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1.
Life Sci Alliance ; 5(10)2022 Oct.
Article in English | MEDLINE | ID: covidwho-1876430

ABSTRACT

By the end of December 2021, coronavirus disease 2019 (COVID-19) produced more than 271 million cases and 5.3 million deaths. Although vaccination is an effective strategy for pandemic control, it is not yet equally available in all countries. Therefore, identification of prognostic biomarkers remains crucial to manage COVID-19 patients. The aim of this study was to evaluate predictors of COVID-19 severity previously proposed. Clinical and demographic characteristics and 120 single-nucleotide polymorphisms were analyzed from 817 patients with COVID-19, who attended the emergency department of the Hospital Universitario de La Princesa during March and April 2020. The main outcome was a modified version of the 7-point World Health Organization (WHO) COVID-19 severity scale (WHOCS); both in the moment of the first hospital examination (WHOCS-1) and of the severest WHOCS score (WHOCS-2). The TMPRSS2 rs75603675 genotype (OR = 0.586), dyslipidemia (OR = 2.289), sex (OR = 0.586), and the Charlson Comorbidity Index (OR = 1.126) were identified as the main predictors of disease severity. Consequently, these variables might influence COVID-19 severity and could be used as predictors of disease development.


Subject(s)
COVID-19 , COVID-19/diagnosis , Comorbidity , Female , Humans , Male , Serine , Serine Endopeptidases/genetics , Severity of Illness Index , Sex Factors
2.
ACS Infect Dis ; 8(6): 1191-1203, 2022 Jun 10.
Article in English | MEDLINE | ID: covidwho-1873405

ABSTRACT

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allows for rapid movement of the existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites by TMPRSS2. Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.


Subject(s)
COVID-19 , Percutaneous Coronary Intervention , Angiotensin-Converting Enzyme 2 , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Humans , Pandemics , SARS-CoV-2 , Serine Endopeptidases
3.
Front Immunol ; 13: 872047, 2022.
Article in English | MEDLINE | ID: covidwho-1855361

ABSTRACT

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Serine Endopeptidases , Animals , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines , Furin/genetics , Furin/metabolism , Humans , Immunity, Cellular , SARS-CoV-2/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/immunology , Spike Glycoprotein, Coronavirus/immunology
4.
BMC Bioinformatics ; 23(1): 180, 2022 May 16.
Article in English | MEDLINE | ID: covidwho-1846793

ABSTRACT

BACKGROUND: The Transmembrane Serine Protease 2 (TMPRSS2) of human cell plays a significant role in proteolytic cleavage of SARS-Cov-2 coronavirus spike protein and subsequent priming to the receptor ACE2. Approaching TMPRSS2 as a therapeutic target for the inhibition of SARS-Cov-2 infection is highly promising. Hence, in the present study, we docked the binding efficacy of ten naturally available phyto compounds with known anti-viral potential with TMPRSS2. The aim is to identify the best phyto compound with a high functional affinity towards the active site of the TMPRSS2 with the aid of two different docking software. Molecular Dynamic Simulations were performed to analyse the conformational space of the binding pocket of the target protein with selected molecules. RESULTS: Docking analysis using PyRx version 0.8 along with AutoDockVina reveals that among the screened phyto compounds, Genistein shows the maximum binding affinity towards the hydrophobic substrate-binding site of TMPRSS2 with three hydrogen bonds interaction ( - 7.5 kcal/mol). On the other hand, molecular docking analysis using Schrodinger identified Quercetin as the most potent phyto compound with a maximum binding affinity towards the hydrophilic catalytic site of TMPRSS2 ( - 7.847 kcal/mol) with three hydrogen bonds interaction. The molecular dynamics simulation reveals that the Quercetin-TMPRSS complex is stable until 50 ns and forms stable interaction with the protein ( - 22.37 kcal/mol of MM-PBSA binding free energy). Genistein creates a weak interaction with the loop residues and hence has an unstable binding and exits from the binding pocket. CONCLUSION: The compounds, Quercetin and Genistein, can inhibit the TMPRSS2 guided priming of the spike protein. The compounds could reduce the interaction of the host cell with the type I transmembrane glycoprotein to prevent the entry of the virus. The critical finding is that compared to Genistein, Quercetin exhibits higher binding affinity with the catalytic unit of TMPRSS2 and forms a stable complex with the target. Thus, enhancing our innate immunity by consuming foods rich in Quercetin and Genistein or developing a novel drug in the combination of Quercetin and Genistein could be the brilliant choices to prevent SARS-Cov-2 infection when we consider the present chaos associated with vaccines and anti-viral medicines.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antiviral Agents/pharmacology , COVID-19/drug therapy , Genistein/pharmacology , Humans , Molecular Docking Simulation , Quercetin/pharmacology , SARS-CoV-2 , Serine Endopeptidases , Virus Internalization
5.
BJOG ; 129(2): 256-266, 2022 01.
Article in English | MEDLINE | ID: covidwho-1831884

