Chemical Modulation of Orphan Nuclear Receptor Function

描述(由申请人提供)：本R01的中心目标是明确定义孕烷X受体(PXR)上新的拮抗剂结合药效团。为此，其他目标包括开发无毒的唑类拮抗剂，用于化学探测不同组织中的PXR活性和表型(S)。在计算机模拟中，随着我们获得有效和特异的PXR抑制剂，参数将不断改进。这些模型可以指导来自不同化学实体的新型小分子拮抗剂的开发。长期目标是最终开发无毒的PXR拮抗剂，可用作癌细胞增殖和耐药性的临床调节剂(例如，PXR激活诱导癌细胞增殖和耐药性)。人们还希望这些拮抗剂能增强特定抗肿瘤药物的活性，并将毒性降至最低(例如，他莫昔芬、紫杉醇是PXR激动剂，在人体内观察到的稳态浓度)。为此，我们已经鉴定并鉴定了两种新的PXR拮抗剂，酮康唑和库美斯特罗，它们可以特异性地破坏激活的(配体结合的)PXR的功能。在随后的研究中，我们发现酮康唑：(I)与受体结合并破坏激活的PXR中辅助调节因子-受体的相互作用；(2)不取代PXR配体结合口袋中的激活药物；(Iii)保留含有配体结合口袋填充突变体的PXR突变形式的拮抗作用；以及(Iv)不能拮抗含有表面辅助调节因子AF-2结合位点变化的PXR突变形式。因此，我们建立了一个PXR拮抗模型，在该模型中，功能的中断要么通过受体的变构修饰，要么通过与辅调节因子结合的竞争来介导。我们现在建议使用结构、分子、生化和遗传系统来评估这一模型，以表征PXR导向的拮抗剂酮康唑和相关化合物抑制受体激活的机制。公共卫生相关性：采用的孤儿核受体孕烷X受体(PXR)在药物相互作用和癌症耐药性中发挥机制作用。该受体的这些功能是新发现的，由于该受体具有器官特有的功能，因此它在其他组织(如肠道)中的许多作用尚不清楚。PXR由激动剂(配体)激活，由拮抗剂失活。我们的实验室和合作研究人员在定义与配体结合口袋外的区域结合的新型PXR拮抗剂方面发挥了主导作用。我们认为，这些拮抗剂将是探索PXR功能和确定PXR在小鼠和人类中的所有表型所必需的。在这个提案中，我们将使用结构、分子、生化和遗传系统来评估我们的拮抗模型。这些研究对于产生无毒的拮抗PXR功能的化合物至关重要--PXR功能是探索细胞中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.