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1.
Int J Mol Sci ; 23(6)2022 Mar 08.
Article in English | MEDLINE | ID: covidwho-1765729

ABSTRACT

Elastases are a broad group of enzymes involved in the lysis of elastin, the main component of elastic fibres. They are produced and released in the human body, mainly by neutrophils and the pancreas. The imbalance between elastase activity and its endogenous inhibitors can cause different illnesses due to their excessive activity. The main aim of this review is to provide an overview of the latest advancements on the identification, structures and mechanisms of action of peptide human neutrophil elastase inhibitors isolated from natural sources, such as plants, animals, fungi, bacteria and sponges. The discovery of new elastase inhibitors could have a great impact on the pharmaceutical development of novel drugs through the optimization of the natural lead compounds. Bacteria produce mainly cyclic peptides, while animals provide for long and linear amino acid sequences. Despite their diverse natural sources, these elastase inhibitors show remarkable IC50 values in a range from nM to µM values, thus representing an interesting starting point for the further development of potent bioactive compounds on human elastase enzymes.


Subject(s)
Leukocyte Elastase , Peptides , Animals , Humans , Leukocyte Elastase/metabolism , Neutrophils/metabolism , Proteinase Inhibitory Proteins, Secretory/pharmacology , Serine Proteinase Inhibitors/pharmacology
2.
Molecules ; 27(6)2022 Mar 14.
Article in English | MEDLINE | ID: covidwho-1742556

ABSTRACT

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.


Subject(s)
COVID-19 , Tandem Mass Spectrometry , Animals , Benzamidines , Chromatography, Liquid/methods , Guanidines , Rats , Rats, Sprague-Dawley , Serine Proteinase Inhibitors/pharmacology , Tandem Mass Spectrometry/methods
3.
Int J Mol Sci ; 23(3)2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1650980

ABSTRACT

TMPRSS2 is a type II transmembrane protease with broad expression in epithelial cells of the respiratory and gastrointestinal tract, the prostate, and other organs. Although the physiological role of TMPRSS2 remains largely elusive, several endogenous substrates have been identified. TMPRSS2 serves as a major cofactor in SARS-CoV-2 entry, and primes glycoproteins of other respiratory viruses as well. Consequently, inhibiting TMPRSS2 activity is a promising strategy to block viral infection. In this review, we provide an overview of the role of TMPRSS2 in the entry processes of different respiratory viruses. We then review the different classes of TMPRSS2 inhibitors and their clinical development, with a focus on COVID-19 treatment.


Subject(s)
COVID-19/drug therapy , Molecular Targeted Therapy/trends , Serine Endopeptidases/physiology , COVID-19/genetics , Humans , Molecular Targeted Therapy/methods , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/therapeutic use , Virus Internalization/drug effects
4.
Biomed Res Int ; 2022: 1558860, 2022.
Article in English | MEDLINE | ID: covidwho-1622112

ABSTRACT

Increasing outbreaks of new pathogenic viruses have promoted the exploration of novel alternatives to time-consuming vaccines. Thus, it is necessary to develop a universal approach to halt the spread of new and unknown viruses as they are discovered. One such promising approach is to target lipid membranes, which are common to all viruses and bacteria. The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has reaffirmed the importance of interactions between the virus envelope and the host cell plasma membrane as a critical mechanism of infection. Metadichol®, a nanolipid emulsion of long-chain alcohols, has been demonstrated as a strong candidate that inhibits the proliferation of SARS-CoV-2. Naturally derived substances, such as long-chain saturated lipid alcohols, reduce viral infectivity, including that of coronaviruses (such as SARS-CoV-2) by modifying their lipid-dependent attachment mechanism to human host cells. The receptor ACE2 mediates the entry of SARS-CoV-2 into the host cells, whereas the serine protease TMPRSS2 primes the viral S protein. In this study, Metadichol® was found to be 270 times more potent an inhibitor of TMPRSS2 (EC50 = 96 ng/mL) than camostat mesylate (EC50 = 26000 ng/mL). Additionally, it inhibits ACE with an EC50 of 71 ng/mL, but it is a very weak inhibitor of ACE2 at an EC50 of 31 µg/mL. Furthermore, the live viral assay performed in Caco-2 cells revealed that Metadichol® inhibits SARS-CoV-2 replication at an EC90 of 0.16 µg/mL. Moreover, Metadichol® had an EC90 of 0.00037 µM, making it 2081 and 3371 times more potent than remdesivir (EC50 = 0.77 µM) and chloroquine (EC50 = 1.14 µM), respectively.


