Chemical Characterization, In-silico Evaluation, and Molecular Docking Analysis of Antiproliferative Compounds Isolated from the Bark of Anthocephalus cadamba Miq.

Abstract

Aim: The present study aimed to isolate and characterize chemical compounds from Anthocephalus cadamba Miq. bark and evaluate their anticancer activity by in silico, molecular docking, and in vitro studies. Background: Anthocephalus cadamba is a traditionally used Indian medicinal plant. The anticancer and phytochemical properties of this plant remain unexplored except for a few studies. Objectives: The objective of the study was to evaluate the antiproliferative activity of extract and fractions against breast cancer and prostate cancer cell lines and isolate and characterize active compounds from bio-active guided fractions. Moreover, the anticancer activity of isolated compounds against breast and prostate cancer cell lines was also evaluated, in addition to in silico and molecular docking interactions of isolated compounds with VEGFR2 and PDGFRα target proteins. Methods: The compounds were isolated and purified with the help of repeated column chromatography, and spectral techniques, such as 1D, 2D NMR, and GC-MS/MS, were used to identify and elucidate the structure of the compounds. Moreover, prediction of activity spectra for substances, physiochemical properties, bioactivity radar prediction, bioactivity score, natural-product likeness, ADME, and toxicity parameters of isolated compounds (AC-1 to AC-4) was performed through various in-silico databases and servers. To evaluate the docking interaction profile and binding energies of compounds, three docking tools were utilized, such as AutoDock, AutoDock Vina, and iGEMDOCK, against two targets VEGFR2 and PDGFRα. MD simulation was performed through ligand and receptor molecular dynamic server (LARMD). Results: It was found that the A. cadamba bark chloroform fraction demonstrated a significant inhibitory effect against MDA-MB-231, MCF-7, and PC-3 cells in a dose-time-dependent manner. The bioassay-guided isolation afforded four molecules AC-1 to AC-4 from chloroform fraction. Moreover, the GC-MS/MS profiling identified fourteen new molecules which were not reported earlier from A. cadamba. The in-silico study showed that the isolated compounds (AC-1 to AC-4) followed Lipinski’s rule and had good oral bioavailability. While compound AC-4 had positive bioactivity scores except for kinase inhibitor activity. The ADMET profiling revealed that AC-4 was non-toxic and easily absorbed in the human intestine, and transportable in the blood-brain barrier compared to AC-1, AC-2, AC-3, and standard drug doxorubicin. Molecular docking and MD simulation assessment also signified AC-4 anticancer activity with dual inhibitory action against the target proteins VEGFR2 and PDGFRα amongst the studied compounds. The in vitro cell viability assay of isolated compounds demonstrated that AC-1 showed IC50 (μg/mL) value of 34.96 ±3.91, 47.76±3.80 69.1±4.96, AC-2; 68.26±4.22, 54.03±5.14, >100, AC-3; 35.34±4.14, 51.5±51.5, 70.8±5.25 and AC-4; 44.2±3.57, 24.2±2.67, 51.2±2.54 for MDA-MB-231, MCF-7, and PC-3 cancer cell lines, respectively and compared with standard drug doxorubicin. Moreover, fluorescence microscopy confirmed the apoptogenic property of compounds. We also found that AC-4 exhibited significant intracellular ROS production in breast cancer cells, thereby inducing apoptosis and eventually cell death. Conclusion: In conclusion, A. cadamba afforded four pure molecules AC-1 to AC-4 with the identification of fourteen new compounds. The entire in-silico studies concluded that the AC-4 compound had better oral bioavailability, bioactivity score, and ADMET profile among studied molecules. Molecular docking analysis and MD simulation also supported AC-4 dual inhibitory action against both VEGFR2 and PDGFRα receptors. Moreover, the isolated molecules AC-1, AC-2, AC-3, and AC-4 were found to be active against MDA-MB-231, MCF-7, and PC-3 cancer cells. The molecule AC-4 was found to induce ROS-mediated apoptosis in breast cancer cells. It was found that the anticancer inhibitory potentiality of AC-4 is directed to its molecular stereochemistry which specifically binds to the target proteins of breast cancer cells with no toxicological effect. Therefore, AC-4 is suggested to be an effective aspirant for novel drug design and discovery.