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，允许使用x射线晶体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。具体来说，我们的方法已经证明，我们可以将晶体结构的命中率和数量增加至少一千倍，并且这可以快速分析这些与化疗药物和途径选择性化合物结合的受体的配体结合域。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展、诊断和治疗有关。糖皮质激素有更广泛的作用，作为几种恶性肿瘤（如白血病和激素难治性前列腺癌）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知之甚少的组织和途径选择性信号。有可能开发组织和途径选择性化合物来改善这些问题，但对这种选择性的结构基础了解甚少。

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.