Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100470
ISSN1083-351X
AutoresEinat B. Vitner, Hagit Achdout, Roy Avraham, Boaz Politi, Lilach Cherry, Hadas Tamir, Yfat Yahalom-Ronen, Nir Paran, Sharon Melamed, Noam Erez, Tomer Israely,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies. The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies. The recent spread of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has created a worldwide public health emergency. In December 2019, the outbreak of this emerging disease (COVID-19) began in Wuhan, China, and rapidly spread. It was declared as a pandemic by the World Health Organization in March 2020 (https://covid19.who.int/). Antiviral drugs which inhibit the replication of SARS-CoV-2 can be used widely to treat patients after infection. Historically, antiviral drug research has mainly focused on targeting viral components because of the perceived specificity of such an approach (1Clercq E.D. Antivirals and antiviral strategies.Nat. Rev. Microbiol. 2004; 2: 704-720Crossref PubMed Scopus (324) Google Scholar). Thus, it is not surprising that the first drug to be approved against SARS-CoV-2, remdesivir (also GS-5734), is a direct-acting antiviral that inhibits the viral RNA-dependent RNA polymerase (2Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295: 6785-6797Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). However, because the viral life cycle is dependent on the host, specific host mechanisms can also be explored as antiviral targets. Sphingolipids (SLs) are biologically active components of cell membranes and as such are tightly linked to all processes involving membrane dynamics, making them potential key regulators in the life cycle of obligatory intracellular pathogens such as viruses. Glucosylceramide (GlcCer) is the backbone of more than 300 structurally different glycosphingolipids (GSLs) including gangliosides and sulfatides. Its accumulation leads to Gaucher diseases accompanied by chronic brain inflammation and activation of the antiviral immune response (3Vitner E.B. Farfel-Becker T. Ferreira N.S. Leshkowitz D. Sharma P. Lang K.S. Futerman A.H. Induction of the type I interferon response in neurological forms of Gaucher disease.J. Neuroinflammation. 2016; 13: 104Crossref PubMed Scopus (48) Google Scholar). Viral-induced elevation of SL levels was shown to be associated with a number of viruses; elevation of GM2-ganglioside and lactosylceramide was shown upon infection with Zika virus and hepatitis C virus, respectively (4Melo C.F. de Oliveira D.N. Lima E.O. Guerreiro T.M. Esteves C.Z. Beck R.M. Padilla M.A. Milanez G.P. Arns C.W. Proença-Modena J.L. Souza-Neto J.A. Catharino R.R. A lipidomics approach in the characterization of Zika-infected mosquito cells: Potential targets for breaking the transmission cycle.PLoS One. 2016; 11e0164377Crossref PubMed Scopus (42) Google Scholar, 5Khan I. Katikaneni D.S. Han Q. Sanchez-Felipe L. Hanada K. Ambrose R.L. Mackenzie J.M. Konan K.V. Modulation of hepatitis C virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2.J. Virol. 2014; 88: 12276-12295Crossref PubMed Scopus (71) Google Scholar). 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Glucosylceramidase maintains influenza virus infection by regulating endocytosis.J. Virol. 2019; 93e00017-19Crossref PubMed Scopus (30) Google Scholar). Drugs targeting SL metabolizing enzymes are currently in use and constantly being developed for treating lysosomal storage diseases (LSDs) and other disorders in which alteration in SL levels are involved in disease pathology (12Gualtierotti R. Guarnaccia L. Beretta M. Navone S.E. Campanella R. Riboni L. Rampini P. Marfia G. Modulation of neuroinflammation in the central nervous system: Role of chemokines and sphingolipids.Adv. Ther. 2017; 34: 396-420Crossref PubMed Scopus (40) Google Scholar, 13Platt F.M. Sphingolipid lysosomal storage disorders.Nature. 