In silico and in vitro applications of drug repositioning

Breast cancer is the most common cancer type among women worldwide. According to the latest cancer statistics; breast cancer has the highest incidence of cancer in women, 30% of which is diagnosed in 2017. Due to its large impact on the population, this disease represents a critical health problem and requires further research at the molecular level for early diagnosis and treatment of breast cancer. Metastasis is one of the most important factors affecting the survival rate of breast cancer patients. Recently, GABAA receptor alpha3 (Gabra3), which is thought to be expressed only in the brain under normal conditions, has been proven to play a critical role in the metastasis of breast cancer cells and it is also known from many studies that Gabra3 is not expressed in normal breast epithelial cells. In this thesis; examination of the Gabra3 expression in MCF-7 and MDA-MB-436 breast cancer cell lines and the effect of flumazenil, a GABA antagonist, against these cancerous cells was aimed. For this purpose, Gabra3/Gabrg2 dimer, which is believed to contain the binding site of flumazenil, was modeled and a molecular docking study with the flumazenil compound was performed for the first time in the literature. Maestro Molecular Modeling Interface 11.3 (Schrödinger Release 2017-3: Maestro, Schrödinger, LLC, New York, NY, 2017) was used as the background for all programs applied in the molecular docking analysis of the Gabra3/Gabrg2 dimer. The molecular docking scores of Flumazenil and Gabra3/Gabrg2 dimer was confirmed by SP and XP glide docking methods as -7.167 and -6.299 respectively. In addition, MCF-7 and MDA-MB-436 cells were cultured to observe this effect with in vitro studies. Then, MDA-MB-436 cells were treated with 0.01 μM, 1 μM, 10 μM and 100 μM flumazenil and DMSO (dimethyl sulfoxide). Quantitative polymerase chain reaction (qPCR) with genomic DNA was carried out after total RNA isolation and cDNA synthesis in all samples respectively and the change of Gabra3 expression was compared. As a result of in vitro experiments; It was determined that the Gabra 3 expression of MDA-MB-436 cells was 20 times more than the Gabra3 expression of MCF-7 cells. 0.01 μM and 1 μM flumazenil administration increased the expression of Gabra3 by about 8 and 10-fold respectively compared to MDA-MB-436 control cells. High concentrations of 10 μM and 100 μM Flumazenil increased the expression of the Gabra3 gene by 0.5 and 1.5, respectively. Depending on the effect of Flumazenil on Gabra3, its use in breast cancer metastasis should be further studied.