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

Abstract

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.

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

“…More likely, favorable patient outcomes are attributable to the beneficial effects of Bromhexine or its main metabolite Ambroxol on lung function, general defense mechanisms against airway infections, and inflammatory response (16–19, 50). 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.…”

“…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.…”

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

“…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 ). The authors further demonstrated using recombinant proteins of the receptor binding domain of spike and ACE2 that ambroxol interferes with the binding of SARS-CoV-2 spike with human ACE2 in vitro ( 39 ).…”

“…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 ). The authors further demonstrated using recombinant proteins of the receptor binding domain of spike and ACE2 that ambroxol interferes with the binding of SARS-CoV-2 spike with human ACE2 in vitro ( 39 ). Thus, ambroxol may prevent infections with SARS-CoV-2 at different levels, i.e.…”

Results of Open-Label non-Randomized Comparative Clinical Trial: “BromhexIne and Spironolactone for CoronаvirUs Infection requiring hospiTalization (BISCUIT)

“…[28,21]. А свежие экспериментальные исследования подтвердили способность бромгексина блокировать активность и основного клеточного рецептора АПФ-2 [29,22]. Более того, только что завершившееся контролируемое исследование с бромгексином на 78 пациентах продемонстрировало статистически значимое снижение перевода больных в отделение реанимации и интенсивной терапии (ОРИТ) (2 / 39 против 11 / 39, р=0,006), необходимость ИВЛ (1 / 39 против 9 / 39, р=0,007) и снижение смертности (0 против 5, р=0,027) [30,23].…”