Chemical Modulation of Orphan Nuclear Receptor Function

项目总结/摘要 本R 01的中心目标是明确定义新型拮抗剂结合 孕烷X受体（PXR）的药效团。在这样做时，其他目标包括 开发无毒的唑类拮抗剂，用于化学探测PXR 不同组织中的活性和表型。计算机模拟建模参数将 随着我们获得有效和特异性的PXR抑制剂，我们将不断改进。这些 模型可以指导新型小分子拮抗剂的开发， 来自不同的化学物质。长期目标是最终发展 可用作癌细胞临床调节剂的PXR的无毒拮抗剂 增殖和耐药性（例如，PXR激活诱导癌细胞增殖 耐药性）。还希望这些拮抗剂将增强活性， 并使毒性最小化，选择的抗肿瘤剂（例如，他莫昔芬，紫杉醇 PXR激动剂在人体稳态时观察到的浓度）。为此， 我们已经鉴定和表征了两种新的PXR拮抗剂，酮康唑和 香豆雌酚，其特异性地破坏激活的（配体结合的）PXR的功能。在 随后的研究表明，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）不取代活化药物 从配体结合口袋的PXR;（iii）保留拮抗突变形式的PXR 含有配体结合口袋填充突变体;和（iv）不能拮抗突变体 PXR的形式包含表面辅助调节因子AF-2结合位点的改变。因此，在本发明中， 我们已经建立了一个PXR拮抗作用的模型，其中功能的破坏是 通过受体的变构修饰或通过与 辅调节子结合。我们现在建议使用结构，分子， 生物化学和遗传系统来表征PXR指导的 拮抗剂酮康唑和相关化合物抑制受体活化。

Project Summary/Abstract 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.