Host Serine Proteases: A Potential Targeted Therapy for COVID-19 and Influenza

The ongoing pandemic illustrates limited therapeutic options for controlling SARS-CoV-2 infections, calling a need for additional therapeutic targets. The viral spike S glycoprotein binds to the human receptor angiotensin-converting enzyme 2 (ACE2) and then is activated by the host proteases. Based on the accessibility of the cellular proteases needed for SARS-S activation, SARS-CoV-2 entrance and activation can be mediated by endosomal (such as cathepsin L) and non-endosomal pathways. Evidence indicates that in the non-endosomal pathway, the viral S protein is cleaved by the furin enzyme in infected host cells. To help the virus enter efficiently, the S protein is further activated by the serine protease 2 (TMPRSS2), provided that the S has been cleaved by furin previously. In this review, important roles for host proteases within host cells will be outlined in SARS-CoV-2 infection and antiviral therapeutic strategies will be highlighted. Although there are at least five highly effective vaccines at this time, the appearance of the new viral mutations demands the development of therapeutic agents. Targeted inhibition of host proteases can be used as a therapeutic approach for viral infection.

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Current Potential Therapeutic Approaches against SARS-CoV-2: A Review

Biomedicines ◽

10.3390/biomedicines9111620 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1620

Author(s):

Dharmendra Kumar Yadav ◽

Desh Deepak Singh ◽

Ihn Han ◽

Yogesh Kumar ◽

Eun-Ha Choi

Keyword(s):

Wide Spectrum ◽

Host Cells ◽

Hiv Protease ◽

Hiv Protease Inhibitors ◽

Therapeutic Approaches ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Nucleotide Analogue ◽

Infection Treatment ◽

Current Potential

The ongoing SARS-CoV-2 pandemic is a serious threat to public health worldwide and, to date, no effective treatment is available. Thus, we herein review the pharmaceutical approaches to SARS-CoV-2 infection treatment. Numerous candidate medicines that can prevent SARS-CoV-2 infection and replication have been proposed. These medicines include inhibitors of serine protease TMPRSS2 and angiotensin converting enzyme 2 (ACE2). The S protein of SARS-CoV-2 binds to the receptor in host cells. ACE2 inhibitors block TMPRSS2 and S protein priming, thus preventing SARS-CoV-2 entry to host cells. Moreover, antiviral medicines (including the nucleotide analogue remdesivir, the HIV protease inhibitors lopinavir and ritonavir, and wide-spectrum antiviral antibiotics arbidol and favipiravir) have been shown to reduce the dissemination of SARS-CoV-2 as well as morbidity and mortality associated with COVID-19.

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Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy

Current Medicinal Chemistry ◽

10.2174/0929867328666210526111318 ◽

2021 ◽

Vol 28 ◽

Author(s):

Patrick Müller ◽

Hannah Maus ◽

Stefan Josef Hammerschmidt ◽

Philip Knaff ◽

Volker Mailänder ◽

...

Keyword(s):

Cathepsin L ◽

Cell Entry ◽

New Drugs ◽

Host Cells ◽

S Protein ◽

Cell Receptors ◽

Current State ◽

Causative Drug ◽

Transmembrane Serine Protease ◽

Global Threat

: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since currently, there is no causative drug against this viral infection available, science is striving for new drugs and approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARS-CoV-2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.

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Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2–Priming Protease TMPRSS2

Pathogens and Immunity ◽

10.20411/pai.v6i1.408 ◽

2021 ◽

Vol 6 (1) ◽

pp. 55-74

Author(s):

Nurit P Azouz ◽

Andrea Klingler ◽

Victoria Callahan ◽

Ivan Akhrymuk ◽

Katarina Elez ◽

...

Keyword(s):

Viral Load ◽

Protease Inhibitor ◽

Serine Protease ◽

Inhibitory Activity ◽

Serine Proteases ◽

Serine Protease Inhibitor ◽

Host Cells ◽

S Protein ◽

Relative Contribution ◽

Alpha 1 Antitrypsin

Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

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Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.007779 ◽

2019 ◽

Vol 294 (25) ◽

pp. 9830-9843 ◽

Cited By ~ 9

Author(s):

Jialin Zhang ◽

Jianfei Chen ◽

Da Shi ◽

Hongyan Shi ◽

Xin Zhang ◽

...

Keyword(s):

Cell Surface ◽

Cell Cultures ◽

Viral Entry ◽

Cathepsin B ◽

Cathepsin L ◽

The United States ◽

Protein Cleavage ◽

S Protein ◽

Endosomal Pathway

Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the genus Deltacoronavirus that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pretreating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Because PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo. Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.

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SARS-CoV-2 Entry inhibitors targeting virus-ACE2 or virus-TMPRSS2 interactions

Current Medicinal Chemistry ◽

10.2174/0929867328666210420103021 ◽

2021 ◽

Vol 28 ◽

Author(s):

Hao Lin ◽

Srinivasulu Cherukupalli ◽

Da Feng ◽

Shenghua Gao ◽

Dongwei Kang ◽

...

Keyword(s):

Life Cycle ◽

Host Cells ◽

Cell Surface Receptor ◽

Target Cells ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Entry Inhibitors ◽

Concise Report ◽

Surface Receptor ◽

Transmembrane Serine Protease

: COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the cleavage effect of type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions, to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.

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D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

10.1101/2020.06.20.161323 ◽

2020 ◽

Cited By ~ 36

Author(s):

Jie Hu ◽

Chang-Long He ◽

Qing-Zhu Gao ◽

Gui-Ji Zhang ◽

Xiao-Xia Cao ◽

...