ABSTRACT

BACKGROUND: Pregnant women have been identified as a potentially at-risk group concerning COVID-19 infection, but little is known regarding the susceptibility of the fetus to infection. Co-expression of ACE2 and TMPRSS2 has been identified as a prerequisite for infection, and expression across different tissues is known to vary between children and adults. However, the expression of these proteins in the fetus is unknown. METHODS: We performed a retrospective analysis of a single cell data repository. The data were then validated at both gene and protein level by performing RT-qPCR and two-colour immunohistochemistry on a library of second-trimester human fetal tissues. FINDINGS: TMPRSS2 is present at both gene and protein level in the predominantly epithelial fetal tissues analysed. ACE2 is present at significant levels only in the fetal intestine and kidney, and is not expressed in the fetal lung. The placenta also does not co-express the two proteins across the second trimester or at term. INTERPRETATION: This dataset indicates that the lungs are unlikely to be a viable route of SARS-CoV2 fetal infection. The fetal kidney, despite presenting both the proteins required for the infection, is anatomically protected from the exposure to the virus. However, the gastrointestinal tract is likely to be susceptible to infection due to its high co-expression of both proteins, as well as its exposure to potentially infected amniotic fluid. TWEETABLE ABSTRACT: This work provides detailed mechanistic insight into the relative protection & vulnerabilities of the fetus & placenta to SARS-CoV-2 infection by scRNAseq & protein expression analysis for ACE2 & TMPRSS2. The findings help to explain the low rate of vertical transmission.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19 , Gene Expression Profiling , Placenta/metabolism , Serine Endopeptidases/genetics , Adult , COVID-19/epidemiology , COVID-19/genetics , COVID-19/transmission , Databases, Nucleic Acid , Disease Susceptibility/metabolism , Female , Fetal Research , Gene Expression Profiling/methods , Gene Expression Profiling/statistics & numerical data , Genetic Testing/methods , Gestational Age , Humans , Immunohistochemistry , Infectious Disease Transmission, Vertical , Pregnancy , Protective Factors , Ribonucleoproteins, Small Cytoplasmic/analysis , SARS-CoV-2/physiology
6.
J Oral Biosci ; 64(2): 229-236, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1804593

ABSTRACT

OBJECTIVES: The oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2. METHODS: To investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis. RESULTS: GEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes. CONCLUSIONS: Periodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes.


Subject(s)
COVID-19 , Periodontitis , Angiotensin-Converting Enzyme 2 , Animals , COVID-19/genetics , Gingiva , Humans , Mice , Peptide Hydrolases , SARS-CoV-2 , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus
7.
Cells ; 11(8)2022 04 08.
Article in English | MEDLINE | ID: covidwho-1785538

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.


Subject(s)
COVID-19 , Epithelial Cells , Histone-Lysine N-Methyltransferase , Serine Endopeptidases , Antiviral Agents/pharmacology , COVID-19/pathology , Epithelial Cells/metabolism , Epithelial Cells/virology , Histone-Lysine N-Methyltransferase/genetics , Humans , SARS-CoV-2/drug effects , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
8.
J Virol ; 96(8): e0012822, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1765079

ABSTRACT

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Serine Endopeptidases , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/pathology , COVID-19/virology , Furin/metabolism , HeLa Cells , Humans , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
9.
Sci Rep ; 12(1): 5207, 2022 03 25.
Article in English | MEDLINE | ID: covidwho-1764204

ABSTRACT

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin-TMPRSS2-AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2-AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.


Subject(s)
COVID-19 , Enoxaparin , COVID-19/drug therapy , Enoxaparin/pharmacology , HEK293 Cells , Humans , SARS-CoV-2 , Serine Endopeptidases
10.
Nature ; 605(7909): 340-348, 2022 05.
Article in English | MEDLINE | ID: covidwho-1764188

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.


