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、免疫应答、受精、血管发生、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酶的不当信号传导导致的病理性细胞反应，这些酶是关键的药物靶标。然而，到目前为止，没有选择性的小分子抑制剂PLC同工酶，主要是由于缺乏一个高通量筛选测定PLC同工酶。在这个建议中，我们描述了一种新的，可溶性的小分子，WH-15，水解与内源性PLC底物，PtdIns（4，5）P2，以产生一个容易检测的荧光产物类似的动力学的发展。该提案的重点是开发一种荧光检测方法，并将其与一系列二级检测方法相结合，以获得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本提案的目标。在具体目标1中，我们将用WH-15优化我们的测定，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LOPAC 1280文库来验证它们。在特定目标2中，我们将开发一系列二级检测，以补充目标1中使用不同的5000种化合物库的荧光检测。通过这些目标的完成，我们将首次拥有一个强大的荧光检测和一个完整的筛选方案，适用于高通量筛选，以确定PLC选择性调节剂。这些小分子可以作为探针来剖析各种疾病状态中的PLC信号传导，包括乳腺癌和前列腺癌的发展和进展，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.