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BioAssay: AID 743328

Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of phospholipase C isozymes (PLC-B3)

Name: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of phospholipase C isozymes (PLC-B3). ..more
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 Tested Compounds
 Tested Compounds
All(157)
 
 
Inactive(157)
 
 
 Tested Substances
 Tested Substances
All(157)
 
 
Inactive(157)
 
 
AID: 743328
Data Source: The Scripps Research Institute Molecular Screening Center (PLCB3_INH_QFRET_1536_3XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2014-03-24
Hold-until Date: 2014-03-27
Modify Date: 2014-03-27

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
720704Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLC-beta3).Screeningdepositor-specified cross reference: Primary assay (PLCB3 Inh in singlicate)
720731Summary of the probe development effort to identify inhibitors of phospholipase C isozymes (PLC-beta 3).Summarydepositor-specified cross reference: PLCB3 summary
743261Fluorescence-based biochemical high throughput confirmation assay to identify inhibitors of phospholipase C isozymes (PLC-B3)Screeningdepositor-specified cross reference: Confirmatio assay (PLCB3 INH in triplicate)
743258Counterscreen for inhibitors of phospholipase C isozymes (PLC-B3): Fluorescence-based biochemical high throughput assay to identify inhibitors of phospholipase C isozymes (PLC-gamma1)Screeningsame project related to Summary assay
743329Counterscreen for inhibitors of phospholipase C isozymes (PLC-B3): Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of phospholipase C isozymes (PLC-gamma1)Confirmatorysame project related to Summary assay
Description:
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: PLCB3_INH_QFRET_1536_3XIC50 DRUN

Name: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of phospholipase C isozymes (PLC-B3).

Description:

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) [1]. 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 [7], 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.

References:

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.

Keywords:

DRUN, dose response, dose, dilution, triplicate, biochemical, endpoint, end-point, phospholipase C, PLCs, PLC-B3, PLCB3, WH-15, fluorogenic reporter, Cholate, biochemical, inhibition, modulators, PLC-G1, 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
Protocol
Assay Overview:

The purpose of this biochemical assay is to determine PLC-B3 inhibitory dose response curves of compounds that confirm activity in a set of previous experiments entitled: "Fluorescence-based biochemical high throughput confirmation assay to identify inhibitors of phospholipase C isozymes (PLC-B3)." (AID 743261). In this assay, PLC-B3 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-B3 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 triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 122.5 uM. (SIDs 14730721, 14742430, 24788424, 24809205, 24841228, 26663487, 49665443, 50085435, 92764458, 93576637, and 93577326 were tested as sextuplicates.)

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-B3 at a final concentration of 0.4ng/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 12.3 uM) 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:

%_Inhibition = 100 *( ( Test_Compound - Median_Low_Control) / ( Median_High_Control - Median_Low_Control) )

Where:

Test_Compound is defined as wells containing PLCB3 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCB3, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 122.5uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 122.5uM.

The activity score range for inactive compounds is 0-0, there are no active compounds.

List of Reagents:

