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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329513

ABSTRACT

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

2.
Virology ; 563: 98-106, 2021 11.
Article in English | MEDLINE | ID: covidwho-1386714

ABSTRACT

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.


Subject(s)
COVID-19/epidemiology , Disease Models, Animal , Obesity/epidemiology , Animals , Chlorocebus aethiops , Comorbidity , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Morbidity , Vero Cells
3.
Viruses ; 13(6)2021 06 03.
Article in English | MEDLINE | ID: covidwho-1259622

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.


Subject(s)
COVID-19/immunology , Chemokine CXCL10/genetics , Chemokine CXCL11/genetics , Chemokine CXCL9/genetics , Proto-Oncogene Proteins c-akt/metabolism , Up-Regulation , Angiotensin-Converting Enzyme 2/genetics , Animals , Cell Line , Chemokine CXCL10/immunology , Chemokine CXCL11/immunology , Chemokine CXCL9/immunology , Cytokine Release Syndrome/genetics , Cytokine Release Syndrome/immunology , Epithelial Cells/immunology , Epithelial Cells/virology , Female , Humans , Inflammation , Lung/cytology , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/genetics , Signal Transduction/immunology
4.
Pathog Immun ; 6(1): 55-74, 2021.
Article in English | MEDLINE | ID: covidwho-1222332

ABSTRACT

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 anti-trypsin (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.

5.
Comput Struct Biotechnol J ; 19: 2246-2255, 2021.
Article in English | MEDLINE | ID: covidwho-1184914

ABSTRACT

To date, the COVID-19 pandemic has claimed over 1 million human lives, infected another 50 million individuals and wreaked havoc on the global economy. The crisis has spurred the ongoing development of drugs targeting its etiological agent, the SARS-CoV-2. Targeting relevant protein-protein interaction interfaces (PPIIs) is a viable paradigm for the design of antiviral drugs and enriches the targetable chemical space by providing alternative targets for drug discovery. In this review, we will provide a comprehensive overview of the theory, methods and applications of PPII-targeted drug development towards COVID-19 based on recent literature. We will also highlight novel developments, such as the successful use of non-native protein-protein interactions as targets for antiviral drug screening. We hope that this review may serve as an entry point for those interested in applying PPIIs towards COVID-19 drug discovery and speed up drug development against the pandemic.

6.
Viruses ; 13(1)2020 12 30.
Article in English | MEDLINE | ID: covidwho-1004763

ABSTRACT

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells.


Subject(s)
COVID-19/virology , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/drug effects , Virion , Animals , Cell Line , HEK293 Cells , HIV-1/drug effects , Humans , Interferon-gamma , Virus Attachment/drug effects , Virus Internalization/drug effects
7.
PeerJ ; 8: e10639, 2020.
Article in English | MEDLINE | ID: covidwho-994191

ABSTRACT

In light of the COVID-19 pandemic, studies that work to understand SARS-CoV-2 are urgently needed. In turn, the less severe human coronaviruses such as HCoV-229E and OC43 are drawing newfound attention. These less severe coronaviruses can be used as a model to facilitate our understanding of the host immune response to coronavirus infection. SARS-CoV-2 must be handled under biosafety level 3 (BSL-3) conditions. Therefore, HCoV-229E and OC43, which can be handled at BSL-2 provide an alternative to SARS-CoV-2 for preclinical screening and designing of antivirals. However, to date, there is no published effective and efficient method to titrate HCoVs other than expensive indirect immunostaining. Here we present an improved approach using an agarose-based conventional plaque assay to titrate HCoV 229E and OC43 with mink lung epithelial cells, Mv1Lu. Our results indicate that titration of HCoV 229E and OC43 with Mv1Lu is consistent and reproducible. The titers produced are also comparable to those produced using human rhabdomyosarcoma (RD) cells. More importantly, Mv1Lu cells display a higher tolerance for cell-cell contact stress, decreased temperature sensitivity, and a faster growth rate. We believe that our improved low-cost plaque assay can serve as an easy tool for researchers conducting HCoV research.

8.
J Med Chem ; 63(6): 3131-3141, 2020 03 26.
Article in English | MEDLINE | ID: covidwho-2598

ABSTRACT

Structure-based stabilization of protein-protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.


Subject(s)
Alkaloids/pharmacology , Antiviral Agents/pharmacology , Indoles/pharmacology , Nucleocapsid Proteins/metabolism , Protein Multimerization/drug effects , Alkaloids/chemistry , Alkaloids/metabolism , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins , Crystallography, X-Ray , Drug Design , Hydrophobic and Hydrophilic Interactions , Indoles/chemistry , Indoles/metabolism , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/drug effects , Molecular Docking Simulation , Nucleocapsid Proteins/chemistry , Protein Binding , Protein Domains , Sequence Alignment , Vero Cells
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