Subject(s)
Fatty Alcohols/pharmacology , SARS-CoV-2/drug effects , Viruses/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cell Line , Chlorocebus aethiops , Esters/pharmacology , Guanidines/pharmacology , Humans , Lipid Metabolism/physiology , Lipids/chemistry , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/drug effects , Serine Endopeptidases/metabolism , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Attachment/drug effects , Virus Internalization/drug effects
5.
J Med Chem ; 65(4): 2971-2987, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1616927

ABSTRACT

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the most common complications in COVID-19. Elastase has been recognized as an important target to prevent ALI/ARDS in the patient of COVID-19. Cyclotheonellazole A (CTL-A) is a natural macrocyclic peptide reported to be a potent elastase inhibitor. Herein, we completed the first total synthesis of CTL-A in 24 linear steps. The key reactions include three-component MAC reactions and two late-stage oxidations. We also provided seven CTL-A analogues and elucidated preliminary structure-activity relationships. The in vivo ALI mouse model further suggested that CTL-A alleviated acute lung injury with reductions in lung edema and pathological deterioration, which is better than sivelestat, one approved elastase inhibitor. The activity of CTL-A against elastase, along with its cellular safety and well-established synthetic route, warrants further investigation of CTL-A as a candidate against COVID-19 pathogeneses.


Subject(s)
Acute Lung Injury/drug therapy , Leukocyte Elastase/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Respiratory Distress Syndrome/drug therapy , Serine Proteinase Inhibitors/pharmacology , Acute Lung Injury/chemically induced , Acute Lung Injury/metabolism , Animals , Bleomycin , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/pathology , Cell Line , Disease Models, Animal , Humans , Leukocyte Elastase/metabolism , Male , Mice , Mice, Inbred C57BL , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Respiratory Distress Syndrome/chemically induced , Respiratory Distress Syndrome/metabolism , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry
6.
J Neuroinflammation ; 19(1): 8, 2022 Jan 06.
Article in English | MEDLINE | ID: covidwho-1613238

ABSTRACT

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.


Subject(s)
Benzamidines , Guanidines , Inflammation/drug therapy , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors , Toll-Like Receptor 7/immunology , Virus Diseases/drug therapy , Animals , Benzamidines/pharmacology , Benzamidines/therapeutic use , COVID-19/complications , COVID-19/drug therapy , Guanidines/pharmacology , Guanidines/therapeutic use , Illness Behavior/drug effects , Imidazoles/administration & dosage , Imidazoles/immunology , Inflammation/metabolism , Inflammation/virology , Male , Mice , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/therapeutic use , Toll-Like Receptor 7/agonists , Virus Diseases/metabolism , Virus Diseases/virology
7.
Nanoscale ; 13(45): 19218-19237, 2021 Nov 25.
Article in English | MEDLINE | ID: covidwho-1521871

ABSTRACT

The global dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously endangered human health. The number of confirmed cases is still increasing; however, treatment options are limited. Transmembrane protease serine 2 (TMPRSS2), as a key protease that primes the binding of SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2), has become an attractive target and received widespread attention. Thus, four potential drugs (bromhexine, camostat, gabexate, and nafamostat) were used to explore the mechanism of binding with TMPRSS2 in this work. A 65 ns molecular dynamics simulation was performed three times for each drug-TMPRSS2 system for reliable energy calculation and conformational analysis, of which the simulations of nafamostat-TMPRSS2 systems were further extended to 150 ns three times due to the discovery of two binding modes. Through the results of calculating binding free energy by nine methods, the binding affinity of camostat, gabexate, and nafamostat to TMPRSS2 showed great advantages compared with bromhexine, where the nafamostat was surprisingly found to present two reasonable binding conformations (forward and reverse directions). Two negatively charged amino acids (Asp435 and Glu299) can clamp the two positively charged groups (guanidinium group and amidinium group) in either forward or reverse fashion, and the forward one is more stable than the reverse. In addition, compared with gabexate, the dimethylamino group in camostat forms more van der Waals interactions with surrounding hot-spots His296 and Val280, resulting in a stronger affinity to TMPRSS2. For bromhexine, multiple binding sites are displayed in the binding pocket due to its small molecular structure, and van der Waals interactions play the dominant role in the binding process. In particular, six typical hot-spots were identified in the last three serine protease inhibitor systems, i.e., Asp435, Ser436, Gln438, Trp461, Ser463, and Gly464. The guanidinium groups of the drugs have powerful interactions with adjacent residues due to the formation of more hydrogen bonds, suggesting that this may be the critical site for drug design against TMPRSS2. This work provides valuable molecular insight into these four drug-TMPRSS2 binding mechanisms and is helpful for designing and screening drugs targeting TMPRSS2.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 , Drug Design , Serine Proteinase Inhibitors/pharmacology , COVID-19/prevention & control , Humans , Molecular Dynamics Simulation , SARS-CoV-2 , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus
9.
J Virol ; 95(19): e0086121, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1486519

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.


