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通过我们开发的一种新技术，利用X射线结晶学快速分析类固醇激素受体，了解类固醇激素受体在癌症发展和治疗中的调节和作用。具体地说，我们的方法已经证明，我们可以将具有晶体结构的命中率和命中率提高至少1000倍，这使得能够快速分析这些受体与化疗药物和途径选择性化合物结合的配体结合域。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(如白血病和激素难治的前列腺癌)的前期治疗药物，以及作为减少其他化疗药物副作用的佐剂。然而，针对这些受体的合成化合物存在重大问题，包括获得性耐药性和不良副作用。它们还显示了在分子和结构水平上鲜为人知的组织和途径选择性信号。开发组织和途径选择性化合物来改善其中一些问题是可能的，但人们对这种选择性的结构基础了解很少。 缺乏良好的组织选择性结构模型是由于制造晶体结构的困难。由于构象异质性和蛋白质错误折叠，类固醇受体配体结合结构域(LBD)很难结晶。在这里，我们建议进一步发展我们的类固醇受体分子分析新技术，我们坚信这将彻底改变X射线结晶学在基础研究和药物发现中的使用，特别是在类固醇受体方面。具体地说，我们已经确定并产生了一系列表面突变，这些突变稳定了激动剂和拮抗剂配体的构象中的雌激素受体。这一进展使我们能够在纯化蛋白的基础上平行添加化合物，并获得载脂蛋白类固醇受体LBD的第一结构。我们建议将这些高通量技术应用于与癌症有关的其他类固醇受体，并使用这种方法来确定糖皮质激素受体(GR)抑制NF-B致癌途径的结构基础。我们相信，这些研究将建立新的和强大的技术，将革命性地使用X射线结晶学来定义小分子如何通过类固醇激素受体控制组织和路径选择信号。这种研究结构群的“类分析”方法是非常新颖的，并且允许将统计能力结合到结构分析中。重要的是，这种方法还将通过迅速提供指导新疗法开发的结构性信息，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.