Keyword(s):

Host Cells ◽

Its Sequence ◽

Elastase Inhibitor ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Functional Differences ◽

Common Receptor ◽

Sequence Variations ◽

Human Igg1 ◽

Major Target

AbstractCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.

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Killing Two Birds with One Stone by Administration of Soluble ACE2: A Promising Strategy to Treat Both Cardiovascular Diseases and SARS-CoV-2 Infection

Viruses ◽

10.3390/v13112243 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2243

Author(s):

Fengling Feng ◽

Jiaoshan Chen ◽

Jin Zhao ◽

Yanjun Li ◽

Minchao Li ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Therapeutic Strategy ◽

Therapeutic Agent ◽

Cardiovascular Complications ◽

Therapeutic Agents ◽

Cell Entry ◽

Host Cells ◽

Angiotensin Converting Enzyme 2 ◽

High Affinity ◽

Promising Strategy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells mainly by the angiotensin converting enzyme 2 (ACE2) receptor, which can recognize the spike (S) protein by its extracellular domain. Previously, recombinant soluble ACE2 (sACE2) has been clinically used as a therapeutic treatment for cardiovascular diseases. Recent data demonstrated that sACE2 can also be exploited as a decoy to effectively inhibit the cell entry of SARS-CoV-2, through blocking SARS-CoV-2 binding to membrane-anchored ACE2. In this study, we summarized the current findings on the optimized sACE2-based strategies as a therapeutic agent, including Fc fusion to prolong the half-life of sACE2, deep mutagenesis to create high-affinity decoys for SARS-CoV-2, or designing the truncated functional fragments to enhance its safety, among others. Considering that COVID-19 patients are often accompanied by manifestations of cardiovascular complications, we think that administration of sACE2 in COVID-19 patients may be a promising therapeutic strategy to simultaneously treat both cardiovascular diseases and SARS-CoV-2 infection. This review would provide insights for the development of novel therapeutic agents against the COVID-19 pandemic.

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Cross-species recognition of SARS-CoV-2 to bat ACE2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020216118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2020216118

Author(s):

Kefang Liu ◽

Shuguang Tan ◽

Sheng Niu ◽

Jia Wang ◽

Lili Wu ◽

...

Keyword(s):

Binding Capacity ◽

Species Recognition ◽

Interaction Network ◽

Binding Mode ◽

Host Cells ◽

Structural Basis ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Protein Receptor ◽

Human Receptor

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2–related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 fromRhinolophus macrotis(bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

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Characterization of spike glycoprotein of 2019-nCoV on virus entry and its immune cross-reactivity with spike glycoprotein of SARS-CoV

10.21203/rs.2.24016/v1 ◽

2020 ◽

Cited By ~ 4

Author(s):

Xiuyuan Ou ◽

Yan Liu ◽

Xiaobo Lei ◽

Pei Li ◽

Dan Mi ◽

...

Keyword(s):

Virus Entry ◽

Cathepsin L ◽

Cross Reactivity ◽

Spike Glycoprotein ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Dependent Cell ◽

Cross Neutralization ◽

Entry Receptor

Abstract Since beginning of this century, there have already been three zoonotic outbreaks caused by beta coronaviruses (CoV), SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly identified 2019-nCoV in late 2019, Wuhan, China. As to Feb 10th, 2020, there are over 40,000 confirmed cases and over 900 deaths. However, little is known about the biology of this newly emerged virus. Here we developed a lentiviral based pseudovirus system for S protein of 2019-nCoV to study virus entry in BSL2 settings. First, we confirmed that human angiotensin converting enzyme 2 (hACE2) is the main entry receptor for 2019-nCoV. Second, we found that 2019-nCoV S protein mediated entry on 293/hACE2 cells was mainly through endocytosis, and PIKfyve, TPC2, and cathepsin L are critical for virus entry. Third, 2019-nCoV S protein is less stable than SARS-CoV, and it could trigger protease-independent and receptor dependent cell-cell fusion, which might help virus rapidly spread from cell to cell. Finally and more importantly, polyclonal anti-SARS S1 antibodies T62 effectively inhibited entry of SARS-CoV S pseudovirions, but almost had no effect on entry of 2019-nCoV S pseudovirions. Further studies using sera from one recovered SARS-CoV patient and five 2019-nCoV patients showed that there was only limited cross-neutralization activities between SARS-CoV and 2019-nCoV sera, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for 2019-nCoV.

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Genetic variations and drug repurposing provides key insights into the disruption of the SARS COV2

10.31219/osf.io/b7y2c ◽

2020 ◽

Cited By ~ 1

Author(s):

Gayatri Panda ◽

Neha Mishra ◽

Arjun Ray

Keyword(s):

Drug Repurposing ◽

Genetic Variations ◽

Protein Docking ◽

Receptor Protein ◽

Energy Estimation ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Drug Molecules ◽

Initial Interaction ◽

Human Receptor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is mediated via the initial interaction of the virus’s spike (S-) protein with a human receptor protein called angiotensin-converting enzyme 2 (hACE2). Interference in this association can have immense therapeutic importance. We used an in-silico combinatorial approach involving homology-based protein modeling, protein-protein docking, binding energy estimation and virtual screening techniques to probe for genetic variations and drug molecules for disruption of the hACE2-CoV2 interaction. Our results identified Ser19Pro variation on hACE2 showed similar structural stability to the native protein while having a destabilizing effect in the hACE2(Ser19Pro)-S-protein complex. We also found several FDA-approved drug molecules that can potentially induce competition-mediated destabilization of the hACE2-CoV2 complex. In conclusion, these findings provide critical insights for intervention strategies targeting the pathogenicity of SARS-CoV-2.

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