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BioAssay: AID 504496

Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) IC50

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) IC50. ..more
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 Tested Compounds
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Active(1)
 
 
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Active(1)
 
 
AID: 504496
Data Source: The Scripps Research Institute Molecular Screening Center (PAFAH2_INH_FLUO_ABPP_INSITU_3XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-21
Hold-until Date: 2011-12-12
Modify Date: 2011-12-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
492956Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Screening depositor-specified cross reference: Primary screen (PAFAH2 inhibitors in singlicate)
492969Summary of the probe development efforts to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Summary1 depositor-specified cross reference: Summary (PAFAH2 inhibitors)
493030Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Screening depositor-specified cross reference: Primary screen (PAFAH2 inhibitors in triplicate)
463082Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH)Screening same project related to Summary assay
504483Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) counterscreen assay to assess selectivity against anti-target pPAFAH in vitroOther same project related to Summary assay
504486Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS/MS assay to assess binding of compounds to active siteOther same project related to Summary assay
504491Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivity to assess carbamate vs. triazole urea scaffoldOther same project related to Summary assay
504494Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory same project related to Summary assay
504495Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) inhibitionOther same project related to Summary assay
504511Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory same project related to Summary assay
504513Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivityOther same project related to Summary assay
504519Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for PAFAH2Other same project related to Summary assay
504527Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) general inhibition and selectivity of serine hydrolasesOther same project related to Summary assay
504531Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) assay to assess selectivity against anti-target pPAFAH in situOther 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 Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R01HL084366
Grant Proposal PI: Brian Bahnson, Univ. of Delaware, Benjamin Cravatt, TSRI
External Assay ID: PAFAH2_INH_FLUO_ABPP_INSITU_3XIC50

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) IC50.

Description:

Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders (1-5). Phospholipids in particular are susceptible to oxidative damage, and (per)oxidized phospholipids can have deleterious effects, including disruption of membrane bilayers and production of toxic byproducts (4, 6-8). One hypothesized pathway for removal of oxidatively damaged species involves hydrolysis by phospholipase A2-type enzymes. Candidate hydrolytic enzymes include the platelet-activating factor acetylhydrolases (PAFAHs) (4,9). The initial role assigned to the PAFAHs was the hydrolysis of platelet activating factor (PAF) (10,11), a potent pro-inflammatory phospholipid signaling molecule (12), which plays a role in myriad physiological processes including inflammation, anaphylaxis, fetal development, and reproduction (4,13). The PAFAHs are subdivided into three classes: plasma (p)PAFAH, and intracellular types 1 and 2. In terms of sequence homology, pPAFAH and PAFAH2 are close homologs and show little similarity to type 1 enzymes. This project aims to develop specific inhibitors for pPAFAH and three counterscreen enzymes: PAFAH2, PAFAH1b2, and PAFAH1b3.

pPAFAH is associated with inflammatory pathways involved in atherosclerosis, asthma, anaphylactic shock, and other allergic reactions (14,15). Numerous studies have also linked increases in pPAFAH concentration and/or activity to an increased risk of various cardiovascular diseases (16,17); however, the biological function of pPAFAH in the development of coronary heart diseases is controversial, with both anti- and proinflammatory roles attributed to it (18,19). Dr. Bahnson and colleagues recently reported the first high-resolution crystal structure of pPAFAH (20), 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 (21), its properties are not suitable for the proposed studies.

PAFAH2 has also been shown to play a role in inflammatory processes via hydrolysis of oxidized phospholipids. Over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death (22,23) and to mediate repair of oxidative-stress induced tissue injury (4). The enzyme also undergoes N-terminal myristoylation and subsequent translocation to the membrane under conditions of oxidative stress (22,23). This evidence suggests that PAFAH2 functions as an important, and perhaps primary, antioxidant enzyme in certain tissues (4); however, its substrate specificity and pathway involvement are far from being fully elucidated. Currently no suitable inhibitors exist for co-crystallization or biochemical studies of PAFAH2.

Given the complex biology of the PAFAH enzymes, chemical tools capable of interrogating enzyme architecture and providing precise temporal control over PAFAH activity are necessary for complete characterization of patho/physiological roles of the PAFAHs in phospholipid metabolism and inflammatory disease processes. Towards that goal, we developed a HTS assay for inhibitor discovery for four PAFAH enzymes: pPAFAH, PAFAH2, PAFAH1b2, and PAFAH1b3, based on their reactivity with the serine-hydrolase-specific fluorophosphonate (FP) (24) activity-based protein profiling (ABPP) probe. This reactivity can be exploited for inhibitor discovery using a competitive-ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling (25). Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP (26). Following the HTS campaign, top inhibitors for each enzyme will be characterized and medchem optimized with the goal of delivering key reagents for elucidating the biology of the PAFAH enzymes.