Subject(s)
Antiviral Agents/pharmacology , Esters/pharmacology , Guanidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/chemistry , Serine Endopeptidases/pharmacology , Serine Proteases/pharmacology , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/prevention & control , Carcinoma, Squamous Cell , Esters/chemistry , Esters/metabolism , Guanidines/chemistry , Guanidines/metabolism , Humans , Molecular Dynamics Simulation , Mouth Neoplasms , Protein Domains , Sequence Alignment , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteases/chemistry , Serine Proteases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Virus Internalization/drug effects
10.
mBio ; 12(4): e0097021, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1338834

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis. IMPORTANCE The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.


Subject(s)
Benzamidines/pharmacology , Esters/pharmacology , Guanidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/drug therapy , Cells, Cultured , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/drug effects , Respiratory Mucosa/pathology , Respiratory Mucosa/virology , Spike Glycoprotein, Coronavirus/metabolism
11.
Pharmacol Res ; 157: 104859, 2020 07.
Article in English | MEDLINE | ID: covidwho-1318929

ABSTRACT

Outbreak and pandemic of coronavirus SARS-CoV-2 in 2019/2020 will challenge global health for the future. Because a vaccine against the virus will not be available in the near future, we herein try to offer a pharmacological strategy to combat the virus. There exists a number of candidate drugs that may inhibit infection with and replication of SARS-CoV-2. Such drugs comprise inhibitors of TMPRSS2 serine protease and inhibitors of angiotensin-converting enzyme 2 (ACE2). Blockade of ACE2, the host cell receptor for the S protein of SARS-CoV-2 and inhibition of TMPRSS2, which is required for S protein priming may prevent cell entry of SARS-CoV-2. Further, chloroquine and hydroxychloroquine, and off-label antiviral drugs, such as the nucleotide analogue remdesivir, HIV protease inhibitors lopinavir and ritonavir, broad-spectrum antiviral drugs arbidol and favipiravir as well as antiviral phytochemicals available to date may limit spread of SARS-CoV-2 and morbidity and mortality of COVID-19 pandemic.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Peptidyl-Dipeptidase A/drug effects , Pneumonia, Viral/drug therapy , Serine Endopeptidases/drug effects , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19 , Coronavirus Infections/epidemiology , Coronavirus Infections/mortality , Humans , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/mortality , SARS-CoV-2 , Serine Proteinase Inhibitors/pharmacology
12.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1285140

ABSTRACT

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS coronavirus 2 (SARS-CoV-2) viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, avoralstat, PCI-27483, antipain, and soybean trypsin inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, avoralstat significantly reduced lung tissue titers and mitigated weight loss when administered prophylactically to mice susceptible to SARS-CoV-2, indicating its potential to be repositioned for coronavirus disease 2019 (COVID-19) prophylaxis in humans.


Subject(s)
COVID-19 , Phylogeny , SARS-CoV-2/physiology , Serine Endopeptidases , Serine Proteinase Inhibitors , Virus Internalization/drug effects , Virus Replication/drug effects , Animals , COVID-19/enzymology , COVID-19/genetics , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Male , Mice , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Vero Cells
13.
Mol Biol Rep ; 48(5): 4667-4675, 2021 May.
Article in English | MEDLINE | ID: covidwho-1237535

ABSTRACT

The transmembrane protease serine 2 (TMPRSS2) is a membrane anchored protease that primarily expressed by epithelial cells of respiratory and gastrointestinal systems and has been linked to multiple pathological processes in humans including tumor growth, metastasis and viral infections. Recent studies have shown that TMPRSS2 expressed on cell surface of host cells could play a crucial role in activation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein which facilitates the rapid early entry of the virus into host cells. In addition, direct suppression of TMPRSS2 using small drug inhibitors has been demonstrated to be effective in decreasing SARS-CoV-2 infection in vitro, which presents TMPRSS2 protease as a potential therapeutic strategy for SARS-CoV-2 infection. Recently, SARS-CoV-2 has been shown to be capable of infecting gastrointestinal enterocytes and to provoke gastrointestinal disorders in patients with COVID-19 disease, which is considered as a new transmission route and target organ of SARS-CoV-2. In this review, we highlight the biochemical properties of TMPRSS2 protease and discuss the potential targeting of TMPRSS2 by inhibitors to prevent the SARS-CoV-2 spreading through gastro-intestinal tract system as well as the hurdles that need to be overcome.


