Partial Agonists at Estrogen Receptor alpha for Breast Cancer Therapy

 描述（由申请人提供）：雌激素受体（ER）是一种药物靶标，在临床上已被选择性雌激素受体调节剂（SERM）所利用，其拮抗乳腺组织中的雌激素效应并在骨中充当激动剂。SERM类药物的原型，他莫昔芬（TAM）已经作为乳腺癌治疗的标准治疗了30年，尽管子宫中的激动剂活性增加了子宫内膜癌的风险。相反，ER α激动剂的治疗潜力尚未被探索或开发。高达80%的乳腺癌是ER+，其中雌二醇（E2）促进肿瘤生长。其中，30-50%对TAM无反应或产生耐药性，几乎没有治疗选择。巧合的是，在引入TAM之前，E2和ER激动剂己烯雌酚都在乳腺癌的治疗中取得了成功，但具有不可接受的副作用，包括乳腺和子宫中的激动剂活性。ER α的部分激动剂在正常妇科组织中没有雌激素作用，但具有消退TAM抗性癌症的能力是一个令人信服的概念，我们在2种动物模型中用2种小分子进行了验证。UI、Greg撒切尔、John Katzenellenbogen、Terry摩尔和Deb Tonetti组成的团队已经准备好了所有的分析和化学方法来实现拟议的目标：目标1。将完成由4个先导系列组成的约350种化合物的文库，并使用FRET测定法筛选SRC 3共激活剂与ER α和ER β的结合。ER与辅激活因子的相互作用被视为表型的最小预测因子。ER的放射性配体置换是一项次要确证性试验。将获得ER α的部分激动剂以及ER α的激动剂/非活性反应，并使用目标2的数据进行迭代优化，以提供计算机辅助设计的SAR开发。目标二。将使用内源性ER α（MCF-7; T47 D）和ER α（β 43）乳腺上皮细胞和子宫内膜细胞中的报告基因测定来研究来自FRET筛选的所选ER α激动剂，以揭示效力和激动剂/拮抗剂药理学。部分激动剂将进一步研究使用PCR测量ERE，AP-1，和Sp1敏感基因。主要的表型测定是TAM抗性MCF 7 -5C细胞的凋亡。反筛选是亲本MCF 7 -5C和T47 D细胞系的增殖。目标3.肝微粒体中的药物代谢、多重CYP抑制和口服血浆生物利用度将用于改进作为化学探针的ER α部分激动剂。我们在SERM代谢方面的丰富经验和所有初步数据中口服给药的使用使我们对这种方法充满信心。DMPK研究将为动物研究提供最终选择。将使用三种不同的TAM耐药异种移植模型来测试化学探针并确定肿瘤消退的治疗指数。将通过子宫生长和雌激素依赖性T47 D异种移植物生长的平行研究预测安全性。支持性作用机制研究仅限于ER α定位和靶点选择性蛋白质组学。这些目标的完成将提供化学探针来研究ER功能的新型部分激动剂和变构调节剂的潜力，并提供先导化合物用于将来在PKCa过表达肿瘤的患者来源的异种移植物中进行测试，作为药物开发的前奏。

 DESCRIPTION (provided by applicant): The estrogen receptor (ER) is a drug target that has been exploited clinically by selective estrogen receptor modulators (SERMs) that antagonize estrogenic effects in breast tissue and act as agonists in bone. The proto- type of the SERM drug class, tamoxifen (TAM) has for 3 decades been the standard of care in breast cancer therapy, although agonist activity in the uterus increases risk of endometrial cancer. Conversely, the therapeutic potential of ERa agonists has not been explored or exploited. Up to 80% of breast cancers are ER+, in which estradiol (E2) fuels tumor growth. Of these, 30-50% are unresponsive to TAM or develop resistance, leaving few treatment options. Paradoxically, prior to the introduction of TAM, both E2 and the ER agonist diethylstilbestrol achieved success in the treatment of breast cancer, but with unacceptable side effects, including agonist activity in breast and uterus. A partial agonist at ERa without estrogenic actions in normal gynecological tissues, but with the ability to regress TAM-resistant cancers is a compelling concept, validated by us in 2 animal models with 2 small molecules. The assembled team at UI, Greg Thatcher, John Katzenellenbogen, Terry Moore, and Deb Tonetti has all assays and chemistry in place to achieve the proposed aims: Aim 1. A library of ~350 com- pounds consisting of 4 lead series will be completed and screened using a FRET assay for SRC3 co-activator binding to ERa and ERß. The ER interaction with co-activator is seen as the minimal predictor of phenotype. Radioligand displacement for ER represents a secondary, confirmatory assay. Partial agonists at ERa with agonist/inactive responses at ERß will be obtained and iteratively optimized using data from Aim 2 to provide SAR development for computer-aided design. Aim 2. Selected ERa agonists from FRET screening will be studied using reporter assays in endogenous ERa (MCF-7; T47D) and in ERß (ß43) mammary epithelial cells and endometrial cells to reveal potency, and agonist/antagonist pharmacology. Partial agonists will be further studied using PCR measurement of ERE, AP-1, and Sp1-sensitive genes. The primary phenotypic assay is apoptosis of TAM-resistant MCF7-5C cells. Counterscreens are proliferation of parent MCF7-5C and T47D cell lines. Aim 3. Drug metabolism in liver microsomes, multiplex CYP-inhibition, and oral plasma bioavailability will be used to refine ERa partial agonists as chemical probes. Our extensive experience with SERM metabolism and use of oral delivery in all preliminary data gives confidence in this approach. DMPK studies will provide final selection for animal studies. Three different TAM-resistant xenograft models will be used to test chemical probes and to determine therapeutic index for tumor regression. Safety will be predicted by uterine growth and parallel study of growth of estrogen-dependent T47D xenografts. Supportive mechanism of action studies are limited to ERa localization and target selectivity proteomics. Completion of these Aims will provide chemical probes to study the potential of novel partial agonists and allosteric modulators of ER function, and provide lead compounds for future testing in patient-derived xenografts of PKCa-overexpressing tumors as a prelude to drug development.