Title: Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells
Abstract: The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019. The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019. Infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic, a massive global health problem. Many infected patients exhibit mild symptoms, but a substantial number of patients, in particular older patients and those with additional risk factors, develop severe disease (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13520) Google Scholar). Severe COVID-19 requires intensive care and ventilator treatment and is associated with a high mortality rate (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13520) Google Scholar). Age, high blood pressure, or overweight are risk factors for severe COVID-19, but even healthy and young individuals develop severe disease (2Serafim R.B. Povoa P. Souza-Dantas V. Andre C. Kalil A.C. Salluh J.I.F. Clinical course and outcomes of critically ill patients with COVID-19 infection: A systematic review.Clin. Microbiol. Infect. 2021; 27: 47-54Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). It is therefore of outstanding interest to develop robust prophylaxis and treatment options. The SARS-CoV-2 spike protein is incorporated into the viral envelope and mediates viral entry into target cells. For this, the surface unit S1 of SARS-CoV-2 spike binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) (3Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. 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Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (84) Google Scholar, 4Lan J. Ge J. Yu J. Shan S. Zhou H. Fan S. Zhang Q. Shi X. Wang Q. Zhang L. Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581: 215-220Crossref PubMed Scopus (3631) Google Scholar, 5Wang Q. Zhang Y. Wu L. Niu S. Song C. Zhang Z. Lu G. Qiao C. Hu Y. Yuen K.Y. Wang Q. Zhou H. Yan J. Qi J. Structural and functional basis of SARS-CoV-2 entry by using human ACE2.Cell. 2020; 181: 894-904Abstract Full Text Full Text PDF PubMed Scopus (1885) Google Scholar). However, the role of membrane lipids for viral entry requires definition. Sphingolipids are not only structural components of cellular membranes and determine their biophysical membrane properties, but they are also involved in cellular signaling transduction, regulation of proliferation and differentiation, apoptosis, membrane trafficking, and the organization of proteins within membranes (6Kolesnick R.N. Goñi F.M. Alonso A. Compartmentalization of ceramide signaling: Physical foundations and biological effects.J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (383) Google Scholar, 7Gulbins E. Kolesnick R. Raft ceramide in molecular medicine.Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (361) Google Scholar, 8Smith E.L. Schuchman E.H. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases.FASEB J. 2008; 22: 3419-3431Crossref PubMed Scopus (181) Google Scholar, 9Nurminen T.A. Holopainen J.M. Zhao H. Paavo K. Kinnunen J. 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Lipid rafts and redox signaling.Antioxid. Redox Signal. 2007; 9: 1411-1415Crossref PubMed Scopus (25) Google Scholar, 12Grassmé H. Jendrossek V. Riehle A. von Kürthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (471) Google Scholar, 13Grassmé H. Henry B. Ziobro R. Becker K.A. Riethmüller J. Gardner A. Seitz A.P. Steinmann J. Lang S. Ward C. Schuchman E.H. Caldwell C.C. Kamler M. Edwards M.J. Brodlie M. et al.β1-Integrin accumulates in cystic fibrosis luminal airway epithelial membranes and decreases sphingosine, promoting bacterial infections.Cell Host Microbe. 