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BioAssay: AID 588769

Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assay for HTS compounds

Name: Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assay for HTS compounds. ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 588769
Data Source: The Scripps Research Institute Molecular Screening Center (pPAFAH_INH_FLUO_GELBASEDABPP_3XIC50_HTSHITS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-11-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
463082Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH)Screening depositor-specified cross reference: Primary screen (pPAFAH inhibitors in singlicate)
463092Summary of probe development efforts to identify inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH)Summary1 depositor-specified cross reference: Summary (pPAFAH inhibitors)
463230Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH; PLA2G7)Screening depositor-specified cross reference: Confirmation screen (pPAFAH inhibitors in triplicate)
588474Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAHOther depositor-specified cross reference: ABPP Screen (pPAFAH inhibitors in singlicate)
588785Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: LC-MS/MS-based biochemical ABPP-MudPIT assay to determine selectivity of test compounds in the mouse brain membrane proteomeOther depositor-specified cross reference
588787Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: LC-MS/MS-based biochemical ABPP-MudPIT assay to determine selectivity of test compounds in the mouse brain soluble proteomeOther depositor-specified cross reference
588788Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: LC-MS/MS-based biochemical assay to determine binding mode of test compoundsOther depositor-specified cross reference
588789Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH in vivoOther depositor-specified cross reference
588817Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH in situOther depositor-specified cross reference
588823Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH and selectivity analysis in vivoOther depositor-specified cross reference
492956Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Screening same project related to Summary assay
588766Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of human pPAFAHConfirmatory same project related to Summary assay
588767Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of PAFAH2Other same project related to Summary assay
588768Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory same project related to Summary assay
588770Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based dose-response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assay for SAR compoundsConfirmatory same project related to Summary assay
588773Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity of HTS compounds in a complex proteomeOther same project related to Summary assay
588774Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity of SAR compounds in a complex proteomeOther same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Providers: Brian Bahnson (Univ. of Delaware); Benjamin Cravatt, (TSRI)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R01HL084366
Grant Proposal PI: Brian Bahnson
External Assay ID: pPAFAH_INH_FLUO_GELBASEDABPP_3XIC50_HTSHITS

Name: Late stage assay provider results from the probe development effort to identify inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH): fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assay for HTS compounds.

Description:

This project aims to develop specific inhibitors of plasma platelet activating factor acetylhydrolase (pPAFAH), and three associated members of the serine hydrolase family of enzymes-PAFAH2, PAFAH1b2, and PAFAH1b3. pPAFAH, an enzyme linked to the inflammatory pathways involved in atherosclerosis, asthma, anaphylactic shock, and other allergic reactions (1,2), is a lipoprotein-associated group VIIA phospholipase A2 that reduces the levels of the signaling molecule platelet activating factor (PAF) (3,4), a potent pro-inflammatory phospholipid signaling molecule (5), and other pro-inflammatory agents, such as oxidized phospholipids, through hydrolysis. A large number of studies have been published over the years since pPAFAH was first discovered linking an increase in pPAFAH concentration and/or activity to an increased risk of various cardiovascular diseases (6,7). The biological function of pPAFAH in the development of coronary heart diseases (CHD) is controversial, with both anti- and pro-inflammatory roles attributed to it (8,9). Dr. Bahnson and colleagues recently reported the first high-resolution crystal structure of the pPAFAH enzyme (10), and would like to expand their studies to co-crystallize pPAFAH with substrate-mimetic inhibitors to further define the active site and substrate specificity of pPAFAH. While one selective pPAFAH inhibitor has been reported (11), its properties are not suitable for the proposed studies. Given the complex biology of the pPAFAH enzymes, a complete characterization of their patho/physiological roles in lipid metabolism is necessary to maximize the success of therapeutic intervention. Towards this goal, development of selective inhibitors would significantly advance our understanding of these enzymes' substrate specificity and contribution to inflammatory disease processes including atherosclerosis, asthma, and rheumatoid arthritis. Pan-PAFAH inhibitors might be of heightened therapeutic value.

References:

1. Karasawa, K., Harada, A., Satoh, N., Inoue, K., and Setaka, M. (2003) Plasma platelet activating factor-acetylhydrolase (PAF-AH), Prog Lipid Res 42, 93-114.
2. Leitinger, N. (2005) Oxidized phospholipids as triggers of inflammation in atherosclerosis, Molecular Nutrition & Food Research 49, 1063-1071.
3. Blank, M. L., Lee, T., Fitzgerald, V., and Snyder, F. (1981) A specific acetylhydrolase for 1-alkyl-2- acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid), J Biol Chem 256, 175-178.
4. Farr, R. S., Cox, C. P., Wardlow, M. L., and Jorgensen, R. (1980) Preliminary studies of an acid labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF), Clin Immunol Immunopathol 15, 318-330.
5. Zimmerman, G. A., McIntyre, T. M., Prescott, S. M., and Stafforini, D. M. (2002) The plateletactivating factor signaling system and its regulators in syndromes of inflammation and thrombosis, Crit Care Med 30, S294-301.
6. Anderson, J. L. (2008) Lipoprotein-associated phospholipase A2: an independent predictor of coronary artery disease events in primary and secondary prevention, Am J Cardiol 101, 23F-33F.
7. Sudhir, K. (2005) Clinical review: Lipoprotein-associated phospholipase A2, a novel inflammatory biomarker and independent risk predictor for cardiovascular disease, J Clin Endocrinol Metab 90, 3100-3105.
8. Wilensky, R. L., and Macphee, C. H. (2009) Lipoprotein-associated phospholipase A(2) and atherosclerosis, Curr Opin Lipidol 20, 415-420.
9. Karabina, S. A., and Ninio, E. (2006) Plasma PAF-acetylhydrolase: an unfulfilled promise?, Biochim Biophys Acta 1761, 1351-1358.
10. Samanta, U., and Bahnson, B. J. (2008) Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis, J Biol Chem 283, 31617-31624.
11. Blackie, J. A., Bloomer, J. C., Brown, M. J. B., Cheng, H. Y., Hammond, B., Hickey, D. M. B., Ife, R. J., Leach, C. A., Lewis, V. A., Macphee, C. H., Milliner, K. J., Moores, K. E., Pinto, I. L., Smith, S. A., Stansfield, I. G., Stanway, S. J., Taylor, M. A., and Theobald, C. J. (2003) The identification of clinical candidate SB-480848: A potent inhibitor of lipoprotein-associated phospholipase A(2), Bioorganic & Medicinal Chemistry Letters 13, 1067-1070.

