36 results on '"Bojkova D"'
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2. COVID-19-related coagulopathy - is transferrin a missing link?
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McLaughlin, K.-M., Bechtel, M., Bojkova, D., Münch, C., Ciesek, S., Wass, M.N., Michaelis, M., Cinatl, J., and Publica
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ddc:610 - Abstract
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term ""blood coagulation"" in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term ""blood coagulation"") was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
- Published
- 2020
3. Correction: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.
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Armstrong LA, Lange SM, Cesare V, Matthews SP, Nirujogi RS, Cole I, Hope A, Cunningham F, Toth R, Mukherjee R, Bojkova D, Gruber F, Gray D, Wyatt PG, Cinatl J, Dikic I, Davies P, and Kulathu Y
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0253364.]., (Copyright: © 2024 Armstrong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2024
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4. Discovery of anti-SARS-CoV-2 secondary metabolites from the heartwood of Pterocarpus santalinus using multi-informative molecular networking.
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Wasilewicz A, Zwirchmayr J, Kirchweger B, Bojkova D, Cinatl J Jr, Rabenau HF, Rollinger JM, Beniddir MA, and Grienke U
- Abstract
A pigment-depleted extract from the heartwood of Pterocarpus santalinus L. f. (PS-DE) showed promising anti-SARS-CoV-2 activity with an IC
50 of 29.9 μg/mL in Caco-2-F03 cells. To determine the potential active constituents within the extract prior to isolation, multi-informative molecular network (MN) was applied. Therefore, the extract was separated by high-performance counter-current chromatography (HPCCC) into 11 fractions which were subsequently tested for anti-SARS-CoV-2 activity and analysed by UPLC-tandem mass spectrometry (MS2 ). The resulting MN combines the bioactivity data of the fractions with the MS2 data. The MN analysis led to the targeted isolation of seven compounds including one pterocarpan (7) reported for the first time as constituent of P. santalinus and four so far undescribed natural products (NPs) that belong to the compound classes of arylpropanes (9) , isoflavanones (10) coumestans (16) and 3-arylcoumarins (17) , respectively. In total, 15 constituents from the heartwood of P. santalinus and one synthetic isoflavonoid that is structurally related to the natural metabolites were tested for anti-SARS-CoV-2 activity. Thereby, the two pterocarpans (-)-homopterocarpin (5) and (-)-medicarpin (2) , the stilbene (E) -pterostilbene (1) and the isoflavonoid 7-O-methylgenistein (11) showed a distinct antiviral activity with IC50 values of 17.2, 33.4, 34.7, and 37.9 µM, respectively, and no cytotoxic effects against Caco-2-F03 cells (CC50 > 100 µM). In addition, a structure-activity relationship (SAR) was proposed indicating structural requirements of pterocarpans for anti-SARS-CoV-2 activity. The herein presented results support the implementation of multi-informative molecular networks as powerful tool for dereplication and targeted isolation of bioactive NPs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wasilewicz, Zwirchmayr, Kirchweger, Bojkova, Cinatl, Rabenau, Rollinger, Beniddir and Grienke.)- Published
- 2023
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5. Silver N-heterocyclic carbene complexes are potent uncompetitive inhibitors of the papain-like protease with antiviral activity against SARS-CoV-2.
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Gil-Moles M, O'Beirne C, Esarev IV, Lippmann P, Tacke M, Cinatl J Jr, Bojkova D, and Ott I
- Abstract
The ongoing SARS-CoV-2 pandemic has caused a high demand for novel innovative antiviral drug candidates. Despite promising results, metal complexes have been relatively unexplored as antiviral agents in general and in particular against SARS-CoV-2. Here we report on silver NHC complexes with chloride or iodide counter ligands that are potent inhibitors of the SARS-CoV-2 papain-like protease (PL
pro ) but inactive against 3C-like protease (3CLpro ) as another SARS-CoV-2 protease. Mechanistic studies on a selected complex confirmed zinc removal from a zinc binding domain of PLpro as relevant factor of their activity. In addition, enzyme kinetic experiments revealed that the complex is an uncompetitive inhibitor and with this rare type of inhibition it offers great pharmacological advantages in terms selectivity. The silver NHC complexes with iodide ligands showed very low or absent host cell toxicity and triggered strong effects on viral replication in cells infected with SARS-CoV-2, making them promising future antiviral drug candidates., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)- Published
- 2023
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6. SARS-CoV-2 Variants Show Different Host Cell Proteome Profiles With Delayed Immune Response Activation in Omicron-Infected Cells.
