Computationally Driven Design and Validation of Piperazine-Bridged Phthalimide Derivatives as Multifunctional Agents Against Alzheimer's Disease

Computer-aided drug design (CADD) facilitates the identification of ligands with optimal binding affinity towards specific targets. A multi-target-directed ligand (MTDL) strategy has been proposed as an effective approach for treating complex multifactorial neurological disorders like Alzheimer's Disease. In the current study we have employed amalgamated ligand- and structure-based screening to identify hits with dual binding affinity against targeted enzymes. A Gaussian field-based 3D-QSAR pharmacophore model of piperazine derivatives against AChE was developed and validated, where in the R2 and Q2 values were found to be statistically significant at 0.9904 and 0.9149, respectively. The best pharmacophore hypothesis model (AHR_1) was used to screen inhouse generated library of 188 phthalimide-piperazine derivatives followed by structure based virtual screening (with AChE and MAO-B) which facilitated the identification of eleven hit molecules. The docking score for these hits were found to be in the range of -11.34 to -8.447 kcal/mol (AChE, donepezil= -10.598 kcal/mol) and -9.505 to -7.594 kcal/mol (MAO-B, Pargyline= -5.596 kcal/mol) and molecular dynamics simulation confirmed the stability of the compound-target complexes. As a proof of concept, the top hits were synthesized in the laboratory and evaluated for their inhibitory potential. Two lead compounds, VPT-2 and VPT-5 were found to display balanced activity profile against AChE and MAO enzymes. Compound VPT-2 displayed IC50 values of 0.897 μM against AChE and 0.519 μM against MAO-B, while VPT-5 showed IC50 values of 3.45 μM against AChE and 1.074 μM against MAO-B, respectively. Additionally, in the cell-based studies both the compounds were found to be neuroprotective against SHSY-5Y cells. Similarly, in ROS inhibition studies, these significantly reduced the intracellular ROS level by 80-90%. Thus, the current research work presents a rational drug design strategy integrating CADD with the in vitro experimental validation, and explored the potential of phthalimide-piperazine derivatives as promising multi-target drug candidates for the treatment of Alzheimer’s disease.