High Throughput Screening Assay Development for Pharmacoperones

描述（由申请人提供）：本提案是对PA-07-320“高通量药物筛选（HTS）测定法（R01）的开发”的回应。该项目公告是分子文库倡议的一个组成部分，是美国国立卫生研究院医学研究路线图的一部分，并支持分子文库“开发可能最终适用于自动筛选的创新检测方法”。我们的具体目标是生产和验证从分子文库中识别药物的筛选，这些药物通过一种新的治疗方法发挥其作用，即控制翻译后蛋白质折叠，从而实现细胞运输和突变体的拯救。G蛋白偶联受体（gpcr）经常是文库筛选的靶标，然而这种方法通常依赖于识别激动剂或拮抗剂的筛选，并且会错过许多将在拟议筛选中识别的药物。我们的方法在治疗方法中识别具有显著新颖性的药物，依赖于目前在分子文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。该提案解决了PA确定为相关的一个特定领域，“对分子伴侣或分子进行检测，以改善翻译后靶向，折叠或蛋白质组装，特别是涉及负责先天性代谢错误的突变蛋白……（或）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检测方法，以改善导致人类疾病的两种突变gpcr的翻译后折叠和靶向性。提议的HTS检测的产品将用于治疗两种罕见疾病，肾源性尿崩症和促性腺功能低下，每一种疾病都是由先天错误引起的。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的治疗分子。许多疾病现在被认为是由蛋白质错误折叠引起的。这种检测方法的发展是重要的和新颖的，因为单独使用激动剂/拮抗剂筛选意味着具有控制贩运能力的有用化学结构（没有受体激动作用或拮抗作用）可能已经存在于现有文库中，但尚未使用现有方法确定。

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.