Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus

Abstract

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 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 ± and ± PFU/ml were detected in and inhibition of virus In addition, the of GZ-161 and GZ-346 to inhibit infection was Vero E6 cells were infected a high = and viral was h after infection, an of viral F.M. de E.D. de E. L. Ferreira et from the first patients in and of a 2020; PubMed Scopus Google Scholar, N.S. Y. L. J. de M. M. I. replication in Vero E6 cells: and Virol. 2020; PubMed Scopus Google Scholar). GZ-161 and GZ-346 viral by and when SARS-CoV-2 were infected with a high (Fig. to a than the To further the antiviral activity and cell of GZ-161 and GZ-346 against SARS-CoV-2 infection, we their and (Fig. Vero E6 cells were infected with SARS-CoV-2 an of in the of of GZ-161 or GZ-346. after infection, the viral in the cell were by the by and cell was the cell assay The of GZ-161 = and GZ-346 = were a The of GZ-161 and GZ-346 were = and = respectively (Fig. of GZ-161 and GZ-346 for inhibition of SARS-CoV-2 infection. the inhibition of SARS-CoV-2 replication and of antivirals. Vero E6 cells were infected in with SARS-CoV-2 a of infection of in the of drug for 24 after which viral was through of SARS-CoV-2 RNA levels by was in but via the data are shown from The is the on an antiviral which virus replication is by in a The is the of an antiviral to of cells in the SARS-CoV-2, severe acute respiratory syndrome coronavirus To that inhibition of GCS reduces viral for was by The reduction in levels 24 h after (Fig. was to reduce viral to the medium by (Fig. The that inhibits SARS-CoV-2 virus, with that viral inhibition is with two GCS inhibitors with different increases the that the antiviral effect of the two is to GCS inhibition than an the of GCS inhibitors to reduce the effect of cells was Vero E6 cells were incubated with 10-μM or 1 h before infection with SARS-CoV-2, and cell was h after infection. with GZ-161 or with 1 h before infection, cell (Fig. these results that GZ-161 and GZ-346 have an antiviral effect on the SARS-CoV-2 clinical in with a to inhibit viral replication 24 to To which of SARS-CoV-2 infection cycle was by GCS a assay was shown in inhibition was when GCS inhibitors were 1 h before infection and was not when 1 h after infection (Fig. GCS inhibitors were after h after viral replication was by (Fig. The of the effect of the that activity be to inhibition of in the SARS-CoV-2 replication To further the which is being by GCS inhibitors in the life cycle of SARS-CoV-2, Vero E6 were infected with high of to infection. were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with SARS-CoV-2. after infection, SARS-CoV-2 levels were (Fig. GZ-161 and GZ-346 the levels of protein in infected Thus, data suggest that GCS inhibitors inhibit SARS-CoV-2 infection cycle after of the virus and before the of It was shown that cells a reduction in the replication of the respiratory RNA virus Influenza A virus, a of the K. Calgi M.P. Harrison W.C. Drews C.M. Costa-Pinheiro P. Shaw J.J.P. Jobe K.A. Han J.D. Fox White J.M. Kester M. Glucosylceramide synthase maintains influenza virus and One. 2020; PubMed Scopus Google Scholar). Thus, we further examined the antiviral activity of GCS inhibitors against Influenza A cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with influenza virus A/PR/8/34 The was harvested h after infection and analyzed by for the of viral RNA in the (Fig. Approximately reduction in viral RNA was in with GZ-161 or GZ-346 with the vehicle In to their to inhibit the of GCS inhibitors to reduce the of cells was further MDCK cells were incubated with 10-μM GZ-161 or GZ-346 1 h before infection with and to the was 24 h after infection as an for cell GZ-161 and GZ-346 by and respectively (Fig. In this study, we that the GCS inhibitors GZ-161 and GZ-346 inhibit viral replication of SARS-CoV-2. 