High Throughput Screening Assay Development for Pharmacoperones

描述（由申请方提供）：本提案是对PA-07-320“高通量药物筛选（HTS）试验的开发（R 01）”的回应。该计划公告是分子库计划的一个组成部分，是NIH医学研究路线图的一部分，并支持“开发最终可能适用于分子库自动筛选的创新检测方法”。我们的具体目标是生产和验证筛选鉴定药物的分子库，发挥其作用的一个新的赞赏治疗方法，即，控制翻译后蛋白质折叠，因此，细胞运输和拯救突变体。G蛋白偶联受体（GPCR）经常在文库筛选中被靶向，然而这种方法通常依赖于鉴定激动剂或拮抗剂的筛选，并且会错过将在所提出的筛选中鉴定的许多药物。我们的方法鉴定了在治疗方法中具有显著新奇的药物，依赖于目前在分子库检测管道中没有代表的细胞机制;这为使用HTS方法提供了一个尚未开发的机会。该提案涉及PA确定为相关的具体领域之一，“分子伴侣或分子的测定，这些分子可改善蛋白质的翻译后靶向、折叠或组装，特别是涉及导致先天性代谢缺陷的突变蛋白质. (or)罕见疾病。“在计划的研究中，我们将开发和表征药理学伴侣的测定方法，这些方法可以改善导致人类疾病的两种突变GPCR的翻译后折叠和靶向。所提出的HTS测定的产品将用于治疗两种罕见疾病，肾源性尿崩症和低促性腺激素性性腺功能减退症，每种疾病均由先天性缺陷引起。这些筛选也将作为鉴定其他治疗分子的原型，特别是那些涉及GPCR的分子。许多疾病现在被认为是由蛋白质错误折叠引起的。这种测定的开发是重要的和新颖的，因为激动剂/拮抗剂筛选的单独广泛使用意味着具有控制运输（没有受体激动或拮抗）能力的有用化学结构可能已经存在于现有文库中，但尚未使用现有方法鉴定。 公共卫生相关性：我们将开发和表征用于药理学分子伴侣的测定，所述药理学分子伴侣改善两种突变受体的翻译后折叠和靶向，所述突变受体引起人类疾病，包括肾源性尿崩症和低促性腺激素性性腺功能减退症，每种疾病均由先天遗传错误引起。这些筛选也将作为识别其他治疗分子的原型，并且很重要，因为现有的筛选可能会错过化学库中已经存在的潜在有价值的药物。

DESCRIPTION (provided by applicant): This proposal is a response to PA-07-320, "Development of Assays for High-throughput Drug Screening (HTS) (R01)." The Program Announcement is a component of the Molecular Libraries Initiative, part of the NIH Roadmap for Medical Research, and supports "the development of innovative assays that may ultimately be adapted for automated screening" of molecular libraries. Our specific aim is to produce and validate screens for identification of drugs from molecular libraries which exert their actions by a newly appreciated therapeutic approach-namely, control of post-translational protein folding and, as a consequence, cellular trafficking and rescue of mutants. G protein coupled receptors (GPCRs) are frequently targeted in library screening, yet this approach generally relies on screens that identify agonists or antagonists and would have missed many of the drugs that will be identified in the proposed screens. Our 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 offers an untapped opportunity for use of the HTS approach. This proposal addresses one of the specific areas that the PA identifies as relevant, "assays for molecular chaperones or molecules that improve the post- translational targeting, folding or assembly of proteins, especially involving mutant proteins responsible for inborn errors of metabolism... (or) rare diseases." In the planned studies we will develop and characterize assays for pharmacological chaperones that improve the post-translational folding and targeting of two mutant GPCRs which cause human disease. The products of the proposed HTS assays will be useful for treatment of two rare diseases, nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn error. These screens will also serve as prototypes for identification of other therapeutic molecules, especially those involving GPCRs. Many diseases are now understood to be caused by protein misfolding. Development of such assays is important and novel since the extensive use of agonist/antagonist screens alone means that useful chemical structures with the ability to control trafficking (without receptor agonism or antagonism) may already be present in existing libraries, but have not been identified using existing methods. PUBLIC HEALTH RELEVANCE: We will develop and characterize assays for pharmacological chaperones that improve the post- translational folding and targeting of two mutant receptors which cause human diseases, including nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn genetic error. These screens will also serve as prototypes for identification of other therapeutic molecules, and are important because existing screens would have missed potentially valuable drugs already present in chemical libraries.