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Metzler M, Tharyan RG, Klann K, Grikscheit K, Bojkova D, Cinatl J, Tascher G, Ciesek S, and Münch C
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- Humans, Proteome, Pandemics, Antiviral Agents, Antibodies, Neutralizing, SARS-CoV-2, COVID-19
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The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remain elusive. Leveraging whole-cell proteomics, we determined host signaling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signaling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signaling pathways causative for the differential pathogenicity of SARS-CoV-2 variants., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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7. Identification of Z-Tyr-Ala-CHN 2 , a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.
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Doijen J, Temmerman K, Van den Eynde C, Diels A, Van den Broeck N, Van Gool M, Heo I, Jaensch S, Zwaagstra M, Diosa Toro M, Chiu W, De Jonghe S, Leyssen P, Bojkova D, Ciesek S, Cinatl J, Verschueren L, Buyck C, Van Kuppeveld F, Neyts J, Van Loock M, and Van Damme E
- Abstract
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN
2 , which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.- Published
- 2023
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8. A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.
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Carius P, Jungmann A, Bechtel M, Grißmer A, Boese A, Gasparoni G, Salhab A, Seipelt R, Urbschat K, Richter C, Meier C, Bojkova D, Cinatl J, Walter J, Schneider-Daum N, and Lehr CM
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- Animals, Humans, SARS-CoV-2, Cell Line, Permeability, Alveolar Epithelial Cells, COVID-19 metabolism
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In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm
2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)- Published
- 2023
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9. Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform.
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Bojkova D, Reus P, Panosch L, Bechtel M, Rothenburger T, Kandler JD, Pfeiffer A, Wagner JUG, Shumliakivska M, Dimmeler S, Olmer R, Martin U, Vondran FWR, Toptan T, Rothweiler F, Zehner R, Rabenau HF, Osman KL, Pullan ST, Carroll MW, Stack R, Ciesek S, Wass MN, Michaelis M, and Cinatl J Jr
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Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)
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- 2023
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10. USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING.
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Karlowitz R, Stanifer ML, Roedig J, Andrieux G, Bojkova D, Bechtel M, Smith S, Kowald L, Schubert R, Boerries M, Cinatl J Jr, Boulant S, and van Wijk SJL
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- Antiviral Agents pharmacology, Humans, Interferons metabolism, Pandemics, SARS-CoV-2, Interferon Lambda, COVID-19, Interferon Type I genetics, Membrane Proteins metabolism, Ubiquitin Thiolesterase metabolism
- Abstract
Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFNs (IFN-λ) exhibit cell-type specific and long-lasting functions in auto-inflammation, tumorigenesis, and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2'3'-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication, and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense., (© 2022. The Author(s).)
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- 2022
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11. SARS-CoV-2 Omicron variant virus isolates are highly sensitive to interferon treatment.
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Bojkova D, Rothenburger T, Ciesek S, Wass MN, Michaelis M, and Cinatl J Jr
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- 2022
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12. SARS-CoV-2 screening strategies for returning international travellers: Evaluation of a rapid antigen test approach.
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Layer E, Hoehl S, Widera M, Bojkova D, Westphal T, Gottschalk R, Boeddinghaus B, Schork J, Ciesek S, and Goetsch U
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- Humans, Quarantine, Sensitivity and Specificity, Travel, COVID-19 diagnosis, SARS-CoV-2
- Abstract
Background: International travel poses the risk of importing SARS-CoV-2 infections and introducing new viral variants into the country of destination. Established measures include mandatory quarantine with the opportunity to abbreviate it with a negative rapid antigen test (RAT)., Methods: A total of 1,488 returnees were tested for SARS-CoV-2 with both PCR and RAT no earlier than 5 days after arrival. We assessed the sensitivity and specificity of the RAT. Positive samples were evaluated for infectivity in vitro in a cell culture outgrowth assay. We tracked if participants who tested negative were reported positive within 2 weeks of the initial test., Results: Potential infectiousness was determined based on symptom onset analysis, resulting in a sensitivity of the antigen test of 89% in terms of infectivity. The specificity was 100%. All positive outgrowth assays were preceded by a positive RAT, indicating that all participants with proven in vitro infectivity were correctly identified. None of the negative participants tested positive during the follow-up., Conclusions: RAT no earlier than the 5th day after arrival was a reliable method for detecting infectious travellers and can be recommended as an appropriate method for managing SARS-CoV-2 travel restrictions. Compliance to the regulations and a high standard of test quality must be ensured., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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13. Reduced interferon antagonism but similar drug sensitivity in Omicron variant compared to Delta variant of SARS-CoV-2 isolates.
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Bojkova D, Widera M, Ciesek S, Wass MN, Michaelis M, and Cinatl J Jr
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- Antiviral Agents pharmacology, Antiviral Agents therapeutic use, Humans, Interferons, SARS-CoV-2, COVID-19 Drug Treatment
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- 2022
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14. Ibuprofen, Flurbiprofen, Etoricoxib or Paracetamol Do Not Influence ACE2 Expression and Activity In Vitro or in Mice and Do Not Exacerbate In-Vitro SARS-CoV-2 Infection.
