High Throughput Screening for Pharmacoperones of the V2 Receptor

描述（由申请人提供）：本提案是对PAR-12-058“高通量筛选（HTS）检测方法征集以发现化学探针（R 01）”的回应。“这个资助机会支持学术，非营利或商业HTS筛选设施之间的合作，这些设施具有必要的专业知识和经验，可以实施用于发现和开发小分子化学探针的HTS就绪检测。拟议的工作将由开发药物操作素概念（和测定）的实验室与另外两个在HTS方面具有丰富经验并可访问大型化学品库的实验室合作进行。我们将使用现有的和经过充分验证的高通量测定来鉴定加压素2型（V2）受体（V2 R）的药物操纵子。该试验将用于筛选约640，000种化合物的库，并将鉴定用于治疗肾源性尿崩症中V2 R的错误路由的治疗分子，这是一种罕见且使人衰弱的疾病，目前没有有效的药物治疗。药物操纵子是靶向特异性的小分子，可扩散到细胞中，拯救错误折叠的蛋白质突变体并使其恢复功能。拯救是基于一个新认识的机制：纠正细胞路由的突变，否则将保留在内质网和无法发挥作用。由于这种类型的屏幕的新颖性，该项目也将作为一个原型，用于识别存在于大型化学图书馆的药物。因此，所提出的方法鉴定了在治疗方法中具有显著新奇的药物，其依赖于目前在分子库测定管道中不存在的细胞机制;该技术为使用HTS方法提供了未开发的机会。这种测定法的开发是重要的和新颖的，因为具有控制细胞凋亡能力的有用化学结构是已知的。 贩运可能已经存在于现有的图书馆中，但尚未使用现有的屏幕进行识别。初步数据显示，该检测试剂盒已成功转移到HTS设施，早期筛选结果显示，所提出的方法很可能成功识别命中。

DESCRIPTION (provided by applicant): This proposal is a response to PAR-12-058, "Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes (R01)." This funding opportunity supports collaboration between academic, nonprofit, or commercial HTS screening facilities that have the requisite expertise and experience to implement an HTS-ready assay for the discovery and development of small molecule chemical probes. The work proposed will be conducted by collaboration between the laboratory that developed the concept of pharmacoperones (and the assay) and two other laboratories with extensive experience in HTS and access to large chemical libraries. We will use an existing and well-validated high throughput assay to identify pharmacoperones of the vasopressin type 2 (V2) receptor (V2R). The assay will be applied to screen a library of approximately 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the V2R in nephrogenic diabetes insipidus, a rare and debilitating disease for which there are presently no effective drug treatments. Pharmacoperones are target-specific and small molecules that diffuse into cells, rescue misfolded protein mutants and restore them to function. Rescue is based on a newly appreciated mechanism: correcting the cellular routing of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function. Because of the newness of this type of screen, this project will also serve as a prototype for the identification of pharmacoperones present in large chemical libraries. Accordingly, the proposed approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this technique offers an untapped opportunity for use of the HTS approach. Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking may already be present in existing libraries, but have not been identified using existing screens. Preliminary data show that the assay was successfully transferred to the HTS facility and early screening results show that the proposed approach is likely to be successful in identification of hits.