Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients
Valeria Inés Segatori, Juan Garona, Lorena Grisel Caligiuri, Juan Bizzotto, Rosario Lavignolle, Ayelén Toro, Pablo Sanchis, Eduardo Spitzer, Alejandro Krolewiecki, Geraldine Gueron, Daniel Fernando Alonso
Viruses, doi:10.3390/v13102084
Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.
Supplementary Materials: The following are available online at www.mdpi.com/article/10.3390/v13102084/s1; Table S1 : Patient demographics for GSE152075 dataset. Figure S1 : Gene expression in COVID-19 and non-COVID-19 patients categorized by sex. Figure S2 : Gene expression of importins and Rho GTPases in A549 cells and ferrets. Figure S3 : Importin and Rho GTPases gene expression analysis in A549 and HMVEC cells. Author Contributions: Conceptualization, D.F.A., G.G., and E.S.; Methodology, V.I.S., J.G., L.G.C., J.B., R.L., A.T., and P.S.; Software, J.B., R.L., A.T., and P.S.; Validation, J.B., R.L., A.T., and P.S.; Formal analysis, V.I.S., J.G.,J.B., R.L., A.T., and P.S.; Investigation, V.I.S., J.G., J.B., R.L., A.T., and P.S.; Data curation, J.B., R.L., A.T., and P.S.; Writing-original draft preparation, V.I.S., J.G., G.G., and D.F.A.; Writing-review & editing, V.I.S., J.G., J.B., R.L., A.T., P.S., E.S., A.K., G.G., and D.F.A.; Supervision, D.F.A. and G.G.; Project administration, V.I.S. and J.G.; Funding acquisition, G.G., D.F.A., and A.K. All authors have read and agreed to the published version of the manuscript.
References
Alonso, Farina, Repurposing of host-based therapeutic agents for the treatment of coronavirus disease 2019 (COVID-19): A link between antiviral and anticancer mechanisms?, Int. J. Antimicrob. Agents,
doi:10.1016/j.ijantimicag.2020.106125Atkinson, Audsley, Lieu, Marsh, Thomas et al., Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V, Sci. Rep,
doi:10.1038/s41598-017-18742-8Audsley, Jans, Moseley, Nucleocytoplasmic trafficking of Nipah virus W protein involves multiple discrete interactions with the nuclear import and export machinery, Biochem. Biophys. Res. Commun,
doi:10.1016/j.bbrc.2016.09.043Bello, Elucidation of the inhibitory activity of ivermectin with host nuclear importin α and several SARS-CoV-2 targets, J. Biomol. Struct. Dyn,
doi:10.1080/07391102.2021.1911857Bizzotto, Sanchis, Abbate, Lage-Vickers, Lavignolle et al., SARS-CoV-2 Infection Boosts MX1 Antiviral Effector in COVID-19 Patients, iScience,
doi:10.1016/j.isci.2020.101585Blanco-Melo, Nilsson-Payant, Liu, Uhl, Hoagland et al., Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19, Cell,
doi:10.1016/j.cell.2020.04.026Boerma, Fu, Wang, Loose, Bartolozzi et al., Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin, Blood Coagul. Fibrinolysis Int. J. Haemost. Thromb,
doi:10.1097/MBC.0b013e32830b2891Chi, Wang, Huang, Stamnes, Chen, Roles of Rho GTPases in Intracellular Transport and Cellular Transformation, Int. J. Mol. Sci,
doi:10.3390/ijms14047089Core, R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing
De Melo, Lazarini, Larrous, Feige, Kornobis et al., Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin, EMBO Mol. Med,
doi:10.15252/emmm.202114122Errecalde, Lifschitz, Vecchioli, Ceballos, Errecalde et al., Safety and Pharmacokinetic Assessments of a Novel Ivermectin Nasal Spray Formulation in a Pig Model, J. Pharm. Sci,
doi:10.1016/j.xphs.2021.01.017Farina, Bublik, Alonso, Gomez, Lovastatin alters cytoskeleton organization and inhibits experimental metastasis of mammary carcinoma cells, Clin. Exp. Metastasis,
doi:10.1023/a:1020355621043Fedson, Opal, Rordam, Hiding in Plain Sight: An Approach to Treating Patients with Severe COVID-19 Infection, mBio,
doi:10.1128/mBio.00398-20Fernández-Iglesias, Fuente, Gil-Peña, Alonso-Duran, García-Bengoa et al., A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes, Sci. Rep,
doi:10.1038/s41598-020-63978-6Fortier, Kent, Ashdown, Poole, Boksa et al., The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats via an interleukin-1-dependent mechanism, Am. J. Physiol. Regul. Integr. Comp. Physiol,
doi:10.1152/ajpregu.00293.2004Garona, Pifano, Orlando, Pastrian, Iannucci et al., The novel desmopressin analogue [V4Q5]dDAVP inhibits angiogenesis, tumour growth and metastases in vasopressin type 2 receptor-expressing breast cancer models, Int. J. Oncol,
doi:10.3892/ijo.2015.2952Garona, Pifano, Pastrian, Gomez, Ripoll et al., Addition of vasopressin synthetic analogue [V(4)Q(5)]dDAVP to standard chemotherapy enhances tumour growth inhibition and impairs metastatic spread in aggressive breast tumour models, Clin. Exp. Metastasis,
doi:10.1007/s10585-016-9799-5Gower, Peeples, Collins, Graham, RhoA Is Activated During Respiratory Syncytial Virus Infection, Virology,
doi:10.1006/viro.2001.0891Gu, Zhao, Jin, Song, Zhi et al., Cytokine Signature Induced by SARS-CoV-2 Spike Protein in a Mouse Model, Front. Immunol,
doi:10.3389/fimmu.2020.621441Guzzo, Furtek, Porras, Chen, Tipping et al., Safety, tolerability, and pharmacokinetics of escalating high doses of ivermectin in healthy adult subjects, J. Clin. Pharmacol,
