly elucidated, studies notified that calpain inhibitor XII is an active inhibitor of cathepsin L. 55 Trypsin Trypsin is a serine endopeptidase, which is expressed mainly in respiratory and digestive cells and it acts as a digestive enzyme. 48 Although trypsin is not highly selective for cleavage sites on S protein, the arginine amino acid residues found in between P6-P6ʹ region of S protein are mainly recognized and cleaved by it. 56 It is an active protease that can be inhibited by a candidate drug, such as camostat. 1 Even though the mechanism of proteolytic activation at the S1/S2 boundary and S2ʹ site is not well-defined, multi-basic S1/S2 site is essential for SARS-CoV-2 viral infection in the cells of the lung. 15 Cleavage at the S1/S2 site of SARS-CoV-2 S protein may be associated with priming due to trypsinlike protease activity. 10 Both trypsin and TMPRSS2 are required for the activation of syncytium formation during SARS-CoV-2 S protein expression. Therefore, S1/S2 sites may be optimized for altered viral pathogenesis and invasion of the virus. 41 In comparisons with various coronaviruses, the cleavage potential of trypsin had a higher efficiency for SARS-CoV-2 than other coronavirus subtypes. 48 Furin Furin is highly expressed in some tissues, including kidney, endocrine organ, and lungs as well as being expressed in moderate amounts in the gastrointestinal tract. 57 Vankadari and Naveen in their 2020 preprint studies express furin as a Ca2+ dependent protease, which is highly expressed in lung cystic fibrosis disorder, and it is involved in the cleavage of SARS-CoV-2 viral glycoprotein. 58 The availability of furinlike cleavage at S1/S2 site of viruses, including SARS-CoV-2 contributes to the pathogenesis of the virus. 57 SARS-CoV-2 utilizes furin for the cleavage of S protein at the S1/S2 site. 59 Furin induces priming of S protein at this site for the fusion of host cell membrane, and viral glycoprotein. 57 The expression of furin in the trans-golgi network hypothesizes its possible involvement in the synthesis of viral S protein. In addition, it mediates multi-basic site cleavage of S protein of SARS-CoV -2. 60 Tang and his coworkers in their preprint studies in 2020 identified the presence of additional amino acid residue, P-R-R-A in SARS-CoV-2 at S1/S2 site that was not notified in SARS-CoV. 61 R-R-A-R amino acid residue at the S1/S2 site of S protein may be sensitive to furin dependent cleavage. 62 The activity of furin resides on this poly basic motif to activate the degradation of S protein to generate S2 and S1 subunits. 52 Comparison of the cleavage potential of both MERS-CoV and SARS-CoV-2 showed it was highly efficient for SARS-CoV-2 rather than MERS-CoV. 63 This amino acid residue is a common nucleotide sequence for the substrate of furin and S1/S2 site of the virus. Therefore, proteolytic activation of the S1/S2 site by furin may lead to syncytium generation. 64 Studies revealed that the fusion of SARS-CoV-2 in S1/S2 mutated S protein was elevated as compared with fusion with mutated furin cleavage activity. 15

