In-silico and in-vitro study reveals ziprasidone as a potential aromatase inhibitor against breast carcinoma

Abstract

Aromatase enzyme plays a fundamental role in the development of estrogen receptors, and due to this functionality, the enzyme has gained significant attention as a therapeutic for reproductive disorders and cancer diseases. The currently employed aromatase inhibitors have severe side effects whereas our novel aromatase inhibitor is more selective and less toxic, therefore has greater potential to be developed as a drug. The research framework of this study is to identify a potent inhibitor for the aromatase target by profiling molecular descriptors of the ligand and to find a functional pocket in the target by docking and MD simulations. For assessing cellular and metabolic activities as indicators of cell viability and cytotoxicity, in-vitro studies were performed by using the colorimetric MTT assay. Aromatase activities were determined by a fluorometric method. Cell morphology was assessed by phase-contrast light microscopy. Flow cytometry and Annexin V-FITC/PI staining assay determined cell cycle distribution and apoptosis. This study reports that CHEMBL708 (Ziprasidone) is the most promising compound that showed excellent aromatase inhibitory activity. By using better drug design methods and experimental studies, our study identified a novel compound that could be effective as a high-potential drug candidate against aromatase enzyme. We conclude that the compound ziprasidone effectively blocks the cell cycle at the G1-S phase and induces cancer cell death. Further, in-vivo studies are vital for developing ziprasidone as an anticancer agent. Lastly, our research outcomes based on the results of the in-silico experiments may pave the way for identifying effective drug candidates for therapeutic use in breast cancer.

Figure 1

A complete computer-aided drug discovery and in-vitro approaches.

Figure 2

(A) 3D Crystal structure of aromatase (PDB ID:3EQM), and (B) Ramachandran plot of prepared structure.

Figure 3

Ligand interaction diagram of the 3EQM and Ziprasidone where VAL369 and VAL368 interact with the O atom and the same VAL370 interacts with the OH atom whereas the PRO429 interacts with the NH atom of the ligand.

Figure 4

MD. Simulation results in (A) Root Mean Square Deviation (RMSD) and (B) Root Mean Square Fluctuations (RMSF) where the blue colour represents the Cα fluctuations while the green line represents the Ziprasidone contacts with residues.

Figure 5

Simulation interaction diagram of Ziprasidone (A) 2-D interactions representation of the protein–ligand and (B) histogram representation of the count of interaction during the simulation period.

Figure 6

Antiproliferative activity of control (5FU) and ziprasidone in MCF-7, MDA MB 231 and T47D cells at 24 and 48 h post-treatment.

Figure 7

Effect of Ziprasidone on cell morphology of MCF-7, MDA MB 231 and T47D cell lines at 24 h post-treatment. Scale of images are displayed on the on the figure at magnification of 20 × tenfold under inverted microscope.

Figure 8

Cell cycle analysis in control and different cell lines (MCF-7, M.D.A. MB 231 and T47D) treated with drugs.

Figure 9

The proapoptotic effect of ziprasidone on MCF-7, MDA-MB 231 and T47D cell lines after 48 h.

Figure 10

(A) Standard curve of Aromatase Substrate metabolite fluorescence. (B) Reaction kinetics of recombinant human aromatase enzyme positive inhibitory control (letrazole) at 37 °C. (C) Dose–response curves for ziprasidone compound with different concentration.