Chemical, structural and molecular rules for fully antagonizing the estrogen receptor

具体目标。我们的目标是利用整合的结构、化学和分子系统生物学方法， 在靶向抗乳腺癌的不同机制中具有改进功效的雌激素受体（ER）拮抗剂 癌症，使更多的个性化医疗。由于70-80%的女性患有ERα阳性疾病，高达50%的女性内分泌治疗失败，疾病复发，大多数死亡是ERα阳性患者，突出了改善治疗的重大未满足的临床需求。重要的是，一种激素治疗失败的患者通常对具有替代作用机制的不同激素治疗有反应。目标1。使用结构和化学系统生物学 在基于ERα的不同耐药机制的背景下，确定拮抗ERα的规则的方法， 以及作为与其他非ER α抗性靶向剂的联合治疗。这些方法旨在克服阻力 在ER（+）乳腺癌中与ERα拮抗剂协同作用。我们开发了一种系统生物学方法， ERα配体结合结构域与许多配体平行结晶。分析了几十种晶体结构， 平行使我们能够实施一种无偏的方法来识别亚微米范围内（在单个结构的噪声内）的细微结构扰动，这些扰动对ERα调节的增殖有显著贡献，我们称之为超分辨率X射线晶体学。我们将使用这种方法来确定驱动转录的结构特征， 抑制、受体降解和不同耐药模型中结构多样的配体的治疗功效， 以及与联合治疗方案的协同作用研究，包括PI 3 K、mTOR和CDK 4/6抑制剂。我们已经开发 用于跟踪受体降解的定量高通量筛选测定。ERα驱动的关键靶基因， 辅调节因子相互作用和定量降解测定将用于将受体结构与抗- 在治疗敏感和耐药乳腺癌模型中的增殖效应，包括组成型活性突变ERα，过度活跃的生长因子信号传导和SRC 3/AIB 1的过表达，我们称之为配体类分析的化学系统生物学方法。目标二。设计和合成具有新药效团的ERα全拮抗剂， 产生不同的受体结构扰动，并表征其在动物体内的药理学 ERα（+）乳腺癌的治疗抗性模型。金刚烷基支架提供了一个稳定的核心， 探索不同的，新的侧链如何扰乱ERα结构，介导转录抑制和克服 阻力因为我们发现替代的OBHS-N核心化合物通过口袋内的扰动作为间接拮抗剂而在没有侧链的情况下产生完全的ER拮抗和降解，所以该OBHS-N核心现在提供了一种新的等排平台，用于添加侧链以获得不同的和组合的直接和间接拮抗机制。我们将光学条形码的耐药模型，允许在体内的配体疗效的多重评估。对于这两种类型，我们将在体内对野生型和他莫昔芬耐药ERα（+）模型进行评估。将使用ERα敲除和ER阴性乳腺癌细胞作为对照验证靶向活性，并验证对体内关键ERα转录物的影响。

Specific Aims. Our goal is use integrated structural, chemical and molecular systems biology approaches to develop estrogen receptor (ER) antagonists with improved efficacy in targeting distinct mechanisms of hormone-resistant breast cancer, enabling more personalized medicine. As 70-80% of women present with ERα positive disease and up to 50% fail on endocrine therapies with disease recurrence, most deaths are of ERα-positive patients, highlighting a significant unmet clinical need for improved therapies. Importantly, patients who fail on one hormone therapy typically respond to a different one having an alternate mechanism of action. Aim 1. Use structural and chemical systems biology approaches to identify rules for antagonizing ERα in the context of different ERα -based mechanisms of resistance, and as co-treatments with other non-ERα resistance targeting agents. These approaches aim to overcome resistance and synergize with ERα antagonists in ER(+) breast cancer. We have developed a systems biology approach enabling crystallization of the ERα ligand-binding domain in parallel with many ligands. Analysis of dozens of crystal structures in parallel allows us to implement an unbiased approach to identify subtle structural perturbations in the sub-Å range (within the noise of the individual structures) that contribute significantly to ERα-regulated proliferation, which we call super-resolution x-ray crystallography. We will use this approach to identify structural features that drive transcriptional repression, receptor degradation, and therapeutic efficacy of structurally diverse ligands in different resistance models, and in synergy studies with co-treatment regimens, including PI3K, mTOR, and CDK4/6 inhibitors. We have developed a quantitative high-throughput screening assay for tracking receptor degradation. Key ERα-driven target genes, coregulator interaction and quantitative degradation assays will be used to mechanistically tie receptor structure to anti- proliferative effects in treatment-sensitive and resistant breast cancer models, including constitutively active mutant ERα, hyperactive growth factor signaling, and overexpression of SRC3/AIB1, in a chemical systems biology approach that we call ligand class analysis. Aim 2. Design and synthesize ERα full antagonists with novel pharmacophores that produce distinct structural perturbations of the receptor, and characterize their in vivo pharmacology in animal models of treatment-resistant ERα(+) breast cancer. The adamantyl scaffold provides a stable core with which we will explore how different, novel side chains perturb ERα structure to mediate transcriptional repression and overcome resistance. Because we found that the alternative OBHS-N core compounds produced full ER antagonism and degradation without side chains, by acting as indirect antagonists through perturbations within the pocket, this OBHS-N core now provides a novel, isosteric platform for adding side chains to access distinct and combined direct and indirect mechanisms of antagonism. We will optically barcode a number of resistance models, allowing for a multiplexed in vivo assessment of ligand efficacy. With both classes, we will evaluate them against wild type and tamoxifen-resistant ERα (+) models in vivo. On-target activity will be verified with ERα knockout and ER-negative breast cancer cells as control, with validation of effects on key ERα transcripts in vivo.