Chemical Characterization, In-silico Evaluation, and Molecular Docking Analysis of
Antiproliferative Compounds Isolated from the Bark of Anthocephalus cadamba Miq.
Authors: Ansari Jamal Akhtar , Ahmad Mohammad Kaleem , Fatima Nishat , Azad Iqbal , Mahdi Abbas Ali , Satyanarayan G. N. V. , Ahmad Naseem 
Published: February 2022

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.