Subject(s)
COVID-19/metabolism , Enterocytes/drug effects , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Evaluation, Preclinical , Enterocytes/metabolism , Enterocytes/virology , Humans , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
14.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1180999

ABSTRACT

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS coronavirus 2 (SARS-CoV-2) viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, avoralstat, PCI-27483, antipain, and soybean trypsin inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, avoralstat significantly reduced lung tissue titers and mitigated weight loss when administered prophylactically to mice susceptible to SARS-CoV-2, indicating its potential to be repositioned for coronavirus disease 2019 (COVID-19) prophylaxis in humans.


Subject(s)
COVID-19 , Phylogeny , SARS-CoV-2/physiology , Serine Endopeptidases , Serine Proteinase Inhibitors , Virus Internalization/drug effects , Virus Replication/drug effects , Animals , COVID-19/enzymology , COVID-19/genetics , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Male , Mice , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Vero Cells
15.
Nat Commun ; 12(1): 1726, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1142436

ABSTRACT

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.


Subject(s)
COVID-19/drug therapy , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , alpha 1-Antitrypsin/pharmacology , Antibodies, Viral/blood , Antiviral Agents/pharmacology , COVID-19/blood , Caco-2 Cells , Humans , Immunoglobulin G/blood , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Virus Replication/drug effects
16.
Molecules ; 26(4)2021 Feb 10.
Article in English | MEDLINE | ID: covidwho-1110462

ABSTRACT

Currently, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected people among all countries and is a pandemic as declared by the World Health Organization (WHO). SARS-CoVID-2 main protease is one of the therapeutic drug targets that has been shown to reduce virus replication, and its high-resolution 3D structures in complex with inhibitors have been solved. Previously, we had demonstrated the potential of natural compounds such as serine protease inhibitors eventually leading us to hypothesize that FDA-approved marine drugs have the potential to inhibit the biological activity of SARS-CoV-2 main protease. Initially, field-template and structure-activity atlas models were constructed to understand and explain the molecular features responsible for SARS-CoVID-2 main protease inhibitors, which revealed that Eribulin Mesylate, Plitidepsin, and Trabectedin possess similar characteristics related to SARS-CoVID-2 main protease inhibitors. Later, protein-ligand interactions are studied using ensemble molecular-docking simulations that revealed that marine drugs bind at the active site of the main protease. The three-dimensional reference interaction site model (3D-RISM) studies show that marine drugs displace water molecules at the active site, and interactions observed are favorable. These computational studies eventually paved an interest in further in vitro studies. Finally, these findings are new and indeed provide insights into the role of FDA-approved marine drugs, which are already in clinical use for cancer treatment as a potential alternative to prevent and treat infected people with SARS-CoV-2.


Subject(s)
Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , SARS-CoV-2/physiology , Serine Proteinase Inhibitors/pharmacology , Catalytic Domain , Depsipeptides/chemistry , Depsipeptides/pharmacology , Drug Repositioning , Furans/chemistry , Furans/pharmacology , Humans , Ketones/chemistry , Ketones/pharmacology , Models, Molecular , Molecular Docking Simulation , Peptides, Cyclic , Quantitative Structure-Activity Relationship , SARS-CoV-2/drug effects , Serine Proteinase Inhibitors/chemistry , Trabectedin/chemistry , Trabectedin/pharmacology , Viral Proteins/antagonists & inhibitors , Virus Replication/drug effects
17.
Chem Biol Interact ; 338: 109428, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1103757

ABSTRACT

Camostat mesylate, a potent inhibitor of the human transmembrane protease, serine 2 (TMPRSS2), is currently under investigation for its effectiveness in COVID-19 patients. For its safe application, the risks of camostat mesylate to induce pharmacokinetic drug-drug interactions with co-administered drugs should be known. We therefore tested in vitro the potential inhibition of important efflux (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2)), and uptake transporters (organic anion transporting polypeptides OATP1B1, OATP1B3, OATP2B1) by camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA). Transporter inhibition was evaluated using fluorescent probe substrates in transporter over-expressing cell lines and compared to the respective parental cell lines. Moreover, possible mRNA induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) was analysed in LS180 cells by quantitative real-time PCR. The results of our study for the first time demonstrated that camostat mesylate and GBPA do not relevantly inhibit P-gp, BCRP, OATP1B1 or OATP1B3. Only OATP2B1 was profoundly inhibited by GBPA with an IC50 of 11 µM. Induction experiments in LS180 cells excluded induction of PXR-regulated genes such as cytochrome P450 3A4 (CYP3A4) and ABCB1 and AhR-regulated genes such as CYP1A1 and CYP1A2 by camostat mesylate and GBPA. Together with the summary of product characteristics of camostat mesylate indicating no inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 in vitro, our data suggest a low potential of camostat mesylate to act as a perpetrator in pharmacokinetic drug-drug interactions. Only inhibition of OATP2B1 by GBPA warrants further investigation.


