Functional Characterization of FKBP52 Interactions with Steroid Hormone Receptors

描述(申请人提供)：Hsp90结合蛋白FKBP52，通过与类固醇激素受体的相互作用，在哺乳动物中发挥重要的生理和潜在的病理作用。FKBP52特异性调节雄激素、孕激素和糖皮质激素受体。因此，FKBP52是治疗激素依赖型癌症(如前列腺癌)的一个有吸引力的治疗靶点。我们已经在了解FKBP52上的功能重要区域方面取得了进展，但我们仍然不知道FKBP52相互作用部位在受体上的什么位置。在这一建议中要检验的主要假设是，FKBP52对受体功能的增强是通过FKBP52的FK1结构域和受体配体结合域之间的直接接触而发生的，从而导致激素结合的增强。受体获得与FKBP52相关联的激素结合构象的动态性质使得使用简单的纯化蛋白系统是不切实际的。因此，我们必须使用更复杂的方法来分析类固醇受体和相关辅助伴侣之间的直接相互作用。下面详述的每个特定目标彼此独立，但都旨在证明FKBP52 FK1结构域和受体LBD之间的作用并针对小分子抑制剂的作用。该项目的长期目标是开发治疗激素依赖型癌症的新药。为了实现这一目标，我们直接的具体目标是：目标1：在酵母中使用先前验证的雄激素受体介导的报告试验，以确定受体内改变受体对FKBP蛋白反应的随机突变。目的#2：利用缺失半胱氨酸的FKBP52突变体的直接光亲和标记结合交联/质谱法鉴定和表征FKBP52-受体相互作用位点。目的#3：筛选选择性FKBP52抑制剂的化合物文库，并鉴定其抑制作用和FKBP52结合位点的特异性。到目前为止，我们已经确定了两种选择性的FKBP52抑制剂。

DESCRIPTION (provided by applicant): The Hsp90 binding protein FKBP52, through interactions with the steroid hormone receptors, plays important physiological and potentially pathological roles in mammals. FKBP52 specifically regulates the androgen, progesterone and glucocorticoid receptors. Thus, FKBP52 represents an attractive therapeutic target for the treatment of hormone-dependent cancers such as prostate cancer. We have made progress in understanding the regions of functional importance on FKBP52, but we still do not know where the FKBP52 interaction site is on the receptors. The main hypothesis to be tested in this proposal is that FKBP52 potentiation of receptor function occurs through direct contacts between the FKBP52 FK1 domain and the receptor ligand binding domain leading to an enhancement of hormone binding. The dynamic nature by which the receptors achieve their hormone bound conformations to which FKBP52 associates makes it impractical to use a simple purified protein system. Thus, we must use more sophisticated approaches to analyze direct interactions between the steroid receptors and associated cochaperones. Each of the specific aims detailed below are independent from each other, but are all aimed at proving the proposed interaction between the FKBP52 FK1 domain and the receptor LBD and targeting that interaction with small molecule inhibitors. The long term goal of this project is the development of novel drugs for the treatment of hormone-dependent cancers. Towards this goal our immediate specific aims are: Aim #1: Use a previously validated androgen receptor-mediated reporter assay in yeast to identify random mutations within the receptor that alter the receptor's response to the FKBP proteins. Aim #2: Use directed photo affinity labeling of a cysteine-lacking FKBP52 mutant in combination with cross linking/mass spectrometry to identify and characterize the FKBP52-receptor interaction site. Aim #3: Screen compound libraries for selective FKBP52 inhibitors and characterize the specificity of inhibition and FKBP52 binding sites. We have identified two selective FKBP52 inhibitors to date.