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)分析方法的发展”(R01)的回应。该计划的宣布是分子图书馆倡议的一部分，也是美国国立卫生研究院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分析”。我们的具体目标是生产和验证用于从分子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作用-即控制翻译后蛋白质折叠，从而控制细胞运输和拯救突变。G蛋白偶联受体(GPCRs)经常是文库筛选的目标，然而这种方法通常依赖于识别激动剂或拮抗剂的筛查，并且可能会错过许多将在建议的筛查中确定的药物。我们的方法在治疗方法上识别出具有相当大程度的新颖性的药物，依赖于目前在分子库分析管道中没有表现出来的细胞机制；这为HTS方法的使用提供了一个尚未开发的机会。这项建议涉及PA认定为相关的特定领域之一，“对分子伴侣或改善蛋白质翻译后靶向、折叠或组装的分子的分析，特别是涉及导致先天新陈代谢错误的突变蛋白质...(或)罕见疾病。”在计划中的研究中，我们将开发和表征药理伴侣的分析方法，以改善导致人类疾病的两个突变GPCR的翻译后折叠和靶向。拟议的HTS检测的产品将用于治疗两种罕见的疾病，肾源性尿崩症和性腺激素减退症，每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GPCRs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。这种分析方法的发展是重要和新颖的，因为激动剂/拮抗剂筛选的广泛使用意味着，现有的文库中可能已经存在具有控制贩运能力的有用的化学结构(没有受体激动剂或拮抗剂)，但还没有使用现有的方法进行鉴定。 公共卫生相关性：我们将开发和表征药理伴侣的分析方法，以改善两种突变受体的翻译后折叠和靶向性，这两种突变受体会导致人类疾病，包括肾源性尿崩症和性腺激素减退症，每一种疾病都是由先天遗传错误引起的。这些筛查也将作为鉴定其他治疗分子的原型，而且很重要，因为现有的筛查可能会错过化学图书馆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.