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de Bruin N, Schneider AK, Reus P, Talmon S, Ciesek S, Bojkova D, Cinatl J, Lodhi I, Charlesworth B, Sinclair S, Pennick G, Laughey WF, Gribbon P, Kannt A, and Schiffmann S
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- Acetaminophen pharmacology, Animals, COVID-19 metabolism, COVID-19 pathology, Caco-2 Cells, Disease Progression, Enzyme Activation drug effects, Etoricoxib pharmacology, Flurbiprofen pharmacology, Gene Expression Regulation, Enzymologic drug effects, Humans, Ibuprofen pharmacology, Male, Mice, Mice, Inbred C57BL, SARS-CoV-2 drug effects, SARS-CoV-2 physiology, Virus Internalization drug effects, Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, Anti-Inflammatory Agents, Non-Steroidal pharmacology, COVID-19 genetics
- Abstract
SARS-CoV-2 uses the human cell surface protein angiotensin converting enzyme 2 (ACE2) as the receptor by which it gains access into lung and other tissue. Early in the pandemic, there was speculation that a number of commonly used medications-including ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs)-have the potential to upregulate ACE2, thereby possibly facilitating viral entry and increasing the severity of COVID-19. We investigated the influence of the NSAIDS with a range of cyclooxygenase (COX)1 and COX2 selectivity (ibuprofen, flurbiprofen, etoricoxib) and paracetamol on the level of ACE2 mRNA/protein expression and activity as well as their influence on SARS-CoV-2 infection levels in a Caco-2 cell model. We also analysed the ACE2 mRNA/protein levels and activity in lung, heart and aorta in ibuprofen treated mice. The drugs had no effect on ACE2 mRNA/protein expression and activity in the Caco-2 cell model. There was no up-regulation of ACE2 mRNA/protein expression and activity in lung, heart and aorta tissue in ibuprofen-treated mice in comparison to untreated mice. Viral load was significantly reduced by both flurbiprofen and ibuprofen at high concentrations. Ibuprofen, flurbiprofen, etoricoxib and paracetamol demonstrated no effects on ACE2 expression or activity in vitro or in vivo. Higher concentrations of ibuprofen and flurbiprofen reduced SARS-CoV-2 replication in vitro.
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- 2022
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15. Angiotensin II receptor blocker intake associates with reduced markers of inflammatory activation and decreased mortality in patients with cardiovascular comorbidities and COVID-19 disease.
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Cremer S, Pilgram L, Berkowitsch A, Stecher M, Rieg S, Shumliakivska M, Bojkova D, Wagner JUG, Aslan GS, Spinner C, Luxán G, Hanses F, Dolff S, Piepel C, Ruppert C, Guenther A, Rüthrich MM, Vehreschild JJ, Wille K, Haselberger M, Heuzeroth H, Hansen A, Eschenhagen T, Cinatl J, Ciesek S, Dimmeler S, Borgmann S, and Zeiher A
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- Adult, Aged, Aged, 80 and over, Angiotensin-Converting Enzyme Inhibitors administration & dosage, Biomarkers blood, Comorbidity, Disease-Free Survival, Female, Humans, Inflammation blood, Inflammation drug therapy, Inflammation mortality, Male, Middle Aged, Severity of Illness Index, Survival Rate, Angiotensin Receptor Antagonists administration & dosage, COVID-19 blood, COVID-19 mortality, Hypertension blood, Hypertension drug therapy, Hypertension mortality, Registries, SARS-CoV-2 metabolism, COVID-19 Drug Treatment
- Abstract
Aims: Patients with cardiovascular comorbidities have a significantly increased risk for a critical course of COVID-19. As the SARS-CoV2 virus enters cells via the angiotensin-converting enzyme receptor II (ACE2), drugs which interact with the renin angiotensin aldosterone system (RAAS) were suspected to influence disease severity., Methods and Results: We analyzed 1946 consecutive patients with cardiovascular comorbidities or hypertension enrolled in one of the largest European COVID-19 registries, the Lean European Open Survey on SARS-CoV-2 (LEOSS) registry. Here, we show that angiotensin II receptor blocker intake is associated with decreased mortality in patients with COVID-19 [OR 0.75 (95% CI 0,59-0.96; p = 0.013)]. This effect was mainly driven by patients, who presented in an early phase of COVID-19 at baseline [OR 0,64 (95% CI 0,43-0,96; p = 0.029)]. Kaplan-Meier analysis revealed a significantly lower incidence of death in patients on an angiotensin receptor blocker (ARB) (n = 33/318;10,4%) compared to patients using an angiotensin-converting enzyme inhibitor (ACEi) (n = 60/348;17,2%) or patients who received neither an ACE-inhibitor nor an ARB at baseline in the uncomplicated phase (n = 90/466; 19,3%; p<0.034). Patients taking an ARB were significantly less frequently reaching the mortality predicting threshold for leukocytes (p<0.001), neutrophils (p = 0.002) and the inflammatory markers CRP (p = 0.021), procalcitonin (p = 0.001) and IL-6 (p = 0.049). ACE2 expression levels in human lung samples were not altered in patients taking RAAS modulators., Conclusion: These data suggest a beneficial effect of ARBs on disease severity in patients with cardiovascular comorbidities and COVID-19, which is linked to dampened systemic inflammatory activity., Competing Interests: The authors have declared that no competing interests exist.
