Targeting Breast Cancer with Small Molecule Inhibitors of Estrogen Receptor

用雌激素受体小分子抑制剂治疗乳腺癌

基本信息

批准号：
8247814
负责人：
DAVID J SHAPIRO
金额：
$ 31.59万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-15 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8247814
关键词：
American Amino Acids Anchorage-Independent Growth Apoptosis Aromatase Inhibitors Binding Biological Assay Breast Breast Cancer Cell Bypass C-terminal Calorimetry Cancer cell line Cells Chemicals Chimera organism Collaborations Collection Complex Consensus Crystallography DNA DNA Binding Domain Detection Development Dimerization Dissociation Dose Drug Delivery Systems Drug resistance Effectiveness Estradiol Estrogen Antagonists Estrogen Receptors Estrogen receptor negative Estrogen receptor positive Estrogens Fluorescein Fluorescence Anisotropy Gene Expression Gene Expression Profile Genes Genetic Transcription Growth Health Human Immune Immunoprecipitation In Vitro Label Lead Ligand Binding Link Longitudinal Studies MAPK3 gene MCF7 cell Malignant Neoplasms Mammary Gland Parenchyma Mediating Methods Microarray Analysis Modeling Mus Mutation Natural Killer Cells Nucleic Acid Regulatory Sequences Nude Mice Pathway interactions Pattern Pharmaceutical Preparations Phosphorylation Phosphorylation Site Photoaffinity Labels Play Point Mutation Pre-Clinical Model Production Proteins Public Health Purinergic P1 Receptors Reporter Genes Reporting Resistance Resistance development Response Elements Role SP1 gene Screening procedure Selective Estrogen Receptor Modulators Signal Transduction Pathway Site Specificity Structure Structure-Activity Relationship Tamoxifen Testing Titrations Toxic effect Toxicity Tests Transcription Factor AP-1 Transfection VP 16 Western Blotting Work Xenograft Model base cell growth chromatin immunoprecipitation efficacy testing follow-up high throughput screening hormone therapy improved inhibitor/antagonist killings malignant breast neoplasm member mouse model mutant neoplastic cell receptor receptor binding research study response restoration scaffold small molecule stable cell line stoichiometry tumor tumor growth

项目摘要

DESCRIPTION (provided by applicant): Estrogenic and antiestrogenic compounds that bind in the estrogen receptor (ER) ligand binding pocket have played key roles in developing our understanding of ER action. The antiestrogenic compound, tamoxifen (Tam), is used extensively to treat breast cancer, but its long-term use is limited because tumors eventually develop tamoxifen resistance. We developed a high throughput screening strategy to identify small molecules that target ER action at the level of ER binding to its DNA response element, rather than the traditional approach of targeting antagonism of estrogen binding. Our lead ER inhibitor, TPSF, potently and specifically inhibits estrogen-ER-mediated gene expression and estrogen-ER-dependent growth of Tam-sensitive MCF-7 cells and three Tam-resistant breast cancer cell lines. However, TPSF has no effect on growth of ER negative cells. The three Specific Aims of this proposal build on our recent identification of TPSF. (Aim 1) Optimize TPSF by using structure-activity relationships to guide synthesis of new compounds for screening and assess inhibitor efficacy, specificity and intracellular actions in cell-based assays using Tam-sensitive and Tam-resistant breast cancer cells. (Aim 2) Identify the site on ER1 that interacts with the inhibitors and their mode of action, and (Aim 3) Test TPSF, and the best inhibitor to emerge from optimization, in mouse xenograft models of Tam-sensitive and Tam-resistant breast cancer. Assays and methods: (Aim 1) We will test the efficacy, potency and specificity of each inhibitor in gene expression assays using ER, AR and GR and examine inhibition of ER actions that do not require direct binding of ER to DNA. Using Tam-sensitive and Tam-resistant breast cancer cells, we will evaluate the inhibitor's ability to alter endogenous gene expression, anchorage-dependent and anchorage-independent cell growth and to re-sensitize breast cancer cells to killing by immune cells. We will test for extranuclear effects on the ERK pathway and other signal transduction pathways and for toxicity in long-term studies. To analyze the lead inhibitor's effects on gene expression patterns, we will perform microarray analysis in ER positive breast cancer cells and in non-tumorigenic breast cells. (Aim 2) To evaluate inhibitor-ER interaction in vitro and in cells, we will use ER mutants containing large ER domains and then do studies with smaller mutations. Photoaffinity labeling, NMR comparison of chemical shifts of free ER and ER-inhibitor complexes, and isothermal titration calorimetry will be used to assess direct interaction of the inhibitors with ER. If feasible, structural studies of ER domain-inhibitor complexes will be performed. We will evaluate potential inhibitor effects on ER dimerization, degradation, phosphorylation and synergy with known antagonists. (Aim 3) Using Tam-sensitive and Tam-resistant breast cancer cells, we will assess the ability of the inhibitors to block tumor growth or induce tumor regression. These small molecule inhibitors are powerful new probes for ER action in breast cancer. PUBLIC HEALTH RELEVANCE: Breast cancers that depend on estrogens bound to estrogen receptor for their growth eventually become resistant to drugs that target estrogen. We have identified new inhibitors that bypasses the site targeted by current drugs and instead target an essential action of the estrogen receptor. Further development of these inhibitors may lead to clinically useful drugs effective against tumors that are resistant to current therapies.