2017; 21: 707-718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The very hydrophobic ceramide molecules associate with each other to spontaneously form distinct membrane domains. These ceramide-enriched membrane domains fuse into large highly hydrophobic, tightly packed, and gel-like ceramide-enriched membrane platforms (10Grassmé H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. CD95 signaling via ceramide-rich membrane rafts.J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 11Li P.L. Gulbins E. Lipid rafts and redox signaling.Antioxid. Redox Signal. 2007; 9: 1411-1415Crossref PubMed Scopus (25) Google Scholar). These platforms serve to cluster receptor molecules and to organize, trap, and concentrate specific signaling molecules (10Grassmé H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. CD95 signaling via ceramide-rich membrane rafts.J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 11Li P.L. Gulbins E. Lipid rafts and redox signaling.Antioxid. Redox Signal. 2007; 9: 1411-1415Crossref PubMed Scopus (25) Google Scholar, 12Grassmé H. Jendrossek V. Riehle A. von Kürthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (471) Google Scholar, 13Grassmé H. Henry B. Ziobro R. Becker K.A. Riethmüller J. Gardner A. Seitz A.P. Steinmann J. Lang S. Ward C. Schuchman E.H. Caldwell C.C. Kamler M. Edwards M.J. Brodlie M. et al.β1-Integrin accumulates in cystic fibrosis luminal airway epithelial membranes and decreases sphingosine, promoting bacterial infections.Cell Host Microbe. 2017; 21: 707-718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We have previously shown an important role of the acid sphingomyelinase/ceramide system for SARS-CoV-2 infections (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We demonstrated that infection of cultured epithelial cells or freshly isolated human nasal epithelial cells with SARS-CoV-2 or vesicular stomatitis virus (VSV) pseudoviral (pp-VSV-SARS-CoV-2) spike particles, respectively, resulted in activation of the acid sphingomyelinase and release of ceramide (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Genetic or pharmacological inhibition of the acid sphingomyelinase as well as consumption or neutralization of ceramide on the cell surface prevented infection of epithelial cells with SARS-CoV-2 (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Several drugs functionally inhibit the acid sphingomyelinase (15Hurwitz R. Ferlinz K. Sandhoff K. The tricyclic antidepressant desipramine causes proteolytic degradation of lysosomal sphingomyelinase in human fibroblasts.Biol. Chem. Hoppe Seyler. 1994; 375: 447-450Crossref PubMed Scopus (219) Google Scholar, 16Kölzer M. Werth N. Sandhoff K. Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine.FEBS Lett. 2004; 559: 96-98Crossref PubMed Scopus (163) Google Scholar, 17Kornhuber J. Tripal P. Reichel M. Terfloth L. Bleich S. Wiltfang J. Gulbins E. Identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model.J. Med. Chem. 2008; 51: 219-237Crossref PubMed Scopus (175) Google Scholar, 18Gulbins E. Palmada M. Reichel M. Lüth A. Böhmer C. Amato D. Müller C.P. Tischbirek C.H. Groemer T.W. Tabatabai G. Becker K.A. Tripal P. Staedtler S. Ackermann T.F. v. Brederode J. et al.Acid sphingomyelinase/ceramide system mediates effects of antidepressant drugs.Nat. Med. 2013; 19: 934-938Crossref PubMed Scopus (270) Google Scholar, 19Gulbins A. Schumacher F. Becker K.A. Wilker B. Soddemann M. Boldrin F. Müller C.P. Edwards M.J. Goodman M. Caldwell C.C. Kleuser B. Kornhuber J. Szabo I. Gulbins E. Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide.Mol. Psychiatry. 2018; 23: 2324-2346Crossref PubMed Scopus (132) Google Scholar, 20Breiden B. Sandhoff K. Emerging mechanisms of drug-induced phospholipidosis.Biol. Chem. 2019; 401: 31-46Crossref PubMed Scopus (66) Google Scholar, 21Kornhuber J. Muehlbacher M. Trapp S. Pechmann S. Friedl A. Reichel M. Mühle C. Terfloth L. Groemer T.W. Spitzer G.M. Liedl K.R. Gulbins E. Tripal P. Identification of novel functional inhibitors of acid sphingomyelinase.PLoS One. 2011; 6e23852Crossref PubMed Scopus (128) Google Scholar). Structural requirements for inhibition are a lipophilic organic ring that integrates into lysosomal membranes, a short spacer, and a charged tertiary amine group that displaces the acid sphingomyelinase from the lysosomal membranes, thereby releasing the enzyme into the lysosomal lumen and causing its partial degradation. Many antidepressants are functional inhibitors of the acid sphingomyelinase (FIASMAs) (15Hurwitz R. Ferlinz K. Sandhoff K. The tricyclic antidepressant desipramine causes proteolytic degradation of lysosomal sphingomyelinase in human fibroblasts.Biol. Chem. Hoppe Seyler. 