Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLC-gamma1).
Name: Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLC-gamma1). ..more
BioActive Compounds: 3123
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute
Assay Provider: Qisheng Zhang, University of North Carolina at Chapel Hill
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01GM098894
Grant Proposal PI: Qisheng Zhang, University of North Carolina at Chapel Hill
External Assay ID: PLCG1_INH_QFRET_1536_1X%INH PRUN
Name: Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLC-gamma1).
Extracellular stimuli including hormones, growth factors, and neurotransmitters promote activation of phospholipase C (PLC) isozymes and cleavage of the membrane lipid phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2) into the classical second messengers, diacylglycerol and inositol 1,4,5- trisphosphate (IP3) . These second messengers coordinately control numerous signaling cascades through the mobilization of intracellular Ca2+ stores and the activation of protein kinase C. Aberrant regulation of PLCs contribute to diverse human diseases including cancer [2-4], cardiovascular diseases [5-6], and neuropathic pain , as well as schizophrenia and epilepsy [5, 8-9]. Consequently, small molecule PLC inhibitors will be valuable pharmacological tools to dissect the roles of PLCs in development and disease, and could potentially serve as candidates for drug development.
1. Harden, T.K. and J. Sondek, Regulation of phospholipase C isozymes by ras superfamily GTPases. Annu Rev Pharmacol Toxicol, 2006. 46: p. 355-79.
2. Bertagnolo, V., et al., Phospholipase C-beta 2 promotes mitosis and migration of human breast cancer-derived cells. Carcinogenesis, 2007. 28(8): p. 1638-45.
3. Sala, G., et al., Phospholipase Cgamma1 is required for metastasis development and progression. Cancer Res, 2008. 68(24): p. 10187-96
4. Shepard, C.R., et al., PLC gamma contributes to metastasis of in situ-occurring mammary and prostate tumors. Oncogene, 2007. 26(21): p. 3020-6.
5. Woodcock, E.A., D.R. Grubb, and P. Iliades, Potential treatment of cardiac hypertrophy and heart failure by inhibiting the sarcolemmal binding of phospholipase Cbeta1b. Curr Drug Targets, 2010. 11(8): p. 1032-40.
6. Zhang, L., et al., Phospholipase C epsilon scaffolds to muscle-specific A kinase anchoring protein (mAKAPbeta) and integrates multiple hypertrophic stimuli in cardiac myocytes. J Biol Chem, 2011. 286(26): p. 23012-21.
7. Kurian, M.A., et al., Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy. Brain, 2010. 133(10): p. 2964-70.
8. Hinkes, B., et al., Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet, 2006. 38(12): p. 1397-405.
9. McOmish, C.E., et al., PLC-beta1 knockout mice as a model of disrupted cortical development and plasticity: behavioral endophenotypes and dysregulation of RGS4 gene expression. Hippocampus, 2008. 18(8): p. 824-34.
10. Bala, G.A., N.R. Thakur, and J.E. Bleasdale, Characterization of the major phosphoinositide-specific phospholipase C of human amnion. Biol Reprod, 1990. 43(4): p. 704-11.
11. Bleasdale, J.E., et al., Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther, 1990. 255(2): p. 756-68.
12. Berven, L.A. and G.J. Barritt, Evidence obtained using single hepatocytes for inhibition by the phospholipase C inhibitor U73122 of store-operated Ca2+ inflow. Biochem Pharmacol, 1995. 49(10): p. 1373-9.
13. Hollywood, M.A., et al., The PI-PLC inhibitor U-73122 is a potent inhibitor of the SERCA pump in smooth muscle. Br J Pharmacol, 2010. 160(6): p. 1293-4.
14. Pulcinelli, F.M., et al., Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets. Biochem Pharmacol, 1998. 56(11): p. 1481-4.
15. Wang, J.P., U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation. Naunyn Schmiedebergs Arch Pharmacol, 1996. 353(6): p. 599-605.
16. Wilsher, N.E., et al., The phosphoinositide-specific phospholipase C inhibitor U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-d ione) spontaneously forms conjugates with common components of cell culture medium. Drug Metab Dispos, 2007. 35(7): p. 1017-22.
17. Burgdorf, C., et al., U73122, an aminosteroid phospholipase C inhibitor, is a potent inhibitor of cardiac phospholipase D by a PIP2-dependent mechanism. J Cardiovasc Pharmacol, 2010. 55(6): p. 555-9.
18. Feisst, C., et al., The aminosteroid phospholipase C antagonist U-73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5- dione) potently inhibits human 5-lipoxygenase in vivo and in vitro. Mol Pharmacol, 2005. 67(5): p. 1751-7.
19. Vickers, J.D., U73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets. J Pharmacol Exp Ther, 1993. 266(3): p. 1156-63.
20. Klein, R.R., et al., Direct activation of human phospholipase C by its well known inhibitor u73122. J Biol Chem, 2011. 286(14): p. 12407-16.
Primary, PRUN, phospholipase C, PLCs, PLC-gamma1, PLCG1, WH-15, fluorogenic reporter, Cholate, biochemical, inhibition, modulators, PLC-gamma1, isozymes, cancer, neurotransmitters, 6-aminoquinoline, quinomethide derivative, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC inhibitor, fluorescence, HTS, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-G1. In this assay, PLC-G1 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-G1 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.
Prior to the start of the assay, 2 microliters of PLC-G1 at a final concentration of 5pg/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.
The percent inhibition for each compound was calculated as follows:
100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Test_Compound is defined as wells containing PLCG1 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCG1, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-11, for inactive 11-0.
List of Reagents:
PLCG1 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
1536-well plates (Corning, part 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)