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.
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.
|Charpentier, Thomas H; Waldo, Gary L; Barrett, Matthew O et al. (2014) Membrane-induced allosteric control of phospholipase C-? isozymes. J Biol Chem 289:29545-57|
|Huang, Weigang; Proctor, Angela; Sims, Christopher E et al. (2014) Fluorous enzymatic synthesis of phosphatidylinositides. Chem Commun (Camb) 50:2928-31|
|Hajicek, Nicole; Charpentier, Thomas H; Rush, Jeremy R et al. (2013) Autoinhibition and phosphorylation-induced activation of phospholipase C-? isozymes. Biochemistry 52:4810-9|
|Huang, Weigang; Barrett, Matthew; Hajicek, Nicole et al. (2013) Small molecule inhibitors of phospholipase C from a novel high-throughput screen. J Biol Chem 288:5840-8|
|Wang, Xiaoyang; Barrett, Matthew; Sondek, John et al. (2012) Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C. Biochemistry 51:5300-6|