Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations

Timmins, Graham S.; Bradfute, Steven B.; Deretic, Vojo; Kumar, Suresh; Clarke, Elizabeth C.; Ye, Chunyan

doi:10.1101/2020.08.11.245100

Cited by 11 publications

(10 citation statements)

References 24 publications

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“…Another recent study by Olaleye et al (21) specifically analyzed the effects of Bromhexine and Ambroxol on the interaction of ACE2 with the SARS-CoV-2 spike receptor binding domain (RBD), and reported a very peculiar behavior of these substances which in part may explain the paradoxic results of our fusion assays and would support a beneficial effect of low-dose Bromhexine, which is converted to Ambroxol in vivo : While Ambroxol weakly inhibited the ACE2-RBD interaction up to 100μM concentration, Bromhexine exhibited a biphasic behavior and was weakly inhibitory below 10μM but increased ACE2-RBD binding at higher concentrations in that study. Both substances were reported to weakly inhibit SARS-CoV-2-mediated cytopathic effect in culture (22), and Ambroxol was also shown to moderately impact replication of SARS-CoV-2 in that report (22), albeit on Vero cells and not lung cells. Our results suggest that Ambroxol can weakly inhibit spike-driven entry of lentiviral pseudotypes into Calu-3 cells at high but potentially attainable concentrations (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…Ambroxol may also exhibit weak but broad anti-viral activity as it was shown to reduce the occurrence of respiratory infections (17) and to inhibit proteolytic activation of influenza virus by triggering release of antiviral factors (18), and it is used to treat acute respiratory distress syndrome in adults and antenatally in infants (19, 20). Further, two recent preprints, one describing modulation of the ACE2-SARS2-S interaction by both Bromhexine and Ambroxol (21) and the other reporting weak inhibitory activity of Ambroxol against SARS-CoV-2 replication (22) in Vero E6 cells, point at a potential utility of these molecules in the therapy of COVID-19.…”

Section: Introductionmentioning

confidence: 99%

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SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

Hörnich

Großkopf

Schlagowski

et al. 2020

Preprint

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The SARS-Coronavirus-2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS-CoV-2-S) with the ACE2 receptor and activation by proteases, in particular TMPRSS2. Viruses can also spread through fusion of infected with uninfected cells. We therefore analyzed cell-cell fusion activity of SARS-CoV-2-S with regard to the requirements for ACE2 expression, proteolytic activation, and sensitivity to inhibitors and compared it to SARS-CoV-S. We compared S-protein-driven fusion with target cells recombinantly overexpressing ACE2, TMPRSS2, or both. SARS-CoV-2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while the reverse observation was made for SARS-CoV-S. TMPRSS2-mediated effects were inhibited by the serine protease inhibitor Camostat. Effector-target-cell fusion by SARS-CoV-2-S was only affected by Camostat when receptor expression was limiting or when the S1/S2 cleavage site was mutated. Mutational ablation of the SARS-CoV-2-S S2’ cleavage site abrogated any effects of TMPRSS2 on fusion. Mutation of the SARS-CoV-2-S S1/S2 cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting. When both factors were abundant, initial target-effector-cell fusion was unaltered, but syncytia remained smaller over time. Overall, its polybasic cleavage site renders SARS-CoV-2-S-mediated cell-cell fusion less dependent on TMPRSS2 activity on target cells. Unexpectedly, we observed enhancement of SARS-CoV-2-S-mediated fusion by Bromhexine, another TMPRSS2 inhibitor. This effect required intact proteolytic cleavage sites, suggesting interference of Bromhexine with proteolytic priming, but not in a therapeutically desired way. Infection with SARS-CoV-2-S-pseudotyped particles clearly differed in the requirements for proteolytic activation from cell-cell fusion. TMPRSS2 strongly enhanced infection, which was reversed by Camostat but not by Bromhexine.IMPORTANCECell-cell fusion allows the virus to infect additional target cells without the need to produce free virus. Fusion likely also contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2’ cleavage site is essential for activation by TMPRSS2 in trans and unravel important differences between SARS-CoV and SARS-CoV-2. Bromhexine, an inhibitor of the TMPRSS2 protease, is currently tested in clinical trials against COVID-19. Our results indicate that Bromhexine does not inhibit SARS-CoV-2-S-mediated particle entry and enhances fusion. We therefore caution against overly optimistic use of Bromhexine in higher dosage in clinical trials or as a therapy, at least until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similar to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on SARS-CoV-2-S-mediated fusion and may therefore currently represent a better choice in therapeutic regimens for COVID-19.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

Hörnich

Großkopf

Schlagowski

et al. 2020

Preprint

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“…Our study is supported by two pre-published studies. A study by Bradfute et al demonstrated that ambroxol blocks Vero-E6 cells infection with SARS-CoV-2 ( 38 ). A study by Olaleye et al revealed that micromolar concentrations of ambroxol prevented cellular infections with SARS-CoV-2 determined as cytopathic effect of the virus ( 39 ).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells

Carpinteiro

Gripp

Hoffmann

et al. 2021

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…et. al 49 ., in which they reported that AMB could inhibit SARS-CoV-2 infection in vitro at high concentrations 49 . Our biochemical findings are most complementary to the report by Hornich, B.F. et.…”

Section: Discussionmentioning

confidence: 97%

“…The IC 50 values that we observed for the reference compounds are consistent with earlier reports [44][45][46][47] . While writing this manuscript, we found that other cellular studies tested BHH and AMB at certain or single concentrations [48][49][50] ; however, this is the first study to our knowledge to conduct the IC 50 evaluation for BHH and AMB against the novel SARS-CoV-2 infection induced CPE.…”

Section: Sars-cov-2 Infection Induced Cytopathic Effect (Cpe) In Veromentioning

confidence: 99%

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein’s Receptor Binding Domain and Recombinant Human ACE2

Kaur

Onyenaka

2020

Preprint

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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein of the novel coronavirus and the host receptor, angiotensin-converting enzyme 2 (ACE2). To date, SARS-CoV-2 has infected up to 26 million people worldwide; yet, there is no clinically approved drug or vaccine available. Therefore, a rapid and coordinated effort to re-purpose clinically approved drugs that prevent or disrupt these critical entry pathways of SARS-CoV-2 spike glycoprotein interaction with human ACE2, could potentially accelerate the identification and clinical advancement of prophylactic and/or treatment options against COVID-19, thus providing possible countermeasures against viral entry, pathogenesis and survival. Herein, we discovered that Ambroxol hydrochloride (AMB), and its progenitor, Bromhexine hydrochloride (BHH), both clinically approved drugs are potent effective modulators of the key interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and human ACE2. We also found that both compounds inhibited SARS-CoV-2 infection-induced cytopathic effect at micromolar concentrations. Therefore, in addition to the known TMPRSS2 activity of BHH; we report for the first time that the BHH and AMB pharmacophore has the capacity to target and modulate yet another key protein-protein interaction essential for the two known SARS-CoV-2 entry pathways into host cells. Altogether, the potent efficacy, excellent safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.

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Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations

Cited by 11 publications

References 24 publications

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein’s Receptor Binding Domain and Recombinant Human ACE2

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