Molecular Interactions, Binding Stability, and Synergistic Inhibition on Acetylcholinesterase Activity of Safranin O in Combination with Quercetin and Gallic Acid: in Vitro and in Silico Study

The clinical management of multifactorial diseases, especially in neurodegenerative disorders, requires the identification of potent synergistic interactions between therapeutic molecules. In this context, the current study was carried out to evaluate the synergistic inhibitory effect on Acetylcholinesterase activity by Safranin O in combination with Quercetin and Gallic acid in vitro and in silico. Anti-AChE activity was assessed using Ellman's colorimetric method, while the Synergistic and antagonistic interactions were calculated by Loewe, Zip, Bliss and HSE statistical models and plotted using synergyfinder plus tool. Molecular docking of complexed ligands was performed by auto-dock vina tool, Discovery studio and MOE softwares. Molecular dynamic simulations were carried out using Desmond Schro center dot dinger software and the essential parameters were analyzed in 100 ns of simulation time. Our results showed potent inhibitory activities for all tested compounds Safranin O (IC50=15.76 +/- 1.15 mu M), Quercetin (IC50=21.41 +/- 0.25 mu M), Gallic acid (IC50=77.59 +/- 0.44 mu M) in comparison with Galantamine (IC50=2,14 +/- 0.07 mu M). The binary combination (1/1 ratio) Exhibited a significant improvement in IC50 values, Safranine O - Quercetin (9.07 +/- 0.82 mu M), Safranine O-Gallic acid (3.46 +/- 0.12 mu M) and became statistically comparable to standard. The interactive dose-response matrixes and the interaction scores were developed and quantified; the latter showed potent synergistic forms in particular doses for each ligand. Moreover, in molecular docking analysis, Quercetin exhibited the highest binding energy (-10,7 Kcal/mol) followed by Safranin O (-9,3 Kcal/mol) and Gallic acid (-6,5 Kcal/mol) respectively in comparison with Reference drugs, Galantamine (-10,2 Kcal/mol), Donepezil (-10,5 Kcal/mol) and Rivastigmine (-7,5 Kcal/mol). In parallel, the studied ligands interacted with both PAS and CAS active site regions of AChE through different stable and strong interactions during molecular dynamic simulations. These findings appreciate the importance of the synergy approach to developing new potent combinations for A.D. management.