2014; 510: 68-75Crossref PubMed Scopus (236) Google Scholar, 14Coutinho M.F. Santos J.I. Alves S. Less is more: Substrate reduction therapy for lysosomal storage disorders.Int. J. Mol. Sci. 2016; 17: 1065Crossref PubMed Scopus (70) Google Scholar). This allows a potential repurposing of these already approved drugs as antivirals. In this study, we examined the antiviral activity of two specific inhibitors of UDP-glucose:ceramide glucosyltransferase (glucosylceramide synthase (GCS)), (EC 2.4.1.80), which catalyze the biosynthesis of GlcCer. These inhibitors block the conversion of ceramide to GlcCer, the first step in the biosynthesis of gangliosides and other GSLs. The following GCS inhibitors were examined: (i) (1R,2R)-nonanoic acid[2-(2′,3′-dihydro-benzo [1,4] dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-l-tartaric acid salt (Genz-123346), termed hereinafter GZ-346. GZ-346 is an analogue of the United States Food and Drug Administration-approved drug eliglustat (Cerdelga), which is indicated for the long-term treatment of adult patients with Gaucher disease type 1 (15Zhao H. Przybylska M. Wu I.-H. Zhang J. Siegel C. Komarnitsky S. Yew N.S. Cheng S.H. Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes.Diabetes. 2007; 56: 1210-1218Crossref PubMed Scopus (203) Google Scholar), and (ii) (S)- quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate (GENZ-667161), termed hereinafter GZ-161. GZ-161 is a specific inhibitor of GCS that can access the central nervous system and has been demonstrated to effectively reduce GSL synthesis (16Ashe K.M. Budman E. Bangari D.S. Siegel C.S. Nietupski J.B. Wang B. Desnick R.J. Scheule R.K. Leonard J.P. Cheng S.H. Marshall J. Efficacy of enzyme and substrate reduction therapy with a novel antagonist of glucosylceramide synthase for Fabry disease.Mol. Med. 2015; 21: 389-399Crossref PubMed Scopus (68) Google Scholar, 17Marshall J. Sun Y. Bangari D.S. Budman E. Park H. Nietupski J.B. Allaire A. Cromwell M.A. Wang B. Grabowski G.A. Leonard J.P. Cheng S.H. CNS-accessible inhibitor of glucosylceramide synthase for substrate reduction therapy of neuronopathic Gaucher disease.Mol. Ther. 2016; 24: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Cabrera-Salazar M.A. Deriso M. Bercury S.D. Li L. Lydon J.T. Weber W. Pande N. Cromwell M.A. Copeland D. Leonard J. Cheng S.H. Scheule R.K. Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.PLoS One. 2012; 7e43310Crossref PubMed Scopus (66) Google Scholar). GZ-161 is an analogue of venglustat which is currently under clinical trials for the LSDs; Gaucher disease, Fabry disease, and Tay–Sachs disease, and is in a phase 3 pivotal trial for autosomal-dominant polycystic kidney disease (16Ashe K.M. Budman E. Bangari D.S. Siegel C.S. Nietupski J.B. Wang B. Desnick R.J. Scheule R.K. Leonard J.P. Cheng S.H. Marshall J. Efficacy of enzyme and substrate reduction therapy with a novel antagonist of glucosylceramide synthase for Fabry disease.Mol. Med. 2015; 21: 389-399Crossref PubMed Scopus (68) Google Scholar, 17Marshall J. Sun Y. Bangari D.S. Budman E. Park H. Nietupski J.B. Allaire A. Cromwell M.A. Wang B. Grabowski G.A. Leonard J.P. Cheng S.H. CNS-accessible inhibitor of glucosylceramide synthase for substrate reduction therapy of neuronopathic Gaucher disease.Mol. Ther. 2016; 24: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Cabrera-Salazar M.A. Deriso M. Bercury S.D. Li L. Lydon J.T. Weber W. Pande N. Cromwell M.A. Copeland D. Leonard J. Cheng S.H. Scheule R.K. Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.PLoS One. 2012; 7e43310Crossref PubMed Scopus (66) Google Scholar). Thus, the antiviral activity of GCS inhibitors was examined for SARS-CoV-2. To test for antiviral activity of GCS inhibitors against SARS-CoV-2, Vero E6 cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with SARS-CoV-2 (multiplicity of infection [MOI] = 0.01). Supernatants were harvested 24 h after infection and analyzed by plaque-forming units (PFU) assay to measure the effect of the drugs on SARS-CoV-2 replication (Fig. 1, A and B). Approximately 1.7e7 ± 1.3e6 PFU/ml were detected in the medium of vehicle dimethyl sulfoxide (DMSO) (untreated) infected cells, whereas only 37 ± 23 and 700 ± 339 PFU/ml were detected in GZ-161– and GZ-346–treated cells, respectively, indicating significant inhibition of virus release (p < 0.0001, p < 0.001, respectively). In addition, the ability of GZ-161 and GZ-346 to inhibit single-cycle infection was studied. Vero E6 cells were infected at a high MOI (MOI = 5), and viral release was determined at 10 h after infection, an early time point of viral release (19Araujo D.B. Machado R.R.G. Amgarten D.E. Malta F.M. de Araujo G.G. Monteiro C.O. Candido E.D. Soares C.P. de Menezes F.G. Pires A.C.C. Santana R.A.F. Viana A.O. Dorlass E. Thomazelli L. Ferreira L.C.S. et al.SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network.Mem. Inst. Oswaldo Cruz. 2020; 115e200342Crossref PubMed Scopus (67) Google Scholar, 20Ogando N.S. Dalebout T.J. Zevenhoven-Dobbe J.C. Limpens R.W.A.L. van der Meer Y. Caly L. Druce J. de Vries J.J.C. Kikkert M. Bárcena M. Sidorov I. Snijder E.J. SARS-coronavirus-2 replication in Vero E6 cells: Replication kinetics, rapid adaptation and cytopathology.J. Gen. Virol. 2020; 101: 925-940Crossref PubMed Scopus (309) Google Scholar). GZ-161 and GZ-346 reduced viral release by 70% and 76%, respectively, even when SARS-CoV-2 were infected with a high MOI (Fig. 1C), albeit to a lesser extent than the low MOI. To further determine the antiviral activity and cell cytotoxicity of GZ-161 and GZ-346 against SARS-CoV-2 infection, we measured their IC50, cytotoxicity concentration 50% (CC50), and selective index (SI) (Fig. 2). Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.01 in the presence of serial dilution of GZ-161 or GZ-346. Twenty four hours after infection, the viral copy numbers in the cell culture supernatant were determined by amplifying the nucleocapsid (N) gene by quantitative real-time PCR, and cell viability was measured using the cell proliferation assay (XTT based). The IC50 values of GZ-161 (IC50 = 2.5 μM) and GZ-346 (IC50 = 2.7 μM) were determined at a low micromolar concentration. The SI (CC50/IC50) values of GZ-161 and GZ-346 were CC50 = 48 μM, SI > 19.2 and CC50 = 46 μM, SI > 17, respectively (Fig. 2).Figure 2Dose–response curves of GZ-161 and GZ-346 for inhibition of SARS-CoV-2 infection. Graphs depict the mean % inhibition of SARS-CoV-2 replication (blue circles, left Y-axis) and % cytotoxicity (red squares, right Y-axis) of antivirals. Vero E6 cells were infected in four replicates with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 in the presence of drug doses for 24 h, after which viral release was measured through quantitation of SARS-CoV-2 RNA levels by real-time PCR. Cytotoxicity was measured in similarly treated but uninfected cultures via the Cell Proliferation Kit (XTT based). Representative data are shown from three independent experiments. The IC50 is the concentration on an antiviral required at which virus replication is inhibited by 50% in a cell-based assay. The cytotoxic concentration 50 (CC50) is the concentration of an antiviral agent required to kill 50% of cells in the uninfected culture. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate that inhibition of GCS directly reduces viral replication, Ugcg (the gene encoding for GCS) was knocked down by Dicer-substrate siRNA (DsiRNA). The resulting ∼90% reduction in Ugcg mRNA expression levels 24 h after transfection (Fig. 3A) was sufficient to reduce viral release to the medium by 50% (Fig. 3B). The fact that knocking down Ugcg inhibits SARS-CoV-2 virus, together with that viral inhibition is observed with two GCS inhibitors with different structures (GZ-161, GZ-346), increases the possibility that the antiviral effect of the two compounds is due to GCS inhibition rather than an off-target effect. Next, the ability of GCS inhibitors to reduce the cytopathic effect (CPE) of SARS-CoV-2–infected