Keywords:

late stage, late stage AID, assay provider, low throughput, secondary, PLA2G7, pPAFAH, serine hydrolase, platelet activating factor acetylhydrolase, inflammation, atherosclerosis, liquids, fluorescence, competitive activity-based protein profiling, ABPP, gel-based, inhibitor, rhodamine-conjugated fluorophosphonate, FP-Rh, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:
The purpose of this assay is to determine the IC50 values of liquid samples of cherry-picked compounds for pPAFAH inhibition. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe is used to label pPAFAH in the presence of test compounds. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as pPAFAH inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Protocol Summary:
Recombinant mouse pPAFAH (50 uL of 0.25 mg/mL membrane proteome preparation from transiently transfected 293T Hek cells overexpressing pPAFAH) in Dulbecco's PBS (DPBS) was treated with test compound (1 uL of a 25x stock in DMSO) or DMSO (1uL) for 30 minutes at 37 C before the addition of FP-Rh (1 uL of 25x stock in DMSO, 1 uM final concentration). The reaction was incubated for 30 minutes at 37 C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the bands. IC50 values for inhibition of pPAFAH were determined from dose-response curves from three replicates at each inhibitor concentration (7-point 1:3 dilution series from 1 uM to 1 nM).
%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as pPAFAH treated with test compound.
High_Control is defined as pPAFAH treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.
PubChem Activity Outcome and Score:
Compounds with IC50 less than or equal to 0.5 uM were considered active. Compounds with IC50 greater than 0.5 uM were considered inactive.
Any compound with a percent activity value less than 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value greater than or equal to 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
List of Reagents:
Overexpressed pPAFAH 293T membrane proteome (Open Biosystems Accession BC010726, supplied by Assay Provider)
FP-Rh (supplied by Assay Provider)
DPBS (Cellgro, part 21-030-CV)
Comment
This assay was performed by the assay provider with liquid cherry-picked compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
2Inhibition at 1 uM [1] (1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 1 uM compound; replicate [1]Float%
3Inhibition at 0.3 uM [1] (0.3μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.3 uM compound; replicate [1]Float%
4Inhibition at 0.1 uM [1] (0.1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.1 uM compound; replicate [1]Float%
5Inhibition at 0.03 uM [1] (0.03μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.03 uM compound; replicate [1]Float%
6Inhibition at 0.01 uM [1] (0.01μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.01 uM compound; replicate [1]Float%
7Inhibition at 0.003 uM [1] (0.003μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.003 uM compound; replicate [1]Float%
8Inhibition at 0.001 uM [1] (0.001μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.001 uM compound; replicate [1]Float%
9Inhibition at 1 uM [2] (1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 1 uM compound; replicate [2]Float%
10Inhibition at 0.3 uM [2] (0.3μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.3 uM compound; replicate [2]Float%
11Inhibition at 0.1 uM [2] (0.1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.1 uM compound; replicate [2]Float%
12Inhibition at 0.03 uM [2] (0.03μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.03 uM compound; replicate [2]Float%
13Inhibition at 0.01 uM [2] (0.01μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.01 uM compound; replicate [2]Float%
14Inhibition at 0.003 uM [2] (0.003μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.003 uM compound; replicate [2]Float%
15Inhibition at 0.001 uM [2] (0.001μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.001 uM compound; replicate [2]Float%
16Inhibition at 1 uM [3] (1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 1 uM compound; replicate [3]Float%
17Inhibition at 0.3 uM [3] (0.3μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.3 uM compound; replicate [3]Float%
18Inhibition at 0.1 uM [3] (0.1μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.1 uM compound; replicate [3]Float%
19Inhibition at 0.03 uM [3] (0.03μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.03 uM compound; replicate [3]Float%
20Inhibition at 0.01 uM [3] (0.01μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.01 uM compound; replicate [3]Float%
21Inhibition at 0.003 uM [3] (0.003μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.003 uM compound; replicate [3]Float%
22Inhibition at 0.001 uM [3] (0.001μM**)The value for percent inhibition of recombinant mouse pPAFAH at 0.001 uM compound; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R01HL084366

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