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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 1 , Isabela de Souza Gomes 2 , Luciana de Souza Fernandes 3 , Luis Adan Flores Andrade 4 , Luciana Ângelo de Souza 5 , Vinícius de Almeida Paiva 2 , Sheila Cruz Araujo 6 7 , Leonardo Henrique Franca de Lima 7 , Roberto Sousa Dias 3 , Raquel Cardoso de Melo-Minardi 8 , Flávio Guimarães da Fonseca 4 , Sérgio Oliveira de Paula 9 , Sabrina de Azevedo Silveira 2 10
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 3CLpro, a cysteine protease that cleavages the viral polyprotein generating 11 proteins that participate in viral pre-replication. Thus, 3CLpro is a promising therapeutic target for SARS-CoV-2 inhibition by new drugs or drug repositioning because 3CLpro 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 1 , Isabela de Souza Gomes 2 , Luciana de Souza Fernandes 3 , Luis Adan Flores Andrade 4 , Luciana Ângelo de Souza 5 , Vinícius de Almeida Paiva 2 , Sheila Cruz Araujo 6 7 , Leonardo Henrique Franca de Lima 7 , Roberto Sousa Dias 3 , Raquel Cardoso de Melo-Minardi 8 , Flávio Guimarães da Fonseca 4 , Sérgio Oliveira de Paula 9 , Sabrina de Azevedo Silveira 2 10
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 3CLpro, a cysteine protease that cleavages the viral polyprotein generating 11 proteins that participate in viral pre-replication. Thus, 3CLpro is a promising therapeutic target for SARS-CoV-2 inhibition by new drugs or drug repositioning because 3CLpro 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.