cells was examined. Vero E6 cells were incubated with 10-μM GZ-161, or GZ-346, 1 h before infection with SARS-CoV-2, and cell cytotoxicity was measured at 48 h after infection. Treatment with GZ-161 or with GZ-346, 1 h before infection, completely eliminated SARS-CoV-2–induced cell cytotoxicity (Fig. 4A). Taken together, these results demonstrate that GZ-161 and GZ-346 have an antiviral effect on the SARS-CoV-2 clinical isolate in vitro, with a single dose able to significantly inhibit viral replication within 24 to 48 h. To determine which stage of SARS-CoV-2 infection cycle was affected by GCS inhibitors, a time-of-addition assay was performed. As shown in Figure 4, A–C, inhibition was most effective when GCS inhibitors were added 1 h before infection and was not effective when given 1 h after infection (Fig. 4C). When GCS inhibitors were added immediately after SARS-CoV-2-attachment (0 h after infection), viral replication was significantly reduced by ∼50% (Fig. 4B). The time dependence of the inhibitory effect of the compound suggests that its anti-SARS-CoV-2 activity may be due to inhibition of early steps in the SARS-CoV-2 replication cycle. To further examine the stage which is being interrupted by GCS inhibitors in the life cycle of SARS-CoV-2, Vero E6 were infected with high MOI of 5 to ensure single-cycle infection. Cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with SARS-CoV-2. Five hours after infection, SARS-CoV-2 nucleocapsid (N) levels were determined (Fig. 4D). GZ-161 and GZ-346 significantly reduced the levels of N protein in infected cells. Thus, our data suggest that GCS inhibitors inhibit SARS-CoV-2 infection cycle after attachment of the virus and before the translation of subgenomic proteins. It was recently shown that Ugcg KO cells demonstrate a reduction in the replication of the respiratory RNA virus Influenza A virus, a member of the family Orthomyxoviridae (21Drews K. Calgi M.P. Harrison W.C. Drews C.M. Costa-Pinheiro P. Shaw J.J.P. Jobe K.A. Han J.D. Fox T.E. White J.M. Kester M. Glucosylceramide synthase maintains influenza virus entry and infection.PLoS One. 2020; 15e0228735Crossref PubMed Scopus (15) Google Scholar). Thus, we further examined the antiviral activity of GCS inhibitors against Influenza A virus. Madin–Darby Canine Kidney (MDCK) cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with mouse-adapted influenza virus A/PR/8/34 (H1N1) (PR8). The supernatant was harvested 8 h after infection and analyzed by qPCR for the detection of viral RNA in the culture media (Fig. 5A). Approximately 90% reduction in viral RNA was measured in samples treated with GZ-161 or GZ-346 compared with the vehicle DMSO (untreated). In addition to their ability to inhibit PR8 replication, the ability of GCS inhibitors to reduce the CPE of PR8-infected cells was further examined. MDCK cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with PR8 (MOI, 0.1) and lactate dehydrogenase release to the supernatant was measured at 24 h after infection as an indication for cell disintegration. Both GZ-161 and GZ-346 significantly reduced PR8-induced cytotoxicity by 65% and 90%, respectively (Fig. 5B). In this study, we demonstrate that the GCS inhibitors GZ-161 and GZ-346 inhibit viral replication of SARS-CoV-2. The importance of GSL biosynthesis in the viral life cycle was demonstrated recently for Influenza virus and severe fever with thrombocytopenia syndrome virus (21Drews K. Calgi M.P. Harrison W.C. Drews C.M. Costa-Pinheiro P. Shaw J.J.P. Jobe K.A. Han J.D. Fox T.E. White J.M. Kester M. Glucosylceramide synthase maintains influenza virus entry and infection.PLoS One. 2020; 15e0228735Crossref PubMed Scopus (15) Google Scholar, 22Drake M.J. Brennan B. Briley Jr., K. Bart S.M. Sherman E. Szemiel A.M. Minutillo M. Bushman F.D. Bates P. 