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- 2021
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16. Targeting the Pentose Phosphate Pathway for SARS-CoV-2 Therapy.
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Bojkova D, Costa R, Reus P, Bechtel M, Jaboreck MC, Olmer R, Martin U, Ciesek S, Michaelis M, and Cinatl J Jr
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SARS-CoV-2 is causing the coronavirus disease 2019 (COVID-19) pandemic, for which effective pharmacological therapies are needed. SARS-CoV-2 induces a shift of the host cell metabolism towards glycolysis, and the glycolysis inhibitor 2-deoxy-d-glucose (2DG), which interferes with SARS-CoV-2 infection, is under development for the treatment of COVID-19 patients. The glycolytic pathway generates intermediates that supply the non-oxidative branch of the pentose phosphate pathway (PPP). In this study, the analysis of proteomics data indicated increased transketolase (TKT) levels in SARS-CoV-2-infected cells, suggesting that a role is played by the non-oxidative PPP. In agreement, the TKT inhibitor benfooxythiamine (BOT) inhibited SARS-CoV-2 replication and increased the anti-SARS-CoV-2 activity of 2DG. In conclusion, SARS-CoV-2 infection is associated with changes in the regulation of the PPP. The TKT inhibitor BOT inhibited SARS-CoV-2 replication and increased the activity of the glycolysis inhibitor 2DG. Notably, metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting SARS-CoV-2 replication. Hence, they may improve COVID-19 therapy outcomes by exerting antiviral and immunomodulatory effects.
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- 2021
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17. Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells.
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Rothenburger T, Thomas D, Schreiber Y, Wratil PR, Pflantz T, Knecht K, Digianantonio K, Temple J, Schneider C, Baldauf HM, McLaughlin KM, Rothweiler F, Bilen B, Farmand S, Bojkova D, Costa R, Ferreirós N, Geisslinger G, Oellerich T, Xiong Y, Keppler OT, Wass MN, Michaelis M, and Cinatl J Jr
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- Cell Line, Tumor, Drug Resistance, Neoplasm, Humans, Leukemia, Myeloid, Acute drug therapy, Nucleosides pharmacology
- Abstract
Background: SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1., Methods: CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation., Results: Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance., Conclusion: Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure., (© 2021. The Author(s).)
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- 2021
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18. A Potential Role of the CD47/SIRPalpha Axis in COVID-19 Pathogenesis.
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McLaughlin KM, Bojkova D, Kandler JD, Bechtel M, Reus P, Le T, Rothweiler F, Wagner JUG, Weigert A, Ciesek S, Wass MN, Michaelis M, and Cinatl J Jr
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- Blood Donors, Blotting, Western methods, Bronchi cytology, COVID-19 pathology, COVID-19 virology, Caco-2 Cells, Epithelial Cells metabolism, Epithelial Cells virology, Healthy Volunteers, Humans, Monocytes metabolism, Monocytes virology, Polymerase Chain Reaction methods, RNA, Viral genetics, SARS-CoV-2 genetics, SARS-CoV-2 isolation & purification, Antigens, Differentiation metabolism, CD47 Antigen metabolism, COVID-19 epidemiology, COVID-19 metabolism, Pandemics, Receptors, Immunologic metabolism, SARS-CoV-2 metabolism, Severity of Illness Index, Signal Transduction immunology
- Abstract
The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air-liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.
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- 2021
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19. Enisamium Inhibits SARS-CoV-2 RNA Synthesis.
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Elli S, Bojkova D, Bechtel M, Vial T, Boltz D, Muzzio M, Peng X, Sala F, Cosentino C, Goy A, Guerrini M, Müller L, Cinatl J, Margitich V, and Te Velthuis AJW
- Abstract
Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.
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- 2021
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20. Differentially conserved amino acid positions may reflect differences in SARS-CoV-2 and SARS-CoV behaviour.
