High-throughput screens to identify modulators of phospholipase C isozymes

描述(申请人提供)：人类表达13种磷脂酶C(PLC)同工酶，根据序列相似性可分为六类(PLC-(、-(和-())。PLC信号通路参与多种细胞和生理过程，包括：细胞运动和迁移、增殖、免疫反应、受精、血管生成、脑发育、肌肉收缩和造血。因此，PLC酶的异常调节会导致多种疾病，如乳腺癌、前列腺癌和胰腺癌、心力衰竭、肾功能衰竭和癫痫。由于通过PLC酶的不正确信号传递导致的病理细胞反应，这些酶是关键的药物靶点。然而，到目前为止，还没有选择性的小分子PLC同工酶抑制剂，这主要是由于缺乏高通量的PLC同工酶筛选方法。在这项提议中，我们描述了一种新型的可溶小分子WH-15的开发，该小分子以与内源PLC底物PtdIns(4，5)P2相似的动力学方式进行水解，以产生易于检测的荧光产物。这项建议的重点是开发一种荧光检测方法，并将其与一系列二次检测相结合，以建立一套完整的高通量筛选方案，以确定PLC活性的调节因子。我们将通过两个目标来实现这项提案的目标。在特定目标1中，我们将使用WH-15优化我们的分析，使其适合于384孔格式的高通量筛选，并通过用PLC-(2和PLC-(1)筛选LOPAC1280文库来验证它们。通过这些目标的完成，我们将第一次拥有一种强大的荧光检测方法和一套适合于高通量筛选的完整的筛选方案，以鉴定PLC选择性调节子。这些小分子可以作为探针来剖析PLC在各种疾病状态下的信号转导，包括乳腺癌和前列腺癌的发生和发展，并作为潜在的药物开发的先导化合物。 与公众健康相关：磷脂酶C(PLC)酶是对生存所需的生理过程至关重要的信号分子，包括：细胞运动、细胞生长、免疫反应、肌肉收缩、血管形成、红细胞发育和脑发育。因此，PLC酶的异常调节会导致多种疾病，如乳腺癌、前列腺癌和胰腺癌、心力衰竭、肾衰竭和癫痫。这项建议的重点是解决目前缺乏有效的针对PLC酶的药物或化合物的问题，方法是优化和执行高通量筛选分析，以确定PLC活性的新调节剂，以i)确定PLC信号在包括乳腺癌和前列腺癌在内的癌症发生和发展中的作用，以及ii)作为潜在的先导化合物开发针对PLC酶的药物治疗。

DESCRIPTION (provided by applicant): Humans express thirteen phospholipase C (PLC) isozymes that can be divided into six classes (PLC-(, -(, -(, -(, -( and -() based upon sequence similarity. PLC signaling cascades are responsible for numerous cellular and physiological processes including: cell motility and migration, proliferation, immune response, fertilization, vasculogenesis, brain development, muscle contraction, and hematopoiesis. Consequently, abnormal regulation of PLC enzymes results in a variety of diseases such as breast, prostate, and pancreatic cancers, cardiac failure, renal failure, and epilepsy. Due to the pathological cellular responses that result from improper signaling through PLC enzymes, these enzymes are key drug targets. However, to date, there are no selective small molecule inhibitors for PLC isozymes, primarily due to the lack of a high-throughput screening assay for PLC isozymes. Within this proposal, we describe the development of a novel, soluble small molecule, WH-15, that is hydrolyzed with similar kinetics as the endogenous PLC substrate, PtdIns (4, 5) P2, to yield an easily detectable fluorescent product. The focus of this proposal is to develop a fluorescent assay and integrate it with a series of secondary assays for a complete set of high-throughput screening protocols to identify modulators of PLC activity. We will accomplish the goals of this proposal through two aims. In Specific Aim 1, we will optimize our assays with WH-15 to enable them suitable for high throughput screens in 384-well format and verify them by screening the LOPAC1280 library with PLC- (2 and PLC- (1. In Specific Aim 2, we will develop a series of secondary assays to complement the fluorescent assay from Aim 1 using a diverse 5000 compound library. Through the completion of these aims, we will for the first time, have a robust, fluorogenic assay and a complete screening protocol suitable for high-throughput screening to identify PLC selective modulators. These small molecules could serve as probes to dissect PLC signaling in various disease states including the development and progression of breast and prostate cancers, and act as potential lead compounds for drug development. PUBLIC HEALTH RELEVANCE: Phospholipase C (PLC) enzymes are signaling molecules that are vital for physiological processes necessary for survival including: cell movement, cell growth, immune response, muscle contraction, blood vessel formation, red blood cell development, and brain development. Consequently, abnormal regulation of PLC enzymes results in a variety of diseases such as breast, prostate, and pancreatic cancers, cardiac failure, kidney failure, and epilepsy. The focus of this proposal is to address the current lack of effective drugs or compounds that target PLC enzymes by optimizing and performing high-throughput screening assays to identify novel modulators of PLC activity to i) determine the role of PLC signaling in the development and progression of cancers including breast and prostate, and ii) serve as potential lead compounds for development of drug therapies targeting PLC enzymes.