tetano
Editor, Senior Moderator
Sci Rep
. 2025 Mar 27;15(1):10606.
doi: 10.1038/s41598-025-94283-9. In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium
Juliana Ângelo de Souza[SUP] 1 [/SUP], Isabela de Souza Gomes[SUP] 2 [/SUP], Luciana de Souza Fernandes[SUP] 3 [/SUP], Luis Adan Flores Andrade[SUP] 4 [/SUP], Luciana Ângelo de Souza[SUP] 5 [/SUP], Vinícius de Almeida Paiva[SUP] 2 [/SUP], Sheila Cruz Araujo[SUP] 6 7 [/SUP], Leonardo Henrique Franca de Lima[SUP] 7 [/SUP], Roberto Sousa Dias[SUP] 3 [/SUP], Raquel Cardoso de Melo-Minardi[SUP] 8 [/SUP], Flávio Guimarães da Fonseca[SUP] 4 [/SUP], Sérgio Oliveira de Paula[SUP] 9 [/SUP], Sabrina de Azevedo Silveira[SUP] 2 10 [/SUP]
Affiliations
The 2019 pandemic of coronavirus disease (COVID-19) caused by SARS-CoV-2 led to millions of deaths worldwide since its emergence. The viral genomic material can code structural and non-structural proteins including the main protease or 3CL[SUP]pro[/SUP], a cysteine protease that cleavages the viral polyprotein generating 11 proteins that participate in viral pre-replication. Thus, 3CL[SUP]pro[/SUP] is a promising therapeutic target for SARS-CoV-2 inhibition by new drugs or drug repositioning because 3CL[SUP]pro[/SUP] is dissimilar to human proteases. We conducted in vitro assays demonstrating the modulation activity of ambenonium, a drug already used in Myasthenia gravis that acts by inhibiting the action of acetylcholinesterase, and had its potential inhibitory activity against viral replication pointed out in a previous in silico study. In concentrations of 100 µM, 50 µM, 25 µM, 10 µM, and 1 µM there was no inhibition in the formation of lysis plates, with a slight increase in the genome copy number at the higher concentrations evaluated. However, in the concentrations of 0,1 µM and 0,01 µM, there was a reduction in the number of lysis plates. This behavior suggests that the ambenonium acts as a modulator of viral activity in vitro. To investigate potential conformational changes in the protein between dimeric and monomeric forms in the presence of the compound, a local docking analysis was performed. Results indicated this conformational shift is possible, though further studies are needed to confirm these findings.
Keywords: Ambenonium; Drug repurposing; In vitro assay; Main protease; Modulation activity; Pandemic; SARS-CoV-2.
. 2025 Mar 27;15(1):10606.
doi: 10.1038/s41598-025-94283-9. In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium
Juliana Ângelo de Souza[SUP] 1 [/SUP], Isabela de Souza Gomes[SUP] 2 [/SUP], Luciana de Souza Fernandes[SUP] 3 [/SUP], Luis Adan Flores Andrade[SUP] 4 [/SUP], Luciana Ângelo de Souza[SUP] 5 [/SUP], Vinícius de Almeida Paiva[SUP] 2 [/SUP], Sheila Cruz Araujo[SUP] 6 7 [/SUP], Leonardo Henrique Franca de Lima[SUP] 7 [/SUP], Roberto Sousa Dias[SUP] 3 [/SUP], Raquel Cardoso de Melo-Minardi[SUP] 8 [/SUP], Flávio Guimarães da Fonseca[SUP] 4 [/SUP], Sérgio Oliveira de Paula[SUP] 9 [/SUP], Sabrina de Azevedo Silveira[SUP] 2 10 [/SUP]
Affiliations
- PMID: 40148508
- PMCID: PMC11950299
- DOI: 10.1038/s41598-025-94283-9
The 2019 pandemic of coronavirus disease (COVID-19) caused by SARS-CoV-2 led to millions of deaths worldwide since its emergence. The viral genomic material can code structural and non-structural proteins including the main protease or 3CL[SUP]pro[/SUP], a cysteine protease that cleavages the viral polyprotein generating 11 proteins that participate in viral pre-replication. Thus, 3CL[SUP]pro[/SUP] is a promising therapeutic target for SARS-CoV-2 inhibition by new drugs or drug repositioning because 3CL[SUP]pro[/SUP] is dissimilar to human proteases. We conducted in vitro assays demonstrating the modulation activity of ambenonium, a drug already used in Myasthenia gravis that acts by inhibiting the action of acetylcholinesterase, and had its potential inhibitory activity against viral replication pointed out in a previous in silico study. In concentrations of 100 µM, 50 µM, 25 µM, 10 µM, and 1 µM there was no inhibition in the formation of lysis plates, with a slight increase in the genome copy number at the higher concentrations evaluated. However, in the concentrations of 0,1 µM and 0,01 µM, there was a reduction in the number of lysis plates. This behavior suggests that the ambenonium acts as a modulator of viral activity in vitro. To investigate potential conformational changes in the protein between dimeric and monomeric forms in the presence of the compound, a local docking analysis was performed. Results indicated this conformational shift is possible, though further studies are needed to confirm these findings.
Keywords: Ambenonium; Drug repurposing; In vitro assay; Main protease; Modulation activity; Pandemic; SARS-CoV-2.