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Bojkova D, McGreig JE, McLaughlin KM, Masterson SG, Antczak M, Widera M, Krähling V, Ciesek S, Wass MN, Michaelis M, and Cinatl J
- Abstract
Motivation: SARS-CoV-2 is a novel coronavirus currently causing a pandemic. Here, we performed a combined in-silico and cell culture comparison of SARS-CoV-2 and the closely related SARS-CoV., Results: Many amino acid positions are differentially conserved between SARS-CoV-2 and SARS-CoV, which reflects the discrepancies in virus behaviour, i.e. more effective human-to-human transmission of SARS-CoV-2 and higher mortality associated with SARS-CoV. Variations in the S protein (mediates virus entry) were associated with differences in its interaction with ACE2 (cellular S receptor) and sensitivity to TMPRSS2 (enables virus entry via S cleavage) inhibition. Anti-ACE2 antibodies more strongly inhibited SARS-CoV than SARS-CoV-2 infection, probably due to a stronger SARS-CoV-2 S-ACE2 affinity relative to SARS-CoV S. Moreover, SARS-CoV-2 and SARS-CoV displayed differences in cell tropism. Cellular ACE2 and TMPRSS2 levels did not indicate susceptibility to SARS-CoV-2. In conclusion, we identified genomic variation between SARS-CoV-2 and SARS-CoV that may reflect the differences in their clinical and biological behaviour., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)
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- 2021
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21. Characterization of ACE Inhibitors and AT 1 R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model.
- Author
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Reus P, Schneider AK, Ulshöfer T, Henke M, Bojkova D, Cinatl J, Ciesek S, Geisslinger G, Laux V, Grättinger M, Gribbon P, and Schiffmann S
- Abstract
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin-renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT
1 receptor (AT1 R)., Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model., Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1 R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate., Conclusion: ACE inhibitors and AT1 R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.- Published
- 2021
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22. Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.
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Mukherjee R, Bhattacharya A, Bojkova D, Mehdipour AR, Shin D, Khan KS, Hei-Yin Cheung H, Wong KB, Ng WL, Cinatl J, Geurink PP, van der Heden van Noort GJ, Rajalingam K, Ciesek S, Hummer G, and Dikic I
- Subjects
- A549 Cells, Binding Sites, Caco-2 Cells, Chemokine CCL2 metabolism, Coronavirus 3C Proteases metabolism, HeLa Cells, Humans, Interferon Regulatory Factor-3 metabolism, Interleukin-6 metabolism, Molecular Docking Simulation, NF-kappa B metabolism, Protein Binding, SARS-CoV-2 physiology, Signal Transduction, Toll-Like Receptor 3 chemistry, Virus Replication, Famotidine pharmacology, Histamine Antagonists pharmacology, SARS-CoV-2 drug effects, Toll-Like Receptor 3 metabolism
- Abstract
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2021
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23. Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.
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Armstrong LA, Lange SM, Dee Cesare V, Matthews SP, Nirujogi RS, Cole I, Hope A, Cunningham F, Toth R, Mukherjee R, Bojkova D, Gruber F, Gray D, Wyatt PG, Cinatl J, Dikic I, Davies P, and Kulathu Y
- Subjects
- A549 Cells, Antigen-Antibody Complex, Humans, Inhibitory Concentration 50, RNA-Dependent RNA Polymerase immunology, RNA-Dependent RNA Polymerase metabolism, Single-Chain Antibodies immunology, Viral Nonstructural Proteins immunology, Viral Nonstructural Proteins metabolism, Antiviral Agents pharmacology, Protease Inhibitors pharmacology, RNA-Dependent RNA Polymerase antagonists & inhibitors, Single-Chain Antibodies pharmacology, Viral Nonstructural Proteins antagonists & inhibitors
- Abstract
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle., Competing Interests: The authors have declared that no competing interests exist.
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- 2021
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24. A method for the rational selection of drug repurposing candidates from multimodal knowledge harmonization.
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Schultz B, Zaliani A, Ebeling C, Reinshagen J, Bojkova D, Lage-Rupprecht V, Karki R, Lukassen S, Gadiya Y, Ravindra NG, Das S, Baksi S, Domingo-Fernández D, Lentzen M, Strivens M, Raschka T, Cinatl J, DeLong LN, Gribbon P, Geisslinger G, Ciesek S, van Dijk D, Gardner S, Kodamullil AT, Fröhlich H, Peitsch M, Jacobs M, Hoeng J, Eils R, Claussen C, and Hofmann-Apitius M
- Subjects
- Adenosine Monophosphate analogs & derivatives, Adenosine Monophosphate therapeutic use, Alanine analogs & derivatives, Alanine therapeutic use, Combined Modality Therapy, Computational Biology, Drug Synergism, Drug Therapy, Combination, GTP Phosphohydrolases therapeutic use, Humans, Knowledge Bases, Nelfinavir therapeutic use, Pandemics, Raloxifene Hydrochloride therapeutic use, Antiviral Agents therapeutic use, Drug Repositioning methods, SARS-CoV-2 physiology, COVID-19 Drug Treatment
- Abstract
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community's massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2/COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.