Subject(s)
COVID-19 , SARS-CoV-2 , Serine Proteinase Inhibitors , Animals , COVID-19/prevention & control , COVID-19/virology , Disease Models, Animal , Humans , Mice , Mice, Transgenic , SARS-CoV-2/drug effects , Serine Endopeptidases , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/therapeutic use , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
11.
Nature ; 603(7902): 706-714, 2022 03.
Article in English | MEDLINE | ID: covidwho-1764186

ABSTRACT

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Subject(s)
COVID-19/pathology , COVID-19/virology , Membrane Fusion , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19 Vaccines/immunology , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Convalescence , Female , Humans , Immune Sera/immunology , Intestines/pathology , Intestines/virology , Lung/pathology , Lung/virology , Male , Middle Aged , Mutation , Nasal Mucosa/pathology , Nasal Mucosa/virology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Tissue Culture Techniques , Virulence , Virus Replication
12.
Curr Drug Targets ; 23(3): 240-259, 2022.
Article in English | MEDLINE | ID: covidwho-1760079

ABSTRACT

Since December 2019, the new Coronavirus disease (COVID-19) caused by the etiological agent SARS-CoV-2 has been responsible for several cases worldwide, becoming pandemic in March 2020. Pharmaceutical companies and academics have joined their efforts to discover new therapies to control the disease since there are no specific drugs to combat this emerging virus. Thus, several tar-gets have been explored; among them, the transmembrane protease serine 2 (TMPRSS2) has gained greater interest in the scientific community. In this context, this review will describe the importance of TMPRSS2 protease and the significant advances in virtual screening focused on discovering new inhibitors. In this review, it was observed that molecular modeling methods could be powerful tools in identifying new molecules against SARS-CoV-2. Thus, this review could be used to guide re-searchers worldwide to explore the biological and clinical potential of compounds that could be promising drug candidates against SARS-CoV-2, acting by inhibition of TMPRSS2 protein.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , Drug Delivery Systems , Humans , Models, Molecular , Pandemics , Serine Endopeptidases
13.
Nat Commun ; 13(1): 1444, 2022 03 17.
Article in English | MEDLINE | ID: covidwho-1751716

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to have devastating consequences worldwide. Recently, great efforts have been made to identify SARS-CoV-2 host factors, but the regulatory mechanisms of these host molecules, as well as the virus per se, remain elusive. Here we report a role of RNA G-quadruplex (RG4) in SARS-CoV-2 infection. Combining bioinformatics, biochemical and biophysical assays, we demonstrate the presence of RG4s in both SARS-CoV-2 genome and host factors. The biological and pathological importance of these RG4s is then exemplified by a canonical 3-quartet RG4 within Tmprss2, which can inhibit Tmprss2 translation and prevent SARS-CoV-2 entry. Intriguingly, G-quadruplex (G4)-specific stabilizers attenuate SARS-CoV-2 infection in pseudovirus cell systems and mouse models. Consistently, the protein level of TMPRSS2 is increased in lungs of COVID-19 patients. Our findings reveal a previously unknown mechanism underlying SARS-CoV-2 infection and suggest RG4 as a potential target for COVID-19 prevention and treatment.


Subject(s)
COVID-19 , Virus Internalization , Animals , Humans , Mice , RNA , SARS-CoV-2 , Serine Endopeptidases/genetics
14.
Int J Mol Sci ; 23(6)2022 Mar 16.
Article in English | MEDLINE | ID: covidwho-1742493

ABSTRACT

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.


Subject(s)
Bone Neoplasms/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/genetics , Organoids/metabolism , Prostatic Neoplasms, Castration-Resistant/genetics , Androgens/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Benzamides/pharmacology , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice , Nitriles/pharmacology , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transplantation, Heterologous , Virus Internalization
15.
Curr Med Chem ; 29(4): 682-699, 2022.
Article in English | MEDLINE | ID: covidwho-1742083

ABSTRACT

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 a 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.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Humans , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/pharmacology , Virus Internalization
16.
J Nippon Med Sch ; 89(1): 95-101, 2022.
Article in English | MEDLINE | ID: covidwho-1736473

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly pathogenic coronavirus that has spread rapidly worldwide and caused an international public health emergency. Patients with hematological cancers are regarded as a high-risk group for coronavirus disease 2019 (COVID-19). However, few reports have investigated factors that might account for the differential severity of COVID-19 disease in these patients. METHODS: Gene expression of SARS-CoV-2 entry-promoting factors and entry-restricting factors and the associated effects on myeloid malignancies were evaluated. Gene expression levels of 11 SARS-CoV-2 entry-promoting factors and 4 SARS-CoV-2 entry-restricting factors were analyzed in patients with myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), and acute myeloid leukemia and its subtypes. RESULTS: Expression levels of promoting and restricting factors were most affected in MDS. Specifically, 4 of the 11 analyzed SARS-CoV-2 entry-promoting factors were significantly increased (TMPRSS4, CD209, CLEC4G, and CTSB), and 2 of the 4 analyzed SARS-CoV-2 entry-restricting factors were significantly decreased (IFITM1 and IFITM2) in MDS. Patients with CML also exhibited a pattern of significant changes in SARS-CoV-2 entry-promoting and entry-restricting factors. Five of the 11 analyzed SARS-CoV-2 entry-promoting factors were significantly increased (ACE2, TMPRSS2, TMPRSS4, ANPEP, CD209), and 1 of the 4 analyzed SARS-CoV-2 entry-restricting factors was significantly decreased (LY6E) in CML. CONCLUSIONS: The present and past results highlight the importance of investigating SARS-CoV-2 entry-promoting factors and entry-restricting factors, because of their crucial role in determining the differential severity of COVID-19 disease.


