Identification of Small Molecules that Influence Trafficking of the GnRH Receptor

项目摘要 本提案是对PAR-14-284“高吞吐量筛选（HTS） 发现化学探针（R 01）。此次融资机会支持了 具有必要专业知识的学术、非营利或商业HTS筛查机构 和经验，以实现HTS准备检测的发现和发展的小 分子化学探针拟议的工作将由以下机构合作进行： Conn实验室开发了药物操作素（和测定）的概念， Scripps研究所的实验室在HTS药物发现方面拥有丰富的经验， 药物化学、药物代谢和药代动力学（DMPK）。我们将使用 现有的和充分验证的高通量测定，以确定药物的 促性腺激素释放激素（GnRH）受体（GnRHR）。 药物操纵子是靶向特异性的小分子，可扩散到细胞中， 错误折叠的蛋白质突变体，并恢复它们的功能。救援是基于一个新的 赞赏机制：纠正突变体的细胞路由，否则将是 滞留在内质网中而不能发挥作用。这些药物的功效 在细胞培养和体内都得到了证实。该测定将用于筛选 超过640，000种化合物的库，并将确定治疗分子， 治疗生殖疾病中GnRHR的错误路由。其中一种疾病是 下丘脑性腺功能减退症（HH）。HH是一个疾病家族，其中一个子集由以下原因引起： GnRHR突变导致内质网滞留。除了 增加突变体在质膜上的表达， 受体增加WT GnRHR的质膜表达。只有50%的合成 人GnRHR通常在质膜上表达，并且该百分比可以是 增加了pharmacoperones。因此，这些药物可用于治疗 这种受体表达不理想的疾病和GnRH是促性腺激素释放激素受体的癌症。 治疗靶点由于这种屏幕的新颖性，该项目还将作为 用于鉴定大型化学文库中存在的药物操纵子的原型。 因此，所提出的方法确定了具有显著新奇的药物， 治疗方法，依赖于细胞机制，目前没有代表在 分子库分析管道;这项技术提供了一个未开发的机会， HTS方法。这种测定法的开发是重要的和新颖的，因为有用的化学物质 有能力控制细胞贩运的结构可能已经存在于现有的 库，但尚未使用现有屏幕识别。初步数据显示， 检测成功转移到HTS设施，早期筛选结果显示， 所提出的方法很可能在识别易处理的命中中是成功的。

Project Summary This proposal is a resubmission in response to PAR-14-284, “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 Conn laboratory that developed the concept of pharmacoperones (and the assay) and the laboratories at Scripps Research Institute with extensive experience in HTS Drug Discovery, Medicinal Chemistry, and Drug Metabolism and Pharmacokinetics (DMPK). We will use an existing and well-validated high throughput assay to identify pharmacoperones of the gonadotropin releasing hormone (GnRH) receptor (GnRHR). 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. The efficacy of these drugs has been demonstrated both in cell cultures and in vivo. The assay will be applied to screen a library of more than 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the GnRHR in diseases of reproduction. One such disease is hypothalamic hypogonadism (HH). HH is a family of disorders, a subset of which is caused by mutations in the GnRHR which result in retention in the endoplasmic reticulum. In addition to increasing the expression of mutants at the plasma membrane, pharmacoperones of this receptor increase plasma membrane expression of WT GnRHR. Only 50% of the synthesized human GnRHR is normally expressed at the plasma membrane and this percent can be increased by pharmacoperones. Accordingly, these drugs may be used in treatment of disorders in which this receptor is sub optimally expressed and cancers in which GnRH is a therapeutic target. 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 tractable hits.