Invastigation of the effects of newly synthesized melatonin analogues on aromatase activity

Endocrine disruptors (EDs) are exogenous substances or mixtures that alter function of endocrine system and consequently cause adverse health effects in an intact organism, or subpopulation. An ED can either interact with receptors and act as an estrogen receptor agonist/antagonist or modulate enzymes that involve in biosynthesis and metabolism of steroid hormones and alter physiologic levels of hormones. These mechanisms of ED actions are also used as therapeutical approaches in breast cancer which is the leading cancer site in women and is the leading cause of death among female cancers. Aromatase, a CYP enzyme highly expressed in the breast tissue and responsible for the local estrogen synthesis especially in postmenopausal women, can be inhibited as a treatment strategy in hormone dependent (estrogen receptor positive; ER +) breast cancer therapy. Recent data show that melatonin, an indolic hormone, inhibits aromatase activity and decreases ER (+) breast cancer cell proliferation. In the present study aromatase inhibitory activity of newly synthesized indol derivative melatonin analogues is investigated with two different assays. The first one is a high throughput screening assay where a fluorescence substrate and recombinant human enzyme is used and the enzyme activity is detected directly by spectrophotometer. In the second assay enzyme activity is indirectly evaluated via proliferation of the MCF-7 BUS cells in an estrogen-free medium in the presence of testosterone. First of all the sensitivity and performance of both assays were evaluated by testing known aromatase inhibitors and partial validation of the methods was performed. Potential aromatase inhibitory effect of newly synthesized indole derivatives were investigated by high throughput screening test. The putative ligand-protein binding interactions of the experimentally confirmed hits were investigated with docking studies. The hits that are determined by high throughput screening was further analysed in cell-based indirect measurement assay. Our findings suggest that both assays, cell-free and cell based are successful in evaluating aromatase inhibitory activity but there are many factors affecting the indirect measurement assay and a reliable evaluation can be made after evaluating all these confounding factors. Gathering all data we obtained, some newly synthesized indole derivatives can be suggested as novel active substances and therefore their cytotoxicity potential is also evaluated as an early stage toxicity indicator. Their cytotoxic potential is investigated both in in human breast cancer cells (MCF-7 BUS) and healthy over cells (Chinese hamster ovary-CHO-K1). Among all tested compounds 2-methyl-1-H-indole-3-carboxyaldehyde (3-chlorophenyl) hydrazone (M5) and 2-methyl-1-H-indole-3- carboxyaldehyde (4-chlorophenyl) hydrazone (M6) were found to have no cytotoxic effects in healthy cells but have aromatase inhibitory potential. As a result these two compounds can be suggested as new therapeutic candidates in preventing development and progression of ER (+) human breast cancer, especially in postmenopausal women. Key Words: Endocrin distrupters; Aromatase enzyme; ER (+); Breast cancer; Aromatase inhibitor, Melatonin