Therapeutic Potential of Inhibiting Entry Mechanism and Proteolytic Activation
The therapeutic strategy plan for human coronavirus depends on host-virus interaction to target host and viral protein of HCov-NL63, MERS-CoV, SARS-CoV, and HCOV-229E. 65 Endoplasmic reticulum stress contributes to the pathogenesis of SARS-CoV-2 and it may be treated with pharmacological chaperones, including GRP78 to enhance protein folding in the ER, but this therapeutic strategy may take a long time. 66 Studies correlated the level of GRP78 with ER stress. Studies confirmed GRP78 serves as a biochemical parameter to evaluate the level of ER stress. 36 Thus, among SARS-CoV -2 infected study groups, the amount of GRP78 was elevated compared with the apparently healthy controls and SARS-COV-2 negative pneumonia patients. 36 Studies done in Turkey hypothesize that cellular ER stress may be elevated because of significant elevation of GRP78 among SARS-CoV-2 infected patients compared with healthy groups 67 ( Table 1). The therapeutic strategy of SARS-CoV-2 is effective by targeting ACE2 and GRP78 host cell surface receptors. 68 The therapeutic strategic mechanism against GRP78 is associated with targeting SBD and nucleotide binding domain of GRP78. Thus, potent inhibitors including epigallocatechin gallate (EGCG) and various bioactipeptides (satpdb18674, satpdb18446, satpdb12488, satpdb14438, and satpdb28899) was tried to inhibit GRP78-S protein complex interaction. 69 Serum protein level of GRP78 was analyzed using case-control observational study in Turkey among 144 adult patients (Table 1). COVID-19 patients showed higher levels of GRP78 compared with control groups and ER stress induces an elevated level of GRP78 67 Morbidity of SARS-CoV-2 was elevated in COVID-19 patients who took angiotensin-receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) because the expression of ACE2 is increased. 70 Similarly, a descriptive study done in China indicates that the severity and infection of SARS-CoV-2 was elevated among chronic renal disease, coronary artery disease, diabetes and hypertensive patients who were under treatment of ACEIs or ARBs. 25 These studies argue that the treatment outcomes induces the elevated expression of ACE2 in various tissues including heart, kidney and intestine and aggravates mortality and morbidity of SARS-CoV-2. On the contrary, the investigation done on animal models notified that ACE2 upregulated as ACEI/ARB demonstrated for therapy; however, ARB/ACEI blocked renin-angiotensin system may prevent COVID-19 illness or severity and it may act as a therapeutic option in COVID-19 patients. 71 Safety of ACEI/ARB is under evaluation by observational cohort study design among 10,000 adult COVID-19 and hypertensive patients (clinicaltrial. gov, 2020) ( Table 1). Although the inhibitors of viral protein are not well-defined and studied, the strategy of SARS-CoV -2 therapy can be associated with the binding interaction of S protein, E protein and N protein with RBD. 72 Inhibiting the binding of SARS-CoV-2 with ACE2 is a potential therapeutic strategy by blocking S1/S2 subunit of RBD of viral spike glycoprotein. 73 The binding affinity of S protein to ACE2 elevated to 10 up to 20 times as compared with their binding interaction in the former SARS-CoV, so the therapeutic target of their interaction has greater potential. 18 TMPRSS2 activates proteolytic cleavage of ACE2, which is a vital host cell receptor for SARS-CoV-2 entry. 73 Studies done on mice indicate that DNA-vaccine development targeting the SARS-CoV-2 S protein inhibits the binding of ACE2 with viral S protein. 74  DovePress macaques. 75 Regarding Th1 vs Th2 polarization, SARS-CoV -2-specific neutralizing antibodies and incidence of adverse events on a Synthetic MVA-based SARSCoV-2 Vaccine was assessed among 129 adult COVID-19 patients in the USA through placebo-randomized interventional clinical trial (clinicaltrial.gov, 2020) ( Table 1). In addition to DNAbased vaccine, pre-clinical trials focus on the investigation of recombinant vaccination of S protein in various research institutions worldwide. 76 A recombinant NVX-CoV2373 nanoparticle vaccine is a potential to evaluate the efficacy in Phase I and the safety of vaccine development in Phase II of the study. 77 Efficacy of recombinant vaccine development is found at a phase I interventional randomized clinical trial among 216 adult Chinese COVID-19 patients (clinicaltrial. gov, 2020) ( Table 1).
Studies done on animal models through 293 T cells showed that blockage of cathepsin L/B activity had their restriction on the entry of the virus to host cells. 16 Studies notified that ER stress is a promising therapeutic strategy to interrupt viral pathogenesis and minimize apoptosis. 37 The entry of coronavirus to the host cell may be blocked by targeting important host proteins, including ACE2 and TMPRSS2, because the former one is crucial for entry, whereas the latter one is critical for the fusion of host and viral membranes. 78 Although there are no well-defined studies done on ACE2 inhibitors, promazine and emodin are expected to interfere with the binding interaction of ACE2 with viral S glycoprotein; however, the effect of these candidate drugs on ACE2-expressed tissue is not well-known. 79 As compared with emodin, promazine is an active inhibitor of the interaction of ACE2 and SARS-CoV S glycoprotein. 80 In addition to host cellular receptors, the viral S protein also is a potential therapeutic target for vaccines, antibody generation and designation of inhibitors. 81 The cleavage of S protein needs the activity of furin and TMPRSS2 priming at the S1/S2 site of viral protein, hence they are a vital therapeutic strategy of serine protease inhibitors to treat the current pandemic of COVID-19. 62 Protease inhibitor (camostat) is under clinical trials of Phases I-III beginning from April 2020 to inhibit protease activity of TMPRSS2; however, the specific target of this protease is not elucidated clearly yet. 82 Clinical trials.gov registered three clinical trials regarding the efficacy of camostat against SARS-CoV-2 in different countries, including Germany, the USA and Denmark. 83 Rubitecan, loprazolam, nafamostat and camostat are some approved drugs that are tried in the clinical trial of treatment of SARS-CoV-2. 84 Transmembrane serine protease 2 is an active serine protease that can be inhibited by camostate mesylate, consequently fusion of host-viral membrane becomes blocked and diminishes entry of virus to host cells. 85 In addition to TMPRSS2, the activity of ACE2 becomes reduced by camostat to inhibit virus-host binding interaction. 86 The safety and efficacy of camostate are under investigation among 114 adult COVID-19 patients through placebo-interventional clinical trial in the USA (clinicaltrial.gov, 2020) ( Table 1). The expression of mRNA TMPRSS2 is downregulated as a result of bicalutamide intervention among COVID-19 patients, which in turn leads to decreased infection of SARS-CoV-2 87 because genetic expression of TMPRSS2 is elevated by androgen receptors. The promoter region of mRNA TMPRSS2 contains 15 base pairs of androgen response elements. Therefore, androgen accounts for post-translational modification as well as transcriptional regulation of TMPRSS2 mRNA. 88 The safety and efficacy of bicalutamide are on Phase III-interventional randomized clinical trial among 100 adult COVID-19 patients in the USA (clinicaltrial.gov, 2020) ( Table 1). Furin-mediated proteolytic cleavage promotes the availability of amino acid motifs RRAR/S found on S protein of SARS-CoV-2. The cleavage at those amino acid residues leads to the fusion of host-cell and viral membrane. 89 In addition to TMPRSS2 and furin, trypsin also contributes to the cleavage of S glycoprotein, which indicates a possible therapeutic target of SARS-CoV-2. 90

Conclusion
Designing therapies for the current SARS-CoV-2 global pandemicis the major challenge of fighting against the virus. Various clinical insights and trials are on the way for the treatment of the virus. This review suggests that targeting the host cell surface receptors had more therapeutic potential than targeting protein required for proteolytic activation of the spike protein. Therefore, targeting ACE2, GRP78 and TMPRSS2 have more therapeutic strategic potential as compared with cathepsins, furin and trypsin. Inhibition of entry mechanism is preferable to blocking proteolytic activation of the virus,because it inhibits fusion and further pathogenesis of the virus.

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