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“高通量筛查(HTS)发现化学探针(R01)的分析征集”的回应。这一融资机会支持学术、非营利或商业HTS筛查机构之间的合作，这些机构拥有必要的专业知识和经验，可以实施HTS就绪测试，以发现和开发小分子化学探测器。拟议的工作将由开发药物操作酮概念(和化验)的实验室与另外两个在高温超导方面拥有丰富经验并可以访问大型化学图书馆的实验室合作进行。我们将使用一种现有的、经过充分验证的高通量试验来鉴定加压素2型(V2)受体(V2R)的药物操纵子。该检测将用于筛选约64万种化合物的库，并将识别用于治疗肾源性尿崩症V2R错误路线的治疗分子。尿崩症是一种罕见的、使人衰弱的疾病，目前还没有有效的药物治疗方法。药物操纵素是靶向特异性的小分子，它扩散到细胞内，拯救错误折叠的蛋白质突变，并使它们恢复功能。挽救是基于一种新认识的机制：纠正突变的细胞路线，否则这些突变将保留在内质网中，无法发挥作用。由于这类筛查的新颖性，该项目还将作为鉴定大型化学图书馆中存在的药物操作酮的原型。因此，建议的方法在治疗方法上识别出具有显著新颖性的药物，依赖于目前在分子文库分析流水线中没有出现的细胞机制；这项技术为HTS方法的使用提供了一个尚未开发的机会。由于有用的化学结构具有控制细胞的能力，因此这种分析方法的发展是重要的和新颖的 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，该分析已成功转移到HTS设施，早期筛选结果表明，所提出的方法很可能在鉴定HITS方面取得成功。

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.