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In Vitro Inhibition of SARS-CoV-2 Infection by Bromhexine hydrochloride

Martins et al., bioRxiv, doi:10.1101/2022.12.23.521817
Dec 2022  
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In Vitro study showing that bromhexine inhibits SARS-CoV-2 infection and replication in vitro by blocking the host cell protease TMPRSS2.
Bromhexine efficacy may vary depending on the degree of TMPRSS-dependent fusion for different variants1,2.
5 preclinical studies support the efficacy of bromhexine for COVID-19:
In Silico studies predict inhibition of SARS-CoV-2 with bromhexine or metabolites via binding to the spikeA,3, MproB,3, RNA-dependent RNA polymeraseC,3, and TMPRSS2D,4 proteins. In Vitro studies demonstrate inhibition of the TMPRSS2D,5 and acid sphingomyelinaseE,6 proteins. Bromhexine is a mucolytic agent that helps thin mucus secretions in the respiratory tract and has been shown to have antiviral properties against respiratory viruses. Bromhexine inhibits the expression of TMPRSS2 which plays an important role in SARS-CoV-2 cell entry and replication4,5,7 and bromhexine metabolite ambroxol inhibits SARS-CoV-2 via inhibition of acid sphingomyelinase in epithelial cells6.
a. The trimeric spike (S) protein is a glycoprotein that mediates viral entry by binding to the host ACE2 receptor, is critical for SARS-CoV-2's ability to infect host cells, and is a target of neutralizing antibodies. Inhibition of the spike protein prevents viral attachment, halting infection at the earliest stage.
b. The main protease or Mpro, also known as 3CLpro or nsp5, is a cysteine protease that cleaves viral polyproteins into functional units needed for replication. Inhibiting Mpro disrupts the SARS-CoV-2 lifecycle within the host cell, preventing the creation of new copies.
c. RNA-dependent RNA polymerase (RdRp), also called nsp12, is the core enzyme of the viral replicase-transcriptase complex that copies the positive-sense viral RNA genome into negative-sense templates for progeny RNA synthesis. Inhibiting RdRp blocks viral genome replication and transcription.
d. Transmembrane protease serine 2 (TMPRSS2) is a host cell protease that primes the spike protein, facilitating cellular entry. TMPRSS2 activity helps enable cleavage of the spike protein required for membrane fusion and virus entry. Inhibition may especially protect respiratory epithelial cells, buy may have physiological effects.
e. Acid sphingomyelinase (ASM) is a lysosomal enzyme that hydrolyzes sphingomyelin into ceramide and phosphorylcholine. ASM activity is upregulated by SARS-CoV-2 infection, leading to ceramide-enriched membrane domains that facilitate viral entry and replication. Inhibiting ASM may disrupt viral entry and reduce infection severity while potentially restoring membrane stability and immune homeostasis.
Martins et al., 26 Dec 2022, preprint, 12 authors. Contact: rb.mj@hotmail.com.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
This PaperBromhexineAll
In Vitro Inhibition of SARS-CoV-2 Infection by Bromhexine hydrochloride
Ronaldo Martins, Iasmin Ferreira, Daniel M M Jorge, Leticia Almeida, Juliano P Souza, Marjorie Pontelli, Italo A Castro, Thais M Lima, Rosa M M Viana, Dario Zamboni, Priscyla D Marcato, Eurico Arruda
doi:10.1101/2022.12.23.521817
The world is enduring the SARS CoV-2 pandemic, and although extensive research has been conducted on the issue, only a few antivirals have been approved to treat patients with COVID-19. Bromhexine hydrochloride was previously identified as a potent inhibitor of TMPRSS2, an essential protease for ACE-2 virus receptor interactions. In the present study, we investigated whether bromhexine treatment could reduce SARS CoV-2 replication in vitro. To evaluate bromhexine's effectiveness against SARS COV-2 infection, viral load was measured using Caco-2 cell lines expressing TMPRSS2. Our molecular docking results indicate that bromhexine displays an affinity with the active site of TMPRSS2. The drug significantly inhibited SARS CoV-2, both parental and P1 variant strains, infection in the Caco-2 cell line, reducing about 40% of SARS-CoV-2 entrance and about 90% of viral progeny in the supernatant 48h postinfection. Furthermore, bromhexine did not exhibit any direct virucidal activity on .
Almeida, Juliano P. Souza, Marjorie Pontelli, Italo A. Castro, Thais M. Lima, and Rosa M. M. Viana designed and executed the experimental SARS-CoV-2 infection. All authors contributed to writting the manuscript. Eurico Arruda, Priscyla D. Marcato, and RBM wrote the final version of the manuscript. All authors have approved the final manuscript. Declaration of Competing Interests The authors declare no conflict of interest.
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