1994; 375: 447-450Crossref PubMed Scopus (219) Google Scholar, 16Kölzer M. Werth N. Sandhoff K. Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine.FEBS Lett. 2004; 559: 96-98Crossref PubMed Scopus (163) Google Scholar, 17Kornhuber J. Tripal P. Reichel M. Terfloth L. Bleich S. Wiltfang J. Gulbins E. Identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model.J. Med. Chem. 2008; 51: 219-237Crossref PubMed Scopus (175) Google Scholar, 18Gulbins E. Palmada M. Reichel M. Lüth A. Böhmer C. Amato D. Müller C.P. Tischbirek C.H. Groemer T.W. Tabatabai G. Becker K.A. Tripal P. Staedtler S. Ackermann T.F. v. Brederode J. et al.Acid sphingomyelinase/ceramide system mediates effects of antidepressant drugs.Nat. Med. 2013; 19: 934-938Crossref PubMed Scopus (270) Google Scholar, 19Gulbins A. Schumacher F. Becker K.A. Wilker B. Soddemann M. Boldrin F. Müller C.P. Edwards M.J. Goodman M. Caldwell C.C. Kleuser B. Kornhuber J. Szabo I. Gulbins E. Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide.Mol. Psychiatry. 2018; 23: 2324-2346Crossref PubMed Scopus (132) Google Scholar, 20Breiden B. Sandhoff K. Emerging mechanisms of drug-induced phospholipidosis.Biol. Chem. 2019; 401: 31-46Crossref PubMed Scopus (66) Google Scholar, 21Kornhuber J. Muehlbacher M. Trapp S. Pechmann S. Friedl A. Reichel M. Mühle C. Terfloth L. Groemer T.W. Spitzer G.M. Liedl K.R. Gulbins E. Tripal P. Identification of novel functional inhibitors of acid sphingomyelinase.PLoS One. 2011; 6e23852Crossref PubMed Scopus (128) Google Scholar). We have proposed the acronym FIASMA (Functional Inhibitor of Acid SphingoMyelinAse) for a compound from this large group of drugs with these properties (21Kornhuber J. Muehlbacher M. Trapp S. Pechmann S. Friedl A. Reichel M. Mühle C. Terfloth L. Groemer T.W. Spitzer G.M. Liedl K.R. Gulbins E. Tripal P. Identification of novel functional inhibitors of acid sphingomyelinase.PLoS One. 2011; 6e23852Crossref PubMed Scopus (128) Google Scholar). Here, we tested whether ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a drug that fits with the aforementioned structural requirements, inhibits acid sphingomyelinase and can thus be repurposed to inhibit SARS-CoV-2 infection. Ambroxol contains a lipophilic organic ring system that is connected to a tertiary amine via a short spacer and, therefore, is a potential functional inhibitor of the acid sphingomyelinase. Ambroxol is a mucolytic drug that is used for the treatment of diseases of the upper and lower respiratory tract. It has almost no side effects. As an infection system, we used replication-deficient VSV pseudoviral particles (pp-VSV) presenting SARS-CoV-2 spike protein on their surface, abbreviated pp-VSV-SARS-CoV-2 spike (22Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (12460) Google Scholar). Several previous studies showed that these particles accurately reflect key aspects of the entry of coronavirus into host cells (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 22Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (12460) Google Scholar). We demonstrate that entry of pp-VSV-SARS-CoV-2 spike into cultured epithelial cells or freshly isolated nasal epithelial cells results in activation of the acid sphingomyelinase and a release of ceramide. These events were blocked by pretreatment with low doses of ambroxol. In accordance, ambroxol prevented cellular entry of pp-VSV-SARS-CoV-2 spike. More importantly, we