Proteomics-Assisted Discovery of Novel Anti-Cancer Agents Targeting The Human Estrogen Receptor Interactome
Asare, Bethany K.
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Estrogen mediates various cellular processes including cell proliferation, differentiation, growth and mammary gland function. Estrogen Receptors (ERs) are present in 70% of breast cancers. Consequently, estrogen mediated ER signaling plays a critical role in breast cancer diagnosis, prognosis, and treatment. Estrogen Receptors are ligand-triggered transcription factors. However, in the absence of a cognate estrogenic ligand, ERs can be activated by a variety of other extracellular signals. Tamoxifen, an anti-estrogen that selectively targets ER, induces substantial regression of breast tumors and an increase in disease-free survival. Tamoxifen mimics estrogen effects in other tissues thereby providing some beneficial effects including reduced risk of osteoporosis. However, breast cancers that initially respond well to tamoxifen tend to develop resistance and resume growth despite the continued presence of the antagonist. In addition to the hormone mediated activation pathway, binding of coregulaors and/or phosphorylation of ER by cellular protein kinases and by binding of cations could mediate ligand-independent activation of ER. Activators of ER have increased cellular expression in cancers, specifically calmodulin (CaM) is significantly overexpressed in several malignancies including breast cancer. Calmodulin antagonists have been shown to down regulate ER and significantly inhibit ER transcriptional activity. Further, corepressors of ER are significantly downregulated in cancers, specifically breast cancer type 1 susceptibility protein (BRCA1) is expressed 30 to 40 % less in sporadic breast cancers. A decrease in BRCA1 is associated with acquired tamoxifen resistance, suggesting that BRCA1 may play a critical role in maintaining breast cancer cellular sensitivity to tamoxifen. The intricate balance between ER coactivators/corepressors is disturbed in breast cancer i.e., normal regulation of ER by corepressors such as, BRCA1, may be shifted in breast cancers to primarily regulation or activation by coactivators like CaM. The major objectives of this study are to identify conformation-sensing regions of ER interacting proteins using state-of-the art proteomics and structure-based discovery tools, and use them to generate structural clues towards novel small molecular therapeutics for breast cancer therapy. To accomplish this, a comprehensive proteomics-assisted drug discovery approach was employed: (i) to detail ER/coregulator interactions; (ii) to identify pharmacophores based on the ER/coregulator interactions; (iii) to design, synthesize structurally divergent scaffolds and evaluate their role as potential anti-cancer agents in vitro. The BR compounds library, a set of structurally divergent ER antagonists that fit the tamoxifen pharmacophore, was designed to target ER Ligand Binding Domain (LBD) and to recruit BRCA1. Two of the lead compounds in the library, BR46 and BR47, were found to inhibit estrogen induced cell proliferation and cell viability. A second library of Coregulator (CORE) targeting compounds was generated based on CaM/ER interface pharmacophores, generated from the proteomics analysis. The CaM/ER interaction was defined by integrating chemical crosslinking, mass spectrometry and computational docking data. For the first time, holistic models of CaM/ER complexes containing all the interaction domains of both ER and CaM and in turn biological units predictive of estrogen receptor mediated signaling were generated. The integrated proteomics-assisted discovery approach employed in this study has resulted in the identification of novel druggable pockets in the ER hinge region and a binding function-3 (BF3)-like allosteric pocket distal to the coregulator binding region. Small molecules targeting these sites have not been described before. Three novel compound classes targeting ER hinge, coregulatory and BF3-like allosteric pockets have been described in this study. The ER hinge targeting compound CORE5 significantly inhibited ER positive breast cancer cell (MCF-7) proliferation and viability but did not alter triple negative breast cancer cells (MDA-MB-231). Discovery of novel druggable ER pockets and lead molecules targeting these alternative binding pockets have provided structural clues towards the development of new generation of small molecule therapeutics that could be used in tamoxifen-resistant breast cancers and as complementary treatments to the already existent anti-estrogen therapies.