Subject(s)
Drug Interactions , Esters/metabolism , Guanidines/metabolism , Serine Proteinase Inhibitors/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Cell Line , Cell Survival/drug effects , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Esters/chemistry , Esters/pharmacology , Guanidines/chemistry , Guanidines/pharmacology , Humans , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/metabolism , Pregnane X Receptor/genetics , Pregnane X Receptor/metabolism , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology
18.
J Chem Inf Model ; 61(2): 1020-1032, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-1065782

ABSTRACT

Currently the entire human population is in the midst of a global pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2). This highly pathogenic virus has to date caused >71 million infections and >1.6 million deaths in >180 countries. Several vaccines and drugs are being studied as possible treatments or prophylactics of this viral infection. M3CLpro (coronavirus main cysteine protease) is a promising drug target as it has a significant role in viral replication. Here we use the X-ray crystal structure of M3CLpro in complex with boceprevir to study the dynamic changes of the protease upon ligand binding. The binding free energy was calculated for water molecules at different locations of the binding site, and molecular dynamics (MD) simulations were carried out for the M3CLpro/boceprevir complex, to thoroughly understand the chemical environment of the binding site. Several HCV NS3/4a protease inhibitors were tested in vitro against M3CLpro. Specifically, asunaprevir, narlaprevir, paritaprevir, simeprevir, and telaprevir all showed inhibitory effects on M3CLpro. Molecular docking and MD simulations were then performed to investigate the effects of these ligands on M3CLpro and to provide insights into the chemical environment of the ligand binding site. Our findings and observations are offered to help guide the design of possible potent protease inhibitors and aid in coping with the COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteases/chemistry , SARS-CoV-2/drug effects , Serine Proteinase Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Computer Simulation , Crystallography, X-Ray , Cysteine Proteases/drug effects , Humans , In Vitro Techniques , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Conformation , SARS-CoV-2/enzymology , Serine Proteases
19.
Eur J Pharmacol ; 890: 173688, 2021 Jan 05.
Article in English | MEDLINE | ID: covidwho-1049783

ABSTRACT

The coronavirus disease-19 (COVID-19) outbreak that is caused by a highly contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a zoonotic pandemic, with approximately 24.5 million positive cases and 8.3 lakhs deaths globally. The lack of effective drugs or vaccine provoked the research for drug candidates that can disrupt the spread and progression of the virus. The identification of drug molecules through experimental studies is time-consuming and expensive, so there is a need for developing alternative strategies like in silico approaches which can yield better outcomes in less time. Herein, we selected transmembrane protease serine 2 (TMPRSS2) enzyme, a potential pharmacological target against SARS-CoV-2, involved in the spread and pathogenesis of the virus. Since 3D structure is not available for this protein, the present study aims at homology modelling and validation of TMPRSS2 using Swiss-model server. Validation of the modelled TMPRSS2 using various online tools confirmed model accuracy, topology and stereochemical plausibility. The catalytic triad consisting of Serine-441, Histidine-296 and Aspartic acid-345 was identified as active binding site of TMPRSS2 using existing ligands. Molecular docking studies of various drugs and phytochemicals against the modelled TMPRSS2 were performed using camostat as a standard drug. The results revealed eight potential drug candidates, namely nafamostat, meloxicam, ganodermanontriol, columbin, myricetin, proanthocyanidin A2, jatrorrhizine and baicalein, which were further studied for ADME/T properties. In conclusion, the study unravelled eight high affinity binding compounds, which may serve as potent antagonists against TMPRSS2 to impact COVID-19 drug therapy.


Subject(s)
Antiviral Agents/pharmacology , Models, Molecular , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Benzamidines , Berberine/analogs & derivatives , Berberine/pharmacology , Binding Sites , COVID-19/drug therapy , Diterpenes/pharmacology , Flavanones/pharmacology , Flavonoids/pharmacology , Guanidines/pharmacology , Lactones/pharmacology , Lanosterol/analogs & derivatives , Lanosterol/pharmacology , Meloxicam/pharmacology , Proanthocyanidins/pharmacology , Protein Binding , SARS-CoV-2
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