Molecular Analysis of Steroid Hormone Receptors with X-ray Crystallography

描述（由申请人提供）：该项目的长期目标是了解类固醇激素受体在癌症发展和治疗中的调节和作用，通过我们开发的新技术，可以使用X射线晶体学快速分析类固醇激素受体。具体地说，我们的方法已经证明，我们可以将晶体结构的命中率和命中数增加至少一千倍，并且这允许快速分析与化疗剂和途径选择性化合物结合的这些受体的配体结合结构域。雌激素和雄激素受体（ER和AR）分别与乳腺癌和前列腺癌的发生、诊断和治疗有关。糖皮质激素具有更广泛的作用，作为治疗几种恶性肿瘤的前期治疗剂（例如，白血病和难治性前列腺癌），以及作为减少其它化疗剂副作用的佐剂。然而，靶向这些受体的合成化合物具有显著的问题，包括获得性抗性和不期望的副作用。它们还显示组织和途径选择性信号传导，这在分子和结构水平上都知之甚少。有可能开发组织和途径选择性的化合物，改善这些问题中的一些，但很少了解这种选择性的结构基础。 缺乏组织选择性的良好结构模型是由于难以产生晶体结构。类固醇受体配体结合域（LBD）已被证明是非常困难的结晶，由于构象异质性和蛋白质错误折叠。在这里，我们建议进一步发展我们的新技术，用于类固醇受体的分子分析，我们坚信这将彻底改变X射线晶体学在基础研究和药物发现中的应用，特别是关于类固醇受体。具体来说，我们已经确定并产生了一系列的表面突变，稳定的激动剂和拮抗剂配体的构象中看到的雌激素受体。这一进展使我们能够将化合物平行添加到纯化的蛋白质中，并获得载脂蛋白类固醇受体LBD的第一个结构。我们建议将这些技术应用于其他涉及癌症的类固醇受体的高通量技术，并使用这种方法来定义糖皮质激素受体（GR）抑制NF-？B致癌途径。我们相信，这些研究将建立新的和强大的技术，将彻底改变X射线晶体学的使用，以确定小分子如何控制组织和路径选择性信号通过类固醇激素受体。这种“类分析”的方法来研究组的结构是非常新颖的，并允许纳入统计权力的结构分析。重要的是，这种方法还将通过快速提供指导新疗法开发的结构信息，直接影响药物发现过程。

DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the regulation and role of steroid hormone receptors in cancer development and therapeutics, through our development of a new technology that allows for the rapid analysis of steroid hormone receptors using X-ray crystallography. Specifically, our approach has demonstrated that we can increase both the rate and numbers of hits with crystal structures by at least a thousand fold, and that this allows rapid analysis of the ligand-binding domain of these receptors bound to chemotherapy agents, and pathway selective compounds. The estrogen and androgen receptors (ER and AR) are implicated in the development, diagnosis, and treatment for breast and prostate cancer, respectively. Glucocorticoids have a broader role, as up-front therapeutics for the treatment of several malignancies (e.g., leukemia and hormone-refractory prostate cancer), and as adjuvants that reduce the side effects of other chemotherapy agents. The synthetic compounds that target these receptors have, however, significant problems, including acquired resistance and undesirable side effects. They also display tissue and pathway selective signaling that is poorly understood, at both the molecular and structural level. It is possible to develop tissue and pathway selective compounds that ameliorate some of these problems, but there is very little understanding of the structural basis for such selectivity. The lack of good structural models for tissue selectivity is due to the difficulty in producing crystal structures. The steroid receptor ligand-binding domain (LBD) has proven very difficult to crystallize, due to conformational heterogeneity and protein misfolding. Here we propose to further develop our new technology for molecular analyses of steroid receptors, which we strongly believe will revolutionize the use of X-ray crystallography in both basic research and drug discovery, especially regarding steroid receptors. Specifically, we have identified and generated a series of surface mutations that stabilize the estrogen receptor in the conformations seen with both agonist and antagonist ligands. This advance has allowed us to add compounds in parallel to the purified protein, and to obtain the first structure of an apo steroid receptor LBD. We propose to apply these techniques to apply this high-throughput technology to other steroid receptors implicated in cancer, and to use this approach to define the structural basis through which the glucocorticoid receptor (GR) inhibits the NF-?B oncogenic pathway. We believe that these studies will establish new and robust techniques that will revolutionize the use of X-ray crystallography in defining how small molecules control tissue- and pathway-selective signaling through steroid hormone receptors. This "class analysis" approach to studying groups of structures is highly novel, and allows for the incorporation of statistical power into structural analysis. Importantly, this approach will also directly impact the drug discovery process, by rapidly providing structural information that will guide the development of new therapeutics.