Phenolic Compounds: Investigating Their Anti-Carbonic Anhydrase, Anti-Cholinesterase, Anticancer, Anticholinergic, and Antiepileptic Properties Through Molecular Docking, MM-GBSA, and Dynamics Analyses

Phenolic compounds are a new class of Carbonic Anhydrase inhibitors (CAIs). Despite numerous advancements in treatment approaches, cancer continues to be a growing health problem worldwide. In our study, we tested the effects of 4-hydroxy-3-methoxyacetophenone (1), doxycycline hydrochloride (2), 5,7-dichloro-8-hydroxyquinoline (3), methyl 3,4,5-trihydroxybenzoate (4), 2-hydroxy-4-methylacetophenone (5), 6-hydroxy-4-methylcoumarin (6), and 2,5-dihydroxyacetophenone (7) on Achetylcholynesterase (AChE), Butrycholynesterase (BChE), and Human Carbonic anhydrase I (hCA I) enzymes. The U2OS human osteosarcoma cell line was used to determine the anticancer potential of these phenolic compounds. The effects of the compounds on proliferation and colony formation were analyzed using the Neutral Red Uptake (NRU) assay and the clonogenic assay. The K_i values of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde, and 3,4-dihydroxy-5-methoxybenzoic acid were 203.80, 1170.00, and 910.00 mM, respectively, for hCA I, and 75.25, 354.00, and 1510.00 mM, respectively, for Human Carbonic anhydrase II (hCA II). Additionally, IC₅₀ values from in vivo studies were found to range from 173.25 to 1360.00 mM for CA I and CA II, respectively, using CO₂-hydratase activity methods. The NRU assay results revealed that the compounds had a dose-dependent cytotoxic effect on U2OS cells. The IC₅₀ values of the compounds in U2OS osteosarcoma cells were determined to be > 100, 93.7, 81.4, 26.9, > 100, 53.1, and > 100 µM, respectively. Notably, methyl 3,4,5-trihydroxybenzoate (4), the compound with the lowest IC₅₀ value, significantly suppressed colony formation at 5 and 10 µM concentrations. These results demonstrated that the phenolic compounds used in in vivo studies could inhibit approximately 30% of the CO2-hydratase activity of the total CA enzyme of rat erythrocytes. Furthermore, the anticancer potential of the tested compounds suggests that these molecules could pave the way for the development of new approaches in cancer treatment. The activities of the seven molecules studied were compared against AChE (PDB ID: 4M0E), BChE (PDB ID: 5NN0), hCA I (PDB ID: 2CAB), and E3 ubiquitin-protein ligase (PDB ID: 4HG7) proteins. The binding free energy of the molecule with the highest docking score is computed using MM/GBSA techniques. Finally, molecular dynamics simulations were performed between 6-hydroxy-4-methylcoumarin and the 4M0E protein over a 0–200 ns interval.

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