PLCB3 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXT Bio)
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)
Comment
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. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Qualifierinhibition Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the inhibition in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
8RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
9Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
10Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
11Response RangeThe range of Y.Float
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.0062 uM [1] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [1]Float%
14Inhibition at 0.0062 uM [2] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [2]Float%
15Inhibition at 0.0062 uM [3] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [3]Float%
16Inhibition at 0.0062 uM [4] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [4]Float%
17Inhibition at 0.0062 uM [5] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [5]Float%
18Inhibition at 0.0062 uM [6] (0.0062μM**)Value of % inhibition at 0.0062 uM compound concentration; replicate [6]Float%
19Inhibition at 0.019 uM [1] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [1]Float%
20Inhibition at 0.019 uM [2] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [2]Float%
21Inhibition at 0.019 uM [3] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [3]Float%
22Inhibition at 0.019 uM [4] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [4]Float%
23Inhibition at 0.019 uM [5] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [5]Float%
24Inhibition at 0.019 uM [6] (0.019μM**)Value of % inhibition at 0.019 uM compound concentration; replicate [6]Float%
25Inhibition at 0.056 uM [1] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [1]Float%
26Inhibition at 0.056 uM [2] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [2]Float%
27Inhibition at 0.056 uM [3] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [3]Float%
28Inhibition at 0.056 uM [4] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [4]Float%
29Inhibition at 0.056 uM [5] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [5]Float%
30Inhibition at 0.056 uM [6] (0.056μM**)Value of % inhibition at 0.056 uM compound concentration; replicate [6]Float%
31Inhibition at 0.17 uM [1] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [1]Float%
32Inhibition at 0.17 uM [2] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [2]Float%
33Inhibition at 0.17 uM [3] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [3]Float%
34Inhibition at 0.17 uM [4] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [4]Float%
35Inhibition at 0.17 uM [5] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [5]Float%
36Inhibition at 0.17 uM [6] (0.17μM**)Value of % inhibition at 0.17 uM compound concentration; replicate [6]Float%
37Inhibition at 0.5 uM [1] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [1]Float%
38Inhibition at 0.5 uM [2] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [2]Float%
39Inhibition at 0.5 uM [3] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [3]Float%
40Inhibition at 0.5 uM [4] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [4]Float%
41Inhibition at 0.5 uM [5] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [5]Float%
42Inhibition at 0.5 uM [6] (0.5μM**)Value of % inhibition at 0.5 uM compound concentration; replicate [6]Float%
43Inhibition at 1.5 uM [1] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [1]Float%
44Inhibition at 1.5 uM [2] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [2]Float%
45Inhibition at 1.5 uM [3] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [3]Float%
46Inhibition at 1.5 uM [4] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [4]Float%
47Inhibition at 1.5 uM [5] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [5]Float%
48Inhibition at 1.5 uM [6] (1.5μM**)Value of % inhibition at 1.5 uM compound concentration; replicate [6]Float%
49Inhibition at 4.5 uM [1] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [1]Float%
50Inhibition at 4.5 uM [2] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [2]Float%
51Inhibition at 4.5 uM [3] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [3]Float%
52Inhibition at 4.5 uM [4] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [4]Float%
53Inhibition at 4.5 uM [5] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [5]Float%
54Inhibition at 4.5 uM [6] (4.5μM**)Value of % inhibition at 4.5 uM compound concentration; replicate [6]Float%
55Inhibition at 13.6 uM [1] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [1]Float%
56Inhibition at 13.6 uM [2] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [2]Float%
57Inhibition at 13.6 uM [3] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [3]Float%
58Inhibition at 13.6 uM [4] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [4]Float%
59Inhibition at 13.6 uM [5] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [5]Float%
60Inhibition at 13.6 uM [6] (13.6μM**)Value of % inhibition at 13.6 uM compound concentration; replicate [6]Float%
61Inhibition at 40.8 uM [1] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [1]Float%
62Inhibition at 40.8 uM [2] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [2]Float%
63Inhibition at 40.8 uM [3] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [3]Float%
64Inhibition at 40.8 uM [4] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [4]Float%
65Inhibition at 40.8 uM [5] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [5]Float%
66Inhibition at 40.8 uM [6] (40.8μM**)Value of % inhibition at 40.8 uM compound concentration; replicate [6]Float%
67Inhibition at 122.5 uM [1] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [1]Float%
68Inhibition at 122.5 uM [2] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [2]Float%
69Inhibition at 122.5 uM [3] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [3]Float%
70Inhibition at 122.5 uM [4] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [4]Float%
71Inhibition at 122.5 uM [5] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [5]Float%
72Inhibition at 122.5 uM [6] (122.5μM**)Value of % inhibition at 122.5 uM compound concentration; replicate [6]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R01GM098894

Data Table (Concise)
Data Table ( Complete ):     View All Data
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