High-throughput screens to identify modulators of phospholipase C isozymes

高通量筛选以确定磷脂酶 C 同工酶的调节剂

• 批准号: 8337320
• 负责人: JOHN E SONDEK
• 金额: $ 28.12万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-26 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8337320
• 关键词: 1,2-diacylglycerol; Address; Benchmarking; Biological Assay; Biological Process; Blood Vessels; Brain; Breast; Calcium; Cell physiology; Cells; Cellular Membrane; Complement; Development; Diglycerides; Disease; Drug Delivery Systems; Eligibility Determination; Enzymes; Epilepsy; Erythrocytes; Event; Family; Fertilization; Fluorescence; Foundations; Future; Goals; Heart Diseases; Heart failure; Hematopoiesis; Human; Hydrolysis; Immune response; Inositol; Isoenzymes; Kidney Failure; Kinetics; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Membrane Lipids; Methodology; Monitor; Muscle Contraction; Outcome; Pharmaceutical Preparations; Pharmacotherapy; Phosphatidylinositols; Phospholipase; Phospholipase C; Physiological Processes; Production; Prostate; Protein Kinase C; Proteins; Protocols documentation; Radioactive; Radiolabeled; Reagent; Regulation; Relative (related person); Role; Screening Result; Screening procedure; Second Messenger Systems; Series; Signal Transduction; Signaling Molecule; Specificity; Testing; Therapeutic; Time; assay development; base; cell growth; cell motility; drug development; high throughput screening; human disease; inhibitor/antagonist; malignant breast neoplasm; migration; novel; phosphatidylinositol phosphate, PtdIns(4,5)P2; prototype; radiotracer; response; second messenger; small molecule; tumor progression; vasculogenesis

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.

描述（由申请人提供）：人类表达13个磷脂酶C（PLC）同工酶，可以分为六个类（plc-（， - （ - （ - （ - （ - （ - （ - （ - （ - （ - ），基于序列相似性。级联反应造成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，血管生成，脑发育，肌肉收缩和造血，因此，PLC酶的异常调节。 ，由于通过PLC酶不正确的信号传导导致的病理细胞反应，这些酶是主要的药物靶标，胰腺癌，心脏衰竭，肾功能衰竭和癫痫病。对于PLC同工酶，主要是由于缺乏对PLC同工酶的高通量筛选测定，我们描述了一种新型的可溶性小分子WH-15的发展底物Ptdins（4，5）P2产生易于检测的荧光产物。该提案的重点是开发荧光测定法，并将其与一系列辅助测定法相结合，以进行一组完整的高通量筛选方案，以识别PLC活动的调节剂。我们将通过两个目标来实现该提案的目标。在特定的目标1中，我们将使用WH-15优化测定法，以使其适合于384孔格式的高吞吐量屏幕，并通过使用PLC-（2和PLC-（1。在特定的AIM 2）筛选LOPAC1280库来验证它们。 ，我们将使用一系列辅助测定法，以通过完成这些目标来补充AIM 1的荧光测定。为了识别PLC选择性调节剂的高通量筛选。