Subject(s)
COVID-19 , Neoplasms , Angiotensin-Converting Enzyme 2 , Cell Line , Humans , Membrane Proteins , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Serine Endopeptidases/genetics
17.
J Virol ; 96(5): e0218621, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1736028

ABSTRACT

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.


Subject(s)
COVID-19/metabolism , Furin/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adaptation, Physiological , Animals , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Furin/genetics , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mutation , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication
18.
Clin Chim Acta ; 531: 4-11, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1734231

ABSTRACT

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.


Subject(s)
COVID-19 , Shock, Septic , Carboxypeptidases , Dipeptidyl Peptidase 4 , Endopeptidases , Gelatinases , Humans , Membrane Proteins , Peptide Hydrolases , Proline , Serine Endopeptidases
19.
J Med Virol ; 93(9): 5487-5504, 2021 09.
Article in English | MEDLINE | ID: covidwho-1733919

ABSTRACT

Along with the control and prevention of coronavirus disease 2019 transmission, infected animals might have potential to carry the virus to spark new outbreaks. However, very few studies explore the susceptibility of animals to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral attachment as a crucial step for cross-species infection requires angiotensin-converting enzyme 2 (ACE2) as a receptor and depends on TMPRSS2 protease activity. Here, we searched the genomes of metazoans from different classes using an extensive BLASTP survey and found ACE2 and TMPRSS2 occur in vertebrates, but some vertebrates lack Tmprss2. We identified 6 amino acids among 25 known human ACE2 residues are highly associated with the binding of ACE2 to SARS-CoV-2 (p value < .01) by Fisher exact test, and following this, calculated the probability of viral attachment within each species by the randomForest function from R randomForest library. Furthermore, we observed that Ace2 selected from seven animals based on the above analysis lack the hydrophobic contacts identified on human ACE2, indicating less affinity of SARS-CoV-2 to Ace2 in animals than humans. Finally, the alignment of 3D structure between human ACE2 and other animals by I-TASSER and TM-align displayed a reasonable structure for viral attachment within these species. Taken together, our data may shed light on the human-to-animal transmission of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Vertebrates/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , COVID-19/metabolism , Disease Susceptibility , Genetic Predisposition to Disease , Humans , Receptors, Virus/metabolism , SARS-CoV-2/classification , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vertebrates/genetics , Virus Attachment , Virus Internalization , Virus Release
20.
Front Immunol ; 13: 811430, 2022.
Article in English | MEDLINE | ID: covidwho-1731772

ABSTRACT

Despite significant research efforts, treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain limited. This is due in part to a lack of therapeutics that increase host defense to the virus. Replication of SARS-CoV-2 in lung tissue is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses that amplify tissue injury. Antagonists of the androgen (AR) and glucocorticoid (GR) receptors have shown efficacy in models of COVID-19 and in clinical studies because the cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2), are transcriptionally regulated by these receptors. We postulated that the GR and AR modulator, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID-19 by down-regulating expression of critical genes regulated through these receptors. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were examined at 3, 5 and 7 days post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the progression of SARS-CoV-2 infection. Results indicated that oral administration of PT150 caused a dose-dependent decrease in replication of SARS-CoV-2 in lung, as well as in expression of ACE2 and TMPRSS2. Lung hypercellularity and infiltration of macrophages and CD4+ T-cells were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular docking studies suggest that PT150 binds to the co-activator interface of the ligand-binding domain of both AR and GR, thereby acting as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR revealed a high degree of sequence identity maintained across multiple species, including humans, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID-19 across variants of SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Glucocorticoids/metabolism , Immunity, Innate/drug effects , Inflammation/drug therapy , Receptors, Androgen/metabolism , Virus Internalization/drug effects , Animals , COVID-19/metabolism , Disease Models, Animal , Female , Inflammation/metabolism , Inflammation/virology , Lung/virology , Male , Mesocricetus , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Load/drug effects
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