描述（由申请人提供）：在雌激素受体（ER）配体结合口袋中结合的雌激素和抗雌激素化合物在发展我们对ER作用的理解方面起着关键作用。抗雌激素化合物他莫昔芬（TAM）被广泛用于治疗乳腺癌，但其长期使用受到限制，因为肿瘤最终会产生他莫昔芬的耐药性。我们制定了高吞吐量筛选策略，以鉴定针对ER结合水平与其DNA响应元件的靶向ER作用的小分子，而不是靶向雌激素结合拮抗作用的传统方法。我们的铅ER抑制剂TPSF，有效，专门抑制雌激素-ER-ER介导的基因表达和雌激素-ER-ER-ER-ER-ER-ER-ER-ER-依赖性MCF-7细胞的生长以及三个抗TAM耐药性乳腺癌细胞系。但是，TPSF对ER负细胞的生长没有影响。该提案的三个特定目标是基于我们最近对TPSF的识别。（AIM 1）通过使用结构活性关系来优化TPSF，以指导新化合物的合成，以筛选和评估使用TAM敏感和抗TAM耐药性乳腺癌细胞在基于细胞的测定中的抑制剂功效，特异性和细胞内作用。（目标2）在ER1上识别与抑制剂及其作用方式相互作用的位点，以及（AIM 3）测试TPSF，以及在TAM敏感和抗TAM耐药性乳腺癌的小鼠异种移植模型中，从优化中出现的最佳抑制剂。测定和方法：（目标1）我们将使用ER，AR和GR测试每个抑制剂在基因表达测定中的功效，效力和特异性，并检查不需要直接结合ER与DNA的ER作用的抑制作用。使用对TAM敏感的和耐TAM的乳腺癌细胞，我们将评估抑制剂改变内源基因表达，锚定依赖性和锚固独立的细胞生长，并将乳腺癌细胞重新敏感到免疫细胞杀死的能力。在长期研究中，我们将测试对ERK途径和其他信号转导途径以及毒性的核外影响。为了分析铅抑制剂对基因表达模式的影响，我们将在ER阳性乳腺癌细胞和非肿瘤乳腺细胞中进行微阵列分析。（目标2）为了评估体外和细胞中抑制剂-ER相互作用，我们将使用含有大ER结构域的ER突变体，然后使用较小的突变进行研究。光性标记，游离ER和ER抑制剂复合物的化学位移的NMR比较以及等温滴定量热法将用于评估抑制剂与ER的直接相互作用。如果可行，将对ER结构域抑制剂复合物进行结构研究。我们将评估潜在的抑制剂对ER二聚化，降解，磷酸化以及与已知拮抗剂的协同作用。（AIM 3）使用对TAM敏感和抗TAM耐药性乳腺癌细胞，我们将评估抑制剂阻断肿瘤生长或诱导肿瘤消退的能力。这些小分子抑制剂是乳腺癌中ER作用的强大新探针。公共卫生相关性：依赖于雌激素受体生长的雌激素的乳腺癌最终对靶向雌激素的药物具有抵抗力。我们已经确定了绕过当前药物靶向的位点的新抑制剂，而是针对雌激素受体的基本作用。这些抑制剂的进一步发展可能会导致临床上有用的药物有效地针对对当前疗法具有抗性的肿瘤。