High-throughput screens to identify modulators of phospholipase C isozymes

描述（由申请人提供）：人类表达13种磷脂酶C （PLC）同工酶，根据序列相似性可分为6类(PLC-(, -(, -(, -(, -(, -), -（和-()）。PLC信号级联负责许多细胞和生理过程，包括：细胞运动和迁移、增殖、免疫反应、受精、血管发生、大脑发育、肌肉收缩和造血。因此，PLC酶的异常调节导致多种疾病，如乳腺癌、前列腺癌和胰腺癌、心力衰竭、肾衰竭和癫痫。由于通过PLC酶的不正确信号传导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，还没有针对PLC同工酶的选择性小分子抑制剂，这主要是由于缺乏针对PLC同工酶的高通量筛选试验。在本提案中，我们描述了一种新型可溶性小分子WH-15的开发，该分子与内源性PLC底物PtdIns (4,5) P2具有相似的水解动力学，以产生易于检测的荧光产物。该提案的重点是开发一种荧光分析方法，并将其与一系列二级分析相结合，形成一套完整的高通量筛选方案，以确定PLC活性的调节剂。我们将通过两个目标来实现这个建议的目标。在Specific Aim 1中，我们将使用wh15优化我们的分析，使其适用于384孔格式的高通量筛选，并通过使用PLC-(2)和PLC-(1)筛选LOPAC1280文库来验证它们。在Specific Aim 2中，我们将使用不同的5000种化合物文库开发一系列二级分析来补充Aim 1中的荧光分析。通过完成这些目标，我们将首次拥有一个强大的荧光分析和一个完整的筛选方案，适用于高通量筛选，以确定PLC选择性调制器。这些小分子可以作为探针来解剖各种疾病状态下的PLC信号，包括乳腺癌和前列腺癌的发生和进展，并作为药物开发的潜在先导化合物。

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.