In vitro investigation of receptor targeted in silico ligand design in ovarian cancer therapy

Human estrogen receptor alpha (ERα) is a nuclear receptor family member that regulates the transcription of many estrogen-inducible genes involved in growth, metabolism, and development. Overexpression of ERα in tissues activates its associated signaling pathways, leading to the accumulation of DNA mutations in the cell, neoplastic transformation of proliferating cells, and tumor progression. ERα expression and activation are of primary importance for developing hormone-dependent cancers. Studies targeting ERα in hormone-dependent cancer therapy have shown that it is a suitable therapeutic target in cancer treatment by inducing cell apoptosis and inhibiting epithelial-mesenchymal transition. However, to overcome the resistance to endocrine therapy that develops over time, alternative approaches to ERα are moving away from the ligand binding domain and focusing on ERα-DNA or ERα-cofactor interactions (the hydrophobic groove of the LxxLL motif of the receptor box (NR)). In targeted therapies, especially in recent years, the inhibition of protein-protein interactions (PPIs), such as ERα-cofactor interactions by therapeutic peptides, has become a general strategy for treating diseases. Cyclic peptides, which bind to the target with a better affinity and perform pharmacologically better than linear peptide motifs, have been extensively investigated as novel and targeted inhibitors in recent years. Therefore, in this thesis, cyclic peptides targeting various α-helix-dominant protein-protein interactions in ERα were designed based on the cofactor binding inhibitor region (LxxLL). Our in silico studies have shown that it is possible to design cyclic peptides in which the most critical amino acids binding to ERα are not restricted but instead released. In this way, newly designed cyclic peptides have been calculated in silico that bind to ERα in a unique and highly bioequivalent manner, providing a major advantage over the motifs presented in the literature. The reference cyclic peptide (SP1) and the novel cyclic peptides derived in this thesis (SP2 and SP3) were synthesized by solid-phase peptide synthesis method using Fmoc chemistry and cyclized by DEAD method. The obtained linear and cyclic crude peptides were analyzed and characterized by LC-MS, purified by reverse phase HPLC, and used in in vitro experiments. In the thesis study, the TPBM commercial small molecule was used as a reference as it has a similar mechanism of action with our peptides against ERα without competing with estrogen as an ERα inhibitor. As a result of our in vitro studies, it was observed that the new cyclic peptides we designed were only effective on ERα (complexed with estrogen (E2)) in the active form, while cyclic peptides in E2-deprived medium did not affect ERα (+) cells. This indicated that the cyclic peptides design binds only to the LxxLL motif exposed in the active form of ERα. At the same time, in an E2-supplemented cell culture medium, SPs did not show any toxic effect on cancer and healthy cell lines that do not express ERα but only showed the antiproliferative effect on ERα-expressing cells, supporting that the peptides inhibit cell growth via ERα. In addition, the antiproliferative and toxic effects of the peptide combinations on cell lines were indicated by cell viability tests. The effect of the most effective combination group, SP2 + SP3 and SP1 + SP2 + SP3, on ERα-related pathways and important apoptosis markers of cells was determined at both the gene and protein level. The data obtained from qPCR and Western blot showed that the altered expression profiles of p53, p21, Bax, and Bcl-2, which are involved in the intrinsic apoptosis pathway, as well as caspase-8, which is involved in the extrinsic apoptosis pathway, showed that the cells were affected through both apoptosis pathways after cyclic peptide combination therapy. Flow cytometry analyses showed that increased caspase 3/7 activity and over-expression of Annexin V markers in cells treated with the effective combinations intensely drove the cells to early and late apoptosis. In addition, flow cytometry analysis of Bcl-2 expression and mitochondrial membrane integrity showed that Bcl-2 inactivation and significant increases in the number of cells with impaired mitochondrial membrane integrity after cyclic peptide combination treatment supported that the Bcl-2 signaling pathway was inactivated and apoptosis occurred through mitochondria. In addition, the decrease in cytoplasmic and nuclear ERα activations distributed in the cell after treatment of cells with only peptides and peptide combinations proved that cyclic peptides were specific for ERα. In addition, cytoplasmic ERα, which plays an important role in the proliferation of MCF-7 cells and has a strong relationship with hormone resistance, decreased dramatically after cyclic peptide treatment, indicating that apoptosis is induced through this pathway. In conclusion, within the scope of this thesis, cyclic peptides for ERα targeting were designed and synthesized, and their inhibitory effect on ERα (+) cells was supported by various in vitro studies. The results show that cyclic peptides induce cell death mechanisms by acting on both the cells' intrinsic and extrinsic apoptosis pathways. It has been demonstrated that the new cyclic peptides we have developed within the scope of this thesis can be further improved with the latest technologies and can be an alternative to current clinical treatments or a helpful approach to creating new peptide combination therapies.