Chemical Modulation of Orphan Nuclear Receptor Function

描述（由申请方提供）：本R 01的中心目标是明确定义孕烷X受体（PXR）的新型拮抗剂结合药效团。在这样做时，另外的目标包括开发无毒的唑类拮抗剂，其将用于化学探测不同组织中的PXR活性和表型。随着我们获得有效和特异性的PXR抑制剂，计算机模拟参数将不断改进。然后，这些模型可以指导开发源自不同化学实体的新型小分子拮抗剂。长期目标是最终开发无毒的PXR拮抗剂，其可用作癌细胞增殖和耐药性的临床调节剂（例如，PXR活化诱导癌细胞增殖和耐药性）。还希望这些拮抗剂将增强所选拮抗剂（例如，他莫昔芬、紫杉醇是在人体稳态下观察到浓度的PXR激动剂）。为此，我们已经确定和表征了两种新的PXR拮抗剂，酮康唑和香豆雌酚，特别是破坏激活（配体结合）PXR的功能。在随后的研究中，我们已经证明了酮康唑：（i）与受体结合并破坏活化PXR中的辅调节因子-受体相互作用;（2）不从PXR的配体结合口袋中置换活化药物;（iii）保留对含有配体结合口袋填充突变体的PXR突变形式的拮抗作用;和（iv）不能拮抗含有表面辅助调节因子AF-2结合位点改变的PXR突变形式。因此，我们已经制定了一个模型PXR拮抗作用，其中功能的破坏是介导的受体的变构修饰或竞争与辅调节结合。我们现在建议使用结构、分子、生物化学和遗传系统来评估该模型，以表征PXR定向拮抗剂酮康唑和相关化合物抑制受体活化的机制。公共卫生相关性：孕烷X受体（Pregnane X Receptor，PXR）是一种孤儿核受体，在药物相互作用和肿瘤耐药中起着重要作用。受体的这些功能是新发现的，并且由于该受体具有器官特异性功能，因此其在其他组织中的许多作用（例如，），是未知的。PXR被激动剂（配体）激活，被拮抗剂灭活。我们的实验室和合作研究者在定义与配体结合口袋外的区域结合的PXR新型拮抗剂方面发挥了主导作用。我们相信，这些拮抗剂将是必要的探针PXR功能和定义的所有表型的PXR在小鼠和人类。在这个建议中，我们将使用结构、分子、生物化学和遗传系统来评估我们的拮抗模型。这些研究对于产生拮抗PXR功能的无毒化合物至关重要-这是探测细胞中PXR功能所必需的工具。

DESCRIPTION (provided by applicant): The central goal of this R01 is to focus on explicitly defining novel antagonist binding pharmacophore on Pregnane X Receptor (PXR). In doing so, additional goals include development of non-toxic azole antagonists that would serve to chemically probe PXR activity and phenotype(s) in different tissues. In silico modeling parameters will continuously be improved as we obtain potent and specific PXR inhibitors. These models could then guide the development of novel small molecule antagonists originating from different chemical entities. The long-term goal is to eventually develop non-toxic antagonists of PXR that can be used as clinical modulators of cancer cell proliferation and drug resistance (e.g., PXR activation induces cancer cell proliferation and drug resistance). It is also hoped that these antagonists will enhance the activity, and minimize the toxicity, of select antineoplastic agents (e.g., tamoxifen, paclitaxel are PXR agonist at concentrations observed at steady-state in humans). Towards this end, we have identified and characterized two novel PXR antagonists, ketoconazole and coumestrol, that specifically disrupt the function of activated (ligand-bound) PXR. In subsequent studies, we have shown that ketoconazole: (i) binds to receptor and disrupts coregulator-receptor interactions in activated PXR; (2) does not displace activating drugs from the ligand-binding pocket of PXR; (iii) retained antagonism of mutant forms of PXR containing ligand-binding pocket filling mutants; and (iv) is unable to antagonize mutant forms of PXR containing alterations in the surface coregulator AF-2 binding site. Thus, we have formulated a model for PXR antagonism in which disruption of function is mediated either by allosteric modification of the receptor or by competition with coregulator binding. We now propose to evaluate this model using structural, molecular, biochemical, and genetic systems to characterize the mechanism by which PXR-directed antagonist ketoconazole and related compounds inhibit receptor activation. PUBLIC HEALTH RELEVANCE: Adopted orphan nuclear receptor, Pregnane X Receptor (PXR), plays a mechanistic role in drug-drug interactions and cancer drug resistance. These functions of the receptor are newly discovered and since this receptor has organ-specific functions, many of its roles in other tissues (e.g., gut) are unknown. PXR is activated by agonists (ligands) and inactivated by antagonists. Our laboratory and co-investigators have taken a lead role in defining novel antagonists of PXR that bind to a region outside the ligand binding pocket. We believe that these antagonists will be necessary to probe PXR function and define all the phenotypes of PXR in mice and humans. In this proposal, we will evaluate our model of antagonism using structural, molecular, biochemical, and genetic systems. These studies will be vital for the generation on non-toxic compounds that antagonize PXR function - a tool necessary to probe PXR function in cells.