obtained nasal epithelial cells from volunteers prior and after inhalation with ambroxol and infected the cells with pp-VSV-SARS-CoV-2 spike. These in vivo/ex vivo experiments demonstrated that inhalation of ambroxol is sufficient to reduce acid sphingomyelinase activity in nasal epithelial cells in vivo and to prevent infection with pp-VSV-SARS-CoV-2 spike ex vivo. Addition of purified acid sphingomyelinase or C16 ceramide restored infection of ambroxol-treated nasal epithelial cells with pp-VSV-SARS-CoV-2 spike. To test whether ambroxol inhibits the acid sphingomyelinase, we incubated Vero-E6 epithelial cells with increasing doses of ambroxol and determined the activity of the acid sphingomyelinase in cell lysates. Ambroxol induced a dose-dependent reduction of the activity of the acid sphingomyelinase in Vero-E6 cells (Fig. 1A). Ambroxol did not show toxicity until 50 μM concentration, whereas higher concentrations such as 75 μM started to show some toxicity as evidenced by flow cytometry studies of untreated and ambroxol-treated cells stained with FITC–annexin V (Roche; Fig. 1B). Next, we investigated whether pp-VSV-SARS-CoV-2 spike induces an activation of the acid sphingomyelinase in Vero-E6 cells and whether this increase is prevented by pretreatment of the cells with ambroxol. Infection of the cells with pp-VSV-SARS-CoV-2 spike resulted in a rapid activation of the acid sphingomyelinase that peaked 30 min after infection (Fig. 2A), consistent with previously reported data (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The increase of acid sphingomyelinase activity upon infection with pp-VSV-SARS-CoV-2 spike was prevented by preincubation with ambroxol (Fig. 2A). Ambroxol had no effect on acid ceramidase activity (not shown). We have previously shown that incubation of epithelial cells with pp-VSV-SARS-CoV-2 spike triggers a release of ceramide that is essential for cellular infection with the virus (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Here, we confirm these data and show that infection of Vero-E6 cells with pp-VSV-SARS-CoV-2 spike results in a release of C16/C18 ceramide as well as C22/C24 ceramide (Fig. 2B). The release of ceramide upon infection is prevented by pretreatment with ambroxol (Fig. 2B). We observed a complete inhibition of infection-induced ceramide formation at 25 μM, but already concentrations of 15 to 20 μM ambroxol reduced or almost abrogated ceramide formation upon infection (Fig. 2B). Ambroxol did not change basal ceramide concentrations of the cells. We have previously shown that ceramide molecules form large ceramide-enriched membrane domains that serve to trap and cluster receptor molecules, thereby facilitating signaling via these receptors (10Grassmé H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. CD95 signaling via ceramide-rich membrane rafts.J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). Here, we demonstrate that infection of Vero-E6 cells with pp-VSV-SARS-CoV-2 spike results in the formation of ceramide-enriched membrane domains that cluster ACE2 (Fig. 3, A and B), suggesting that they serve as platforms to allow infection of the cells. We have previously shown that sphingosine also inhibits infection of human cells with SARS-CoV-2 (23Edwards M.J. Becker K.A. Gripp B. Hoffmann M. Keitsch S. Wilker B. Soddemann M. Gulbins A. Carpinteiro E. Patel S.H. Wilson G.C. Pöhlmann S. Walter S. Fassbender K. Ahmad S.A. et al.Sphingosine prevents binding of SARS-CoV-2 spike to its cellular receptor ACE2.J. Biol. Chem. 2020; 295: 15174-15182Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We therefore tested whether ambroxol alters cellular sphingosine concentrations. The results demonstrate that even high concentrations of ambroxol did not change cellular sphingosine (not shown). The blockade of pp-VSV-SARS-CoV-2 spike–dependent activation of acid sphingomyelinase activation and ceramide release by ambroxol