Journal of Biological Chemistry
Volume 296, January–June 2021, 100701
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Research Article
Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells

https://doi.org/10.1016/j.jbc.2021.100701Get rights and content
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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.

Keywords

ambroxol
SARS-CoV-2
acid sphingomyelinase
ceramide
infection
human nasal epithelial cells

Abbreviations

ACE2
angiotensin-converting enzyme 2
ATCC
American Type Culture Collection
COVID-19
coronavirus disease 2019
DMEM
Dulbecco's modified Eagle's medium
DTPA
diethylenetriaminepentaacetic acid
eGFP
enhanced GFP
FCS
fetal calf serum
FIASMAs
functional inhibitors of the acid sphingomyelinase
H/S
Hepes/saline
MEM
minimum essential medium
NP-40
Nonidet P40
OGP
n-octyl-d-glucopyranoside
PFA
paraformaldehyde
pp-VSV-SARS-CoV-2
vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface
SARS-CoV-2
severe acute respiratory syndrome coronavirus 2
TMPRSS2
transmembrane serine protease 2

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