Bioactivity-Guided Synthesis: In Silico and In Vitro Studies of β-Glucosidase Inhibitors to Cope with Hepatic Cytotoxicity

The major cause of hyperglycemia can generally be attributed to beta-glucosidase as per its involvement in non-alcoholic fatty liver disease. This clinical condition leads to liver carcinoma (HepG2 cancer). The phthalimides and phthalamic acid classes possess inhibitory potential against glucosidase, forming the basis for designing new phthalimide and phthalamic acid analogs to test their ability as potent inhibitors of beta-glucosidase. The study also covers in silico (molecular docking and MD simulations) and in vitro (beta-glucosidase and HepG2 cancer cell line assays) analyses. The phthalimide and phthalamic acid derivatives were synthesized, followed by spectroscopic characterization. The mechanistic complexities associated with beta-glucosidase inhibition were identified via the docking of the synthesized compounds inside the active site of the protein, and the results were analyzed in terms of the best binding energy and appropriate docking pose. The top-ranked compounds were subjected to extensiveMD simulation studies to understand the mode of interaction of the synthesized compounds and binding energies, as well as the contribution of individual residues towards binding affinities. Lower RMSD/RMSF values were observed for 2c and 3c, respectively, in the active site, confirming more stabilized, ligand-bound complexes when compared to the free state. An anisotropic network model was used to unravel the role of loop fluctuation in the context of ligand binding and the dynamics that are distinct to the bound and free states, supported by a 3D surface plot. An in vitro study revealed that 1c (IC50 = 1.26 mu M) is far better than standard acarbose (2.15 mu M), confirming the potential of this compound against the target protein. Given the appreciable potential of the candidate compounds against beta-glucosidase, the synthesized compounds were further tested for their cytotoxic activity against hepatic carcinoma on HepG2 cancer cell lines. The cytotoxicity profile of the synthesized compounds was performed against HepG2 cancer cell lines. The resultant IC50 value (0.048 mu M) for 3c is better than the standard (thalidomide: IC50 0.053 mu M). The results promise the hypothesis that the synthesized compounds might become potential drug candidates, given the fact that the beta-glucosidase inhibition of 1c is 40% better than the standard, whereas compound 3c holds more anti-tumor activity (greater than 9%) against the HepG2 cell line than the known drug.

Automated summary

This study focuses on the synthesis of phthalimide and phthalamic acid analogs to test their inhibitory activity against beta-glucosidase and their cytotoxic activity against HepG2 cancer cells. Molecular docking and MD simulations were performed to understand the mode of interaction and binding affinities. Compound 1c showed better inhibitory activity against beta-glucosidase than the standard drug, while compound 3c exhibited stronger anti-tumor activity against the HepG2 cell line than the known drug.