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Unlike the iminosugar NB-DNJ, eliglustat is a ceramide analogue that inhibits UDP-GCS without inhibiting of intestinal glycosidases (lactase, maltase, sucrase), α-glucosidase I and II, as was evaluated in in vitro cell-based and cell-free assays (36McEachern K.A. Fung J. Komarnitsky S. Siegel C.S. Chuang W.-L. Hutto E. Shayman J.A. Grabowski G.A. Aerts J.M.F.G. Cheng S.H. Copeland D.P. Marshall J. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.Mol. Genet. Metab. 2007; 91: 259-267Crossref PubMed Scopus (140) Google Scholar, 37Shayman J.A. ELIGLUSTAT TARTRATE: Glucosylceramide synthase inhibitor treatment of type 1 Gaucher disease.Drugs Future. 2010; 35: 613-620Crossref PubMed Scopus (87) Google Scholar). The mechanism by which GCS inhibitors block viral replication is not fully resolved. SLs play a significant role in endocytosis and thus may play a major role in virus penetration to the cell. Previous works showed that knocking out Ugcg impaired the entry of Influenza virus and thrombocytopenia syndrome virus by endocytosis (21Drews K. Calgi M.P. Harrison W.C. Drews C.M. Costa-Pinheiro P. Shaw J.J.P. Jobe K.A. Han J.D. Fox T.E. White J.M. Kester M. Glucosylceramide synthase maintains influenza virus entry and infection.PLoS One. 2020; 15e0228735Crossref PubMed Scopus (15) Google Scholar, 22Drake M.J. Brennan B. Briley Jr., K. Bart S.M. Sherman E. Szemiel A.M. Minutillo M. Bushman F.D. Bates P. A role for glycolipid biosynthesis in severe fever with thrombocytopenia syndrome virus entry.PLoS Pathog. 2017; 13e1006316Crossref PubMed Scopus (27) Google Scholar). This is consistent with our data showing interruption of early stages of SARS-CoV-2 replication. Whether the same mechanism of inhibition is relevant to SARS-CoV-2 needs to be elucidated. Moreover, whether the antiviral effect of GCS inhibitors is due to decreased levels of GlcCer and/or other GSLs or due to elevated levels of ceramide needs to be further explored. The antiviral effect of GCS inhibitors on viruses of three different families (i.e., Bunyaviridae, Orthomyxoviridae, and Coronaviridae) suggests a key role of the GSL synthesis pathway in viral infection. One advantage of targeting host proteins used by multiple viruses over targeting specific viral proteins is it being less prone to the development of resistance to the drug through mutations. Although side effects may be of particular concern for such treatments, another advantage of targeting host protein is the availability of many approved drugs against host proteins, allowing for drug repurposing. The main advantage of repurposing approved drugs is that they have already proven to be sufficiently safe, they have successfully passed clinical trials and regulatory scrutiny, and they have already undergone postmarketing surveillance (38Talevi A. Bellera C.L. Challenges and opportunities with drug repurposing: Finding strategies to find alternative uses of therapeutics.Expert Opin. Drug Discov. 2020; 15: 397-401Crossref PubMed Scopus (144) Google Scholar). Although the inhibitor NB-DNJ affects multiple host targets, specific inhibition of GCS is now possible using GCS inhibitors that are currently available. Eliglustat is an oral therapy approved in the European Union (2015) and the United States (2014) as a first-line treatment for adults with type 1 Gaucher disease who have compatible CYP2D6 metabolism phenotypes. Phase I studies in healthy volunteers revealed limited toxicity with an excellent pharmacodynamic response (37Shayman J.A. ELIGLUSTAT TARTRATE: Glucosylceramide synthase inhibitor treatment of type 1 Gaucher disease.Drugs Future. 2010; 35: 613-620Crossref PubMed Scopus (87) Google Scholar). A phase 3 trial in which patients received eliglustat daily (50 or 100 mg twice daily) for 9 months showed mild or moderate adverse events (39Mistry P.K. Lukina E. Ben Turkia H. Amato D. Baris H. Dasouki M. Ghosn M. Mehta A. Packman S. Pastores G. Petakov M. Assouline S. Balwani M. Danda S. Hadjiev E. et al.Effect of oral eliglustat on splenomegaly in patients with Gauc
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