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- 2021
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25. Human Mesenchymal Stromal Cells Are Resistant to SARS-CoV-2 Infection under Steady-State, Inflammatory Conditions and in the Presence of SARS-CoV-2-Infected Cells.
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Schäfer R, Spohn G, Bechtel M, Bojkova D, Baer PC, Kuçi S, Seifried E, Ciesek S, and Cinatl J
- Subjects
- Angiotensin-Converting Enzyme 2 metabolism, COVID-19 metabolism, Caco-2 Cells, Cell Line, Tumor, Humans, Indoleamine-Pyrrole 2,3,-Dioxygenase metabolism, Inflammation metabolism, Mesenchymal Stem Cells metabolism, Serine Endopeptidases metabolism, COVID-19 Drug Treatment, COVID-19 virology, Inflammation virology, Mesenchymal Stem Cells virology, SARS-CoV-2 pathogenicity
- Abstract
Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2021
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26. A SARS-CoV-2 cytopathicity dataset generated by high-content screening of a large drug repurposing collection.
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Ellinger B, Bojkova D, Zaliani A, Cinatl J, Claussen C, Westhaus S, Keminer O, Reinshagen J, Kuzikov M, Wolf M, Geisslinger G, Gribbon P, and Ciesek S
- Subjects
- Benzamidines, COVID-19, Caco-2 Cells, Cetylpyridinium, Drug Evaluation, Preclinical, Esters, Guanidines, Humans, Lopinavir, Mefloquine, Papaverine, Antiviral Agents pharmacology, Drug Repositioning, SARS-CoV-2 drug effects
- Abstract
SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.
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- 2021
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27. SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.
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Bojkova D, Wagner JUG, Shumliakivska M, Aslan GS, Saleem U, Hansen A, Luxán G, Günther S, Pham MD, Krishnan J, Harter PN, Ermel UH, Frangakis AS, Milting H, Zeiher AM, Klingel K, Cinatl J, Dendorfer A, Eschenhagen T, Tschöpe C, Ciesek S, and Dimmeler S
- Subjects
- Adenosine Monophosphate analogs & derivatives, Adenosine Monophosphate pharmacology, Alanine analogs & derivatives, Alanine pharmacology, Angiotensin-Converting Enzyme 2 metabolism, Antiviral Agents pharmacology, COVID-19 metabolism, COVID-19 pathology, Caco-2 Cells, Cathepsins metabolism, Heart Diseases drug therapy, Heart Diseases metabolism, Heart Diseases pathology, Host-Pathogen Interactions, Humans, Myocytes, Cardiac drug effects, Myocytes, Cardiac metabolism, Myocytes, Cardiac pathology, Reactive Oxygen Species metabolism, SARS-CoV-2 drug effects, Signal Transduction, COVID-19 Drug Treatment, Apoptosis drug effects, COVID-19 virology, Heart Diseases virology, Myocytes, Cardiac virology, SARS-CoV-2 pathogenicity
- Abstract
Aims: Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection., Methods and Results: Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir., Conclusion: This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)
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- 2020
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28. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.
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Shin D, Mukherjee R, Grewe D, Bojkova D, Baek K, Bhattacharya A, Schulz L, Widera M, Mehdipour AR, Tascher G, Geurink PP, Wilhelm A, van der Heden van Noort GJ, Ovaa H, Müller S, Knobeloch KP, Rajalingam K, Schulman BA, Cinatl J, Hummer G, Ciesek S, and Dikic I
- Subjects
- Animals, Coronavirus Papain-Like Proteases antagonists & inhibitors, Cytokines chemistry, Cytokines metabolism, Deubiquitinating Enzymes antagonists & inhibitors, Deubiquitinating Enzymes chemistry, Deubiquitinating Enzymes metabolism, Humans, Interferon Regulatory Factor-3 metabolism, Interferons immunology, Interferons metabolism, Mice, Models, Molecular, Molecular Dynamics Simulation, NF-kappa B immunology, NF-kappa B metabolism, Protein Binding, SARS-CoV-2 drug effects, SARS-CoV-2 physiology, Ubiquitination, Ubiquitins chemistry, Ubiquitins metabolism, COVID-19 Drug Treatment, COVID-19 immunology, COVID-19 virology, Coronavirus Papain-Like Proteases chemistry, Coronavirus Papain-Like Proteases metabolism, Immunity, Innate, SARS-CoV-2 enzymology, SARS-CoV-2 immunology
- Abstract
The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread
1,2 . PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5 . Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.- Published
- 2020
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29. Aprotinin Inhibits SARS-CoV-2 Replication.
