Study On Bioactivity And Anti-Breast Cancer Effect Of Novel Dual-Target Selective Estrogen Receptor Modulators

Breast cancer is the most common type of noncutaneous malignancy and the leading cause of cancer-related death in women worldwide. Approximately 75% of breast cancer are classified as estrogen receptor-alpha (ERa)-positive. Estrogen and estrogen receptors play key role in proliferation, survival and invasion of bresast tumour cells. Selcetive estrogen receptor modulators (SERMs) are the most recently approved class of first line drugs for the treatment of breast cancer. Unfortunately, some disadvantages severely limit their therapeutical use. For example, tamoxifen, which is the first clinically used SERM for treatment of breast cancer, is associated with uterine hypertrophy and an increased risk of endometrial cancer; addtionally, tamoxifen is not effective in ER-breast cancer patients, it is also ineffective in a significant fraction of ER+ patients, and even when initially effective, resistance to tamoxifen therapy often develops with time.In another aspect, histone deacetylases (HDACs), which are an important kind of epigenetic regulators and post-transcriptional modifiers, have recently emerged as important targets for cancer therapy, including breast cancer. Several HDAC inhibitors (HDACi) are in various stages of clinical trials, with approximately 500 clinical trials initiated over the past decade. However, in clinical studies, HDACi have failed to show considerable anti-tumor activity as single agents in breast tumors.Emerging evidences suggest simultaneous ER and HDAC inhibition could be a promising approach in breast cancer therapy. In this study, we discribe a novel kind of SERM-HDACi hybrid compound dual targeting ER and HDAC. One of the parent compouds is a new tyle of ER ligand with three-dimensional core scaffold, another is the first FDA approved HDACi (SAHA). These conjugates are divided into 3 series, called OBHS-HDACi, OBHSA-HDACi, FcOBHS-HDACi, according to their different parent ER ligands.To begain with, we tested the ER binding affinity of the SERM-HDACi conjugates by developing a novel fluorescence polarization method. In general, these three series conjugates showed remarkable differences in receptor binding ability, which is significant effected by the type and position of substituents. Compounds 22a, 28g and 33b stand out in these conjugates for having very high binding affinity for ERa and ER subtype selectivity, the relative binding affinity (RBA)values are 12.2%, 13.07% and 3.28%, respectively.In the second part, we first tested all conjugates for modulation of ER transcriptional activity in mammalian cell culture using a dual luciferase reporter system. Then, in an attempt to understand the structural basis for the observed ERa antagonist effects of SERM-HDACi, in silico docking analysis was performed using Autodock 4.2D. The OBHS-HDACi conjugates exhibited multiple transcriptional activities for ERa, with 9 agonists and 17 antagonists. Among them, compound 24q represents a novel mechanism to generate a full ERa antagonist, with its suberic acid side chain destroying the transcriptional coactivator-binding site and thereby reducing the AF2-meditated activity of ERa. The OBHSA-HDACi conjugates, however, didn't activate ERa at all, possiblely due to the full antagonist activity of their parent compound OBHSA. While most of FcOBHS-HDACi conjugates examined are ERa agonists, with only three antagonists.In the third part, all dual-acting conjugates were screened against breast cancer cells to obtain their antitumor activity. A number of compounds, like 22a,24g,25h, 28a,28g,33b,35, were found to be more potent than 4OH-tamoxifen (4OHT) against breast cancer cells, even equipotent to SAHA. Cell apoptosis assays also proved 3 representative compounds 22a,28g,33b could induce cellular apoptosis in breast cancer MCF-7 cells. Additionally, all conjugates are nontoxic to healthy cells, while SAHA and 40HT showed considerable toxicity.As a final test, an array of representative OBHS-HDACi conjugates with significant antibreast tumour effects were assayed for HDAC inhibition activity against HDAC1 and HDAC6. We found these compounds could actually inhibit HDAC 1 or HDAC6, and the HDAC inhibition activity was very much dependent on the type and position of substituents. Morever, immunoblotting analysis also comfirmed these SERM-HDACi conjugates could act as HDAC inhibitor at the cellular level.