doi:10.1177/009127002401382731Haji Aghajani, Moradi, Azhdari Tehrani, Amini, Pourheidar et al., Promising effects of atorvastatin on mortality and need for mechanical ventilation in patients with severe COVID-19; a retrospective cohort study, Int. J. Clin. Pract,
doi:10.1111/ijcp.14434Harris, Sridhar, Peng, Phillips, Cohn et al., Double-stranded RNA induces molecular and inflammatory signatures that are directly relevant to COPD, Mucosal Immunol,
doi:10.1038/mi.2012.86Hill, Garratt, Levi, Falconer, Ellis et al., Expression of Concern: "Meta-analysis of Randomized Trials of Ivermectin to Treat SARS-CoV-2 Infection, Open Forum Infect. Dis,
doi:10.1093/ofid/ofab394Huang, Stuart, Takeda, D'agnillo, Golding et al., I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5, PLoS ONE,
doi:10.1371/journal.pone.0160875Kassambara, Based Publication Ready Plots. R package version 0
Kern, Schöning, Chaccour, Hammann, Modeling of SARS-CoV-2 Treatment Effects for Informed Drug Repurposing, Front. Pharmacol,
doi:10.3389/fphar.2021.625678Krolewiecki, Lifschitz, Moragas, Travacio, Valentini et al., Antiviral effect of high-dose ivermectin in adults with COVID-19: A proof-of-concept randomized trial, EClinicalMedicine,
doi:10.1016/j.eclinm.2021.100959Lever, Park, Mulhern, Jackson, Comolli et al., Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model, Physiol. Rep,
doi:10.14814/phy2.12334Lieberman, Peddu, Xie, Shrestha, Huang et al., In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age, PLoS Biol,
doi:10.1371/journal.pbio.3000849Livak, Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods,
doi:10.1006/meth.2001.1262López-Medina, López, Hurtado, Dávalos, Ramirez et al., Effect of Ivermectin on Time to Resolution of Symptoms Among Adults With Mild COVID-19: A Randomized Clinical Trial, JAMA,
doi:10.1001/jama.2021.3071Menna, Parera, Cardama, Alonso, Gomez et al., Enhanced cytostatic activity of statins in mouse mammary carcinoma cells overexpressing β2-chimaerin, Mol. Med. Rep,
doi:10.3892/mmr_00000068Miyamoto, Saiwaki, Yamashita, Yasuda, Kotera et al., Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block, J. Cell Biol,
doi:10.1083/jcb.200312008Poon, Oro, Dias, Zhang, Jans, Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells, Cancer Res,
doi:10.1158/0008-5472.can-05-1370Pushpakom, Iorio, Eyers, Escott, Hopper et al., Drug repurposing: Progress, challenges and recommendations, Nat. Rev. Drug Discov,
doi:10.1038/nrd.2018.168Sanchis, Lavignolle, Abbate, Lage-Vickers, Vazquez et al., Analysis workflow of publicly available RNA-sequencing datasets, STAR Protoc,
doi:10.1016/j.xpro.2021.100478Schloerke, Cook, Larmarange, Francois, Marbach et al., Extension to "ggplot2
Serafin, Bottega, Foletto, Da Rosa, Hörner et al., Drug repositioning is an alternative for the treatment of coronavirus COVID-19, Int. J. Antimicrob. Agents,
doi:10.1016/j.ijantimicag.2020.105969Shukla, Misra, Antiviral Effects of Ivermectin in COVID-19-Clinically Plausible?, Int. J. Infect. Dis. IJID Off. Publ. Int. Soc. Infect. Dis,
doi:10.1016/j.ijid.2021.06.048Siedner, Ivermectin for the treatment of COVID-19 disease: Too good to pass up or too good to be true? Open, Forum Infect. Dis,
doi:10.1093/ofid/ofab318Tay, Fraser, Chan, Moreland, Rathore et al., Nuclear localization of dengue virus (DENV) 1-4 non-structural protein 5; protection against all 4 DENV serotypes by the inhibitor Ivermectin, Antivir. Res,
doi:10.1016/j.antiviral.2013.06.002Wagstaff, Sivakumaran, Heaton, Harrich, Jans, Ivermectin is a specific inhibitor of importin α/βmediated nuclear import able to inhibit replication of HIV-1 and dengue virus, Biochem. J,
doi:10.1042/bj20120150Wickham, Elegant Graphics for Data Analysis
Yan, Ci, Chen, Chen, Li et al., Anti-inflammatory effects of ivermectin in mouse model of allergic asthma, Inflamm. Res,
doi:10.1007/s00011-011-0307-8Zeidler, Karpinski, SARS-CoV, MERS-CoV, SARS-CoV-2 Comparison of Three Emerging Coronaviruses, Jundishapur J. Microbiol,
doi:10.5812/jjm.103744Zhang, Song, Ci, An, Ju et al., Ivermectin inhibits LPS-induced production of inflammatory cytokines and improves LPS-induced survival in mice, Inflamm. Res,
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'abstract': '<jats:p>Nuclear transport and vesicle trafficking are key cellular functions involved in the '
'pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, '
'ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin '
'(ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. '
'In this work, we first analyzed the response to infection in nasopharyngeal swabs from '
'SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective '
'host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in '
'KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. '
'An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded '
'RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin '
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'confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic '
'importin α distribution, alone or in combination. Results showed a significant inhibition of '
'importin α nuclear accumulation under IVM and ATV treatments. These findings confirm '
'transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point '
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