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Bojkova D, Bechtel M, McLaughlin KM, McGreig JE, Klann K, Bellinghausen C, Rohde G, Jonigk D, Braubach P, Ciesek S, Münch C, Wass MN, Michaelis M, and Cinatl J Jr
- Subjects
- Animals, Antiviral Agents pharmacology, COVID-19 metabolism, Caco-2 Cells, Chlorocebus aethiops, Epithelial Cells drug effects, Humans, Pandemics, SARS-CoV-2 physiology, Serine Proteinase Inhibitors pharmacology, Vero Cells, Aprotinin pharmacology, SARS-CoV-2 drug effects, Virus Replication drug effects, COVID-19 Drug Treatment
- Abstract
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
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- 2020
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30. Growth Factor Receptor Signaling Inhibition Prevents SARS-CoV-2 Replication.
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Klann K, Bojkova D, Tascher G, Ciesek S, Münch C, and Cinatl J
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- Adrenal Cortex Hormones therapeutic use, Angiotensin-Converting Enzyme Inhibitors therapeutic use, Antibodies, Neutralizing therapeutic use, Betacoronavirus immunology, Betacoronavirus pathogenicity, Caco-2 Cells, Gene Expression Regulation, Host-Pathogen Interactions drug effects, Host-Pathogen Interactions genetics, Humans, Mitogen-Activated Protein Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinases metabolism, Phosphatidylinositol 3-Kinase metabolism, Phosphoproteins antagonists & inhibitors, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphorylation, Receptors, Growth Factor antagonists & inhibitors, Receptors, Growth Factor metabolism, SARS-CoV-2, Signal Transduction, Viral Proteins antagonists & inhibitors, Viral Proteins metabolism, Virus Replication drug effects, Antiviral Agents therapeutic use, Betacoronavirus drug effects, Mitogen-Activated Protein Kinases genetics, Phosphatidylinositol 3-Kinase genetics, Receptors, Growth Factor genetics, Viral Proteins genetics
- Abstract
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment., Competing Interests: Declaration of Interests The authors filed a patent application on the use of GFR signaling inhibitors for the treatment of COVID-19., (Copyright © 2020 Elsevier Inc. All rights reserved.)
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- 2020
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31. Lack of antiviral activity of darunavir against SARS-CoV-2.
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De Meyer S, Bojkova D, Cinatl J, Van Damme E, Buyck C, Van Loock M, Woodfall B, and Ciesek S
- Subjects
- Antiviral Agents therapeutic use, COVID-19, Cell Line, Cell Survival drug effects, Humans, SARS-CoV-2, Betacoronavirus drug effects, Coronavirus Infections drug therapy, Darunavir therapeutic use, Pandemics, Pneumonia, Viral drug therapy
- Abstract
Objectives: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives., Methods: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed., Results: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC
50 > 100 μM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 μM)., Conclusions: Overall, the data do not support the use of DRV for the treatment of COVID-19., (Copyright © 2020. Published by Elsevier Ltd.)- Published
- 2020
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32. COVID-19-Related Coagulopathy-Is Transferrin a Missing Link?
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McLaughlin KM, Bechtel M, Bojkova D, Münch C, Ciesek S, Wass MN, Michaelis M, and Cinatl J Jr
- Abstract
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term "blood coagulation" in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term "blood coagulation") was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
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- 2020
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33. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.
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Bojkova D, Klann K, Koch B, Widera M, Krause D, Ciesek S, Cinatl J, and Münch C
- Subjects
- Betacoronavirus genetics, Betacoronavirus growth & development, COVID-19, Caco-2 Cells, Carbon metabolism, Coronavirus Infections genetics, Coronavirus Infections virology, Drug Evaluation, Preclinical, Host-Pathogen Interactions drug effects, Host-Pathogen Interactions genetics, Humans, In Vitro Techniques, Kinetics, Pandemics, Pneumonia, Viral genetics, Pneumonia, Viral virology, Protein Biosynthesis drug effects, Proteome metabolism, Proteostasis, RNA Splicing, SARS-CoV-2, Time Factors, Virus Replication drug effects, COVID-19 Drug Treatment, Betacoronavirus drug effects, Betacoronavirus metabolism, Coronavirus Infections drug therapy, Coronavirus Infections metabolism, Molecular Targeted Therapy, Pneumonia, Viral drug therapy, Pneumonia, Viral metabolism, Proteomics
- Abstract
A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and has been rapidly spreading around the globe
1,2 . SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome3 and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.- Published
- 2020
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34. Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs.