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Full Text PDF PubMed Google In this is used as an inhibitor of to reduce of that to a in GlcCer E.B. Futerman A.H. forms of Gaucher PubMed Scopus Google Scholar). Thus, the antiviral activity of can also be by inhibition of the GSL synthetic be therapeutic targets for a of viral infection. the eliglustat is a ceramide analogue that inhibits of I and as was in in vitro and K.A. J. Komarnitsky S. Siegel C.S. E. J.A. Grabowski G.A. Cheng S.H. Copeland D.P. Marshall J. A specific and inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.Mol. 2007; PubMed Scopus Google Scholar, J.A. Glucosylceramide synthase inhibitor treatment of type 1 Gaucher PubMed Scopus Google Scholar). The by which GCS inhibitors block viral replication is not SLs a in and a major in virus to the that the of Influenza virus and syndrome virus by K. Calgi M.P. Harrison W.C. Drews C.M. Costa-Pinheiro P. Shaw J.J.P. Jobe K.A. Han J.D. Fox White J.M. Kester M. 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This is with data of of SARS-CoV-2 the of inhibition is to SARS-CoV-2 to be Moreover, whether the antiviral effect of GCS inhibitors is to levels of GlcCer other or to levels of ceramide to be further The antiviral effect of GCS inhibitors on of different and a key of the GSL synthesis in viral infection. of targeting host used by targeting specific viral is it being to the of to the drug through be of for such of targeting host protein is the of approved drugs against host for drug The of repurposing approved drugs is that have already to be have clinical trials and and have already A. and with drug to of Drug 2020; PubMed Scopus Google Scholar). the inhibitor host specific inhibition of GCS is GCS inhibitors that are currently is an therapy approved in the and the United States as a treatment for with type 1 Gaucher disease who have I in with an response J.A. Glucosylceramide synthase inhibitor treatment of type 1 Gaucher PubMed Scopus Google Scholar). A phase 3 trial in which patients eliglustat or for or E. H. D. H. M. M. A. S. G. M. S. M. S. E. et of eliglustat on in patients with Gaucher disease type The clinical 2015; PubMed Scopus Google Scholar, N. M. C. P. D. E. P. M. C. J. T.M. S. T.M. and for the use of eliglustat in with type 1 Gaucher disease in J. Med. 2017; Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We have shown that United States Food and Drug Administration-approved inhibitors specific for GCS have an antiviral activity against both SARS-CoV-2 and Influenza We suggest that the of these drugs in as as against other can be and novel treatments for viral infection. Vero E6 and MDCK cells were by Mandelboim were in medium with and under a SARS-CoV-2 was by of were by infection of Vero E6 cells for 2 a in which were and by before being for storage of the was by assay on Vero E6 cell Influenza virus was by Mandelboim The GZ-161 and GZ-346 acid[2-(2′,3′-dihydro-benzo [1,4] dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide-l-tartaric acid were from The were as and solutions in or in The was in a for and RNA was by the viral RNA as the RNA in the was by was with and analyzed with the The was from the from virus and for the of SARS-CoV-2 were from and for the of Vero E6 cells were a of cells in cells were in with GZ-161 or GZ-346. were infected 1 h with SARS-CoV-2 or as The was 24 h after infection or h after infection for and for Vero E6 cells were a of cells in cell were infected with of and to of virus as control and incubated for h the of GCS inhibitors was by the of with was h after infection by the to The of inhibition was by the of the and cells from involving SARS-CoV-2 were in a in with the for Vero E6 cells were a of in a and to in the of cells were with of or The was to the in a of medium were to for 1 and were analyzed for or infected with SARS-CoV-2. cells were used as was by to were with and in with a inhibitor were for to and for to were on and a membrane 2 The membrane was with in and incubated with a the membrane was incubated with for 1 h The membrane was with and developed system and by for MDCK cells were a of cells in cells were in with GZ-161 or GZ-346. cells were infected in a medium with Supernatants were h after infection for was 24 h after infection by to The of inhibition was by the of and cells from were with a test or test by as indicated in the are indicated by in the as and with a of or were The of is in the for all are as the ± were data for the are the other data are from the are in United States synthase inhibitors for and treatment of viral The that have of with the of this We for and from for GZ-161 and for the and Mandelboim for cells and Influenza virus E. B. the E. B. H. R. B. L. H. Y. Y. N. S. N. and T. the E. B. the results and on the before R. A. is by the E. B. is by the GZ-161 and GZ-346 from through an

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