key: cord-0719923-hqslodlu authors: Carpinteiro, Alexander; Gripp, Barbara; Hoffmann, Markus; Pöhlmann, Stefan; Hoertel, Nicolas; Edwards, Michael J.; Kamler, Markus; Kornhuber, Johannes; Becker, Katrin Anne; Gulbins, Erich title: Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells date: 2021-04-23 journal: J Biol Chem DOI: 10.1016/j.jbc.2021.100701 sha: 13aceb756f8cb08db9cd3c3c78b33d11625975e0 doc_id: 719923 cord_uid: hqslodlu The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance rhinovirus or 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, i.e. 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 spike protein pseudotyped viral particles (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 [(14)C]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 COVID-19. platforms (10, 11) . These platforms serve to cluster receptor molecules and to organize, trap and concentrate specific signaling molecules (10) (11) (12) (13) . We have previously shown an important role of the acid sphingomyelinase/ceramide system for SARS-CoV-2 infections (14) . We CoV-2 (14) . Several drugs functionally inhibit the acid sphingomyelinase (15) (16) (17) (18) (19) (20) (21) . 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 (15) (16) (17) (18) (19) (20) (21) . We have proposed the acronym FIASMA (Functional Inhibitor of Acid SphingoMyelinAse) for a compound from this large group of drugs with these properties (21) . Here, we tested whether ambroxol, i.e. Several previous studies showed that these particles accurately reflect key aspects of the entry of coronavirus into host cells (14, 22) . We demonstrate that entry of pp-VSV- spike was prevented by pre-incubation 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 (14) . Here, we confirm these data and show . Pseudotyped viral particles were generated according to a previously published protocol (40) . The authors declare no competing financial interests. 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