promoted us to investigate whether ambroxol also inhibits entry of pp-VSV-SARS-CoV-2 spike. Vero-E6 cells were pretreated with ambroxol for 60 min, washed, and then infected with pp-VSV-SARS-CoV-2 spike for 60 min. The results indicate that 10 μM or 15 to 20 μM ambroxol reduced viral entry by approximately 50% or 80 to 90%, respectively, and 25 μM ambroxol completely blocked entry (Fig. 4, A and B). Infection with higher concentrations of VSV-SARS-CoV-2 spike was also blocked by ambroxol (Fig. 4B). Addition of purified acid sphingomyelinase that generates endogenous ceramide or 10 μM exogenous C16 ceramide restored viral entry (Fig. 4A) indicating that ambroxol acted via inhibition of the acid sphingomyelinase and the release of ceramide. In contrast, addition of sphingomyelin did not alter infection with pp-SARS-CoV-2 (Fig. 4B). Next, we neutralized ceramide with two different anti-ceramide antibodies or by incubation with ceramidase and show that these treatments also prevent viral uptake (Fig. 4B). Treatment of Caco-2 cells with ambroxol or genetic downregulation of the acid sphingomyelinase in Caco-2 cells also resulted in an inhibition of pp-VSV-SARS-CoV-2 uptake, which was restored by C16 ceramide (Fig. 4C). Addition of ambroxol to C16 ceramide–treated acid sphingomyelinase downregulated cells did not further affect viral uptake supporting the notion that ambroxol specifically acts by targeting the acid sphingomyelinase. Controls with pp-VSV-G, which does not activate the acid sphingomyelinase/ceramide system and does not employ this system for cellular infection as previously shown (14Carpinteiro A. Edwards M.J. Hoffmann M. Kochs G. Gripp B. Weigang S. Adams C. Carpinteiro E. Gulbins A. Keitsch S. Sehl C. Soddemann M. Wilker B. Kamler M. Bertsch T. et al.Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells.Cell Rep. Med. 2020; 1: 100142Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), demonstrated that pp-VSV-G entry was not modulated by ambroxol (Fig. 4A), indicating that ambroxol specifically blocks entry of pp-VSV-SARS-CoV-2 spike by inhibition of the acid sphingomyelinase/ceramide system. We next examined whether our findings made with a cell line are recapitulated with primary human respiratory epithelial cells. To this end, we isolated nasal epithelial cells from volunteers, treated them with 25 μM ambroxol for 60 min, or left the cells untreated and determined the activity of the acid sphingomyelinase and infection of the cells. Ambroxol reduced the activity of the acid sphingomyelinase in uninfected cells by approximately 50% and prevented the stimulation of the acid sphingomyelinase upon exposure to pp-VSV-SARS-CoV-2 (Fig. 5A). Ambroxol blocked the entry of pp-VSV-SARS-CoV-2 spike into freshly isolated nasal epithelial cells (Fig. 5B) and addition of purified acid sphingomyelinase, or 10 μM C16 ceramide restored entry of the viral particles into ambroxol-treated cells (Fig. 5B). To simulate the effects of ambroxol on infection as closely as possible to treatment of patients, we isolated nasal epithelial cells from volunteers. The volunteers then inhaled 2 ml ambroxol (7.5 mg/ml), and nasal epithelial cells were again isolated from the opposite nasal cavity 60 min after inhalation and inoculated with pp-VSV-SARS-CoV-2 spike ex vivo for 60 min. Cells were washed, and infectious entry was determined after incubation for 24 h. In addition, we determined the activity of the acid sphingomyelinase in nasal epithelial cells prior and after inhalation of ambroxol. These studies revealed that inhalation of ambroxol in vivo markedly reduced the activity of the acid sphingomyelinase in nasal epithelial cells (Fig. 6A). Infection with pp-VSV-SARS-CoV-2 for 30 min resulted in an activation of the acid sphingomyelinase (Fig. 6A) and a release of ceramide (Fig. 6B), events that were blocked by a previous ambroxol inhalation. Infection also resulted in clustering of ACE2