- Author
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Toptan T, Hoehl S, Westhaus S, Bojkova D, Berger A, Rotter B, Hoffmeier K, Cinatl J Jr, Ciesek S, and Widera M
- Subjects
- Animals, Betacoronavirus genetics, COVID-19 Testing, Caco-2 Cells, Chlorocebus aethiops, Clinical Laboratory Techniques economics, Clinical Laboratory Techniques standards, Coronavirus Infections diagnosis, Coronavirus Infections economics, Coronavirus M Proteins, Costs and Cost Analysis, Humans, RNA, Viral chemistry, RNA, Viral genetics, Reverse Transcriptase Polymerase Chain Reaction economics, Reverse Transcriptase Polymerase Chain Reaction standards, SARS-CoV-2, Sensitivity and Specificity, Vero Cells, Viral Matrix Proteins genetics, Clinical Laboratory Techniques methods, Reverse Transcriptase Polymerase Chain Reaction methods
- Abstract
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
- Published
- 2020
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35. Sofosbuvir Activates EGFR-Dependent Pathways in Hepatoma Cells with Implications for Liver-Related Pathological Processes.
- Author
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Bojkova D, Westhaus S, Costa R, Timmer L, Funkenberg N, Korencak M, Streeck H, Vondran F, Broering R, Heinrichs S, Lang KS, and Ciesek S
- Subjects
- Antiviral Agents pharmacology, Humans, Liver pathology, Risk Factors, Sofosbuvir pharmacology, Antiviral Agents therapeutic use, Hepatitis C, Chronic drug therapy, Liver drug effects, Sofosbuvir therapeutic use
- Abstract
Direct acting antivirals (DAAs) revolutionized the therapy of chronic hepatitis C infection. However, unexpected high recurrence rates of hepatocellular carcinoma (HCC) after DAA treatment became an issue in patients with advanced cirrhosis and fibrosis. In this study, we aimed to investigate an impact of DAA treatment on the molecular changes related to HCC development and progression in hepatoma cell lines and primary human hepatocytes. We found that treatment with sofosbuvir (SOF), a backbone of DAA therapy, caused an increase in EGFR expression and phosphorylation. As a result, enhanced translocation of EGFR into the nucleus and transactivation of factors associated with cell cycle progression, B-MYB and Cyclin D1, was detected. Serine/threonine kinase profiling identified additional pathways, especially the MAPK pathway, also activated during SOF treatment. Importantly, the blocking of EGFR kinase activity by erlotinib during SOF treatment prevented all downstream events. Altogether, our findings suggest that SOF may have an impact on pathological processes in the liver via the induction of EGFR signaling. Notably, zidovudine, another nucleoside analogue, exerted a similar cell phenotype, suggesting that the observed effects may be induced by additional members of this drug class.
- Published
- 2020
- Full Text
- View/download PDF
36. Omeprazole Increases the Efficacy of Acyclovir Against Herpes Simplex Virus Type 1 and 2.
- Author
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Michaelis M, Kleinschmidt MC, Bojkova D, Rabenau HF, Wass MN, and Cinatl J Jr
- Abstract
Omeprazole was shown to improve the anti-cancer effects of the nucleoside analogue 5-fluorouracil. Here, we combined omeprazole with the antiviral nucleoside analogues ribavirin and acyclovir. Omeprazole did not affect the antiviral effects of ribavirin in non-toxic concentrations up to 80 μg/mL but increased the acyclovir-mediated effects on herpes simplex virus 1 and 2 (HSV-1 and -2) replication in a dose-dependent manner. Omeprazole alone reduced HSV-1 and -2 titers [but not HSV-induced formation of cytopathogenic effects (CPE)] at concentrations ≥40 μg/mL. However, it exerted substantially stronger effects on acyclovir activity and also increased acyclovir activity at lower concentrations that did not directly interfere with HSV replication. Omeprazole 80 μg/mL caused a 10.8-fold (Vero cells) and 47.7-fold (HaCaT cells) decrease of the acyclovir concentrations that reduced HSV-1-induced CPE formation by 50% (IC
50 ). In HSV-2-infected cells, omeprazole 80 μg/mL reduced the acyclovir IC50 by 7.3- (Vero cells) and 12.9-fold (HaCaT cells). In HaCaT cells, omeprazole 80 μg/mL reduced the HSV-1 titer in the presence of acyclovir 1 μg/mL by 1.6 × 105 -fold and the HSV-2 titer in the presence of acyclovir 2 μg/mL by 9.2 × 103 -fold. The proton pump inhibitors pantoprazole, rabeprazole, lansoprazole, and dexlansoprazole increased the antiviral effects of acyclovir in a similar fashion as omeprazole, indicating this to be a drug class effect. In conclusion, proton pump inhibitors increase the anti-HSV activity of acyclovir and are candidates for antiviral therapies in combination with acyclovir, in particular for topical preparations for the treatment of immunocompromised individuals who are more likely to suffer from severe complications., (Copyright © 2019 Michaelis, Kleinschmidt, Bojkova, Rabenau, Wass and Cinatl.)- Published
- 2019
- Full Text
- View/download PDF
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