References:

1. Ames, B.N., Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science, 1983. 221(4617): p. 1256-64.
2. Halliwell, B. and J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol., 1990. 186: p. 1-85.
3. Harman, D., The aging process. Proc. Natl. Acad. Sci. U. S. A., 1981. 78(11): p. 7124-8.
4. Kono, N., et al., Protection against oxidative stress-induced hepatic injury by intracellular type II platelet-activating factor acetylhydrolase by metabolism of oxidized phospholipids in vivo. J. Biol. Chem., 2008. 283(3): p. 1628-36.
5. Southorn, P.A. and G. Powis, Free radicals in medicine. II. Involvement in human disease. Mayo. Clin. Proc., 1988. 63(4): p. 390-408.
6. Kinnunen, P.K., On the principles of functional ordering in biological membranes. Chem. Phys. Lipids, 1991. 57(2-3): p. 375-99.
7. Uchida, K., 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res., 2003. 42(4): p. 318-43.
8. Fruhwirth, G.O., A. Loidl, and A. Hermetter, Oxidized phospholipids: from molecular properties to disease. Biochim. Et Biophys. Acta, 2007. 1772(7): p. 718-36.
9. Nigam, S. and T. Schewe, Phospholipase A(2)s and lipid peroxidation. Biochim. Et Biophys. Acta, 2000. 1488(1-2): p. 167-81.
10. Blank, M.L., et al., A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid). J. Biol. Chem., 1981. 256(1): p. 175-8.
11. Farr, R.S., et al., Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF). Clin. Immunol. Immunopathol., 1980. 15(3): p. 318-330.
12. Zimmerman, G.A., et al., The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis. Crit. Care Med., 2002. 30(5 Suppl): p. S294-301.
13. Prescott, S.M., et al., Platelet-activating factor and related lipid mediators. Annu. Rev. Biochem., 2000. 69: p. 419-45.
14. Karasawa, K., et al., Plasma platelet activating factor-acetylhydrolase (PAF-AH). Prog. Lipid Res., 2003. 42(2): p. 93-114.
15. Leitinger, N., Oxidized phospholipids as triggers of inflammation in atherosclerosis. Mol. Nutr. Food Res., 2005. 49(11): p. 1063-71.
16. Anderson, J.L., Lipoprotein-associated phospholipase A2: an independent predictor of coronary artery disease events in primary and secondary prevention. Am. J. Cardiol., 2008. 101(12A): p. 23F-33F.
17. Sudhir, K., Clinical review: Lipoprotein-associated phospholipase A2, a novel inflammatory biomarker and independent risk predictor for cardiovascular disease. J. Clin. Endocrinol. Metab., 2005. 90(5): p. 3100-5.
18. Wilensky, R.L. and C.H. Macphee, Lipoprotein-associated phospholipase A(2) and atherosclerosis. Curr. Opin. Lipidol., 2009. 20(5): p. 415-20.
19. Karabina, S.A. and E. Ninio, Plasma PAF-acetylhydrolase: an unfulfilled promise? Biochim. Et Biophys. Acta, 2006. 1761(11): p. 1351-8.
20. Samanta, U. and B.J. Bahnson, Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis. J. Biol. Chem., 2008. 283(46): p. 31617-24.
21. Blackie, J.A., et al., The identification of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospholipase A2. Bioorg. Med. Chem. Lett., 2003. 13(6): p. 1067-70.
22. Matsuzawa, A., et al., Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II. J. Biol. Chem., 1997. 272(51): p. 32315-20.
23. Marques, M., et al., Identification of platelet-activating factor acetylhydrolase II in human skin. J. Invest. Dermatol., 2002. 119(4): p. 913-9.
24. Jessani, N., et al., Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A., 2002. 99(16): p. 10335-40.
25. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
26. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol., 2009. 27(4): p. 387-94.

Keywords:

late stage, late stage AID, assay provider, powders, platelet-activating factor acetylhydrolase, PAFAH, PAF-AH, plasma platelet-activating factor acetylhydrolase, pPAFAH, platelet-activating factor acetylhydrolase type II, PAFAH2, PAFAHII, cancer, inflammation, atherosclerosis, serine hydrolase, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, inhibitor, in situ, cell-based assay, BW5147, murine T cells, T cells, Scripps, 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 powder samples of test compounds for PAFAH2 inhibition in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. 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 PAFAH2 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:

BW5147-derived murine T-cells (5 mL total volume; supplemented with 10% FCS) were treated with DMSO or test compound (5 uL of a 1000x stock in DMSO) for 4 hours at 37 C. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 C, quenched with 2x SDS-PAGE loading buffer, 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 PAFAH2 band relative to a DMSO-only (no compound) control. IC50 values for inhibition of PAFAH2 were determined from dose-response curves from three replicates at each inhibitor concentration (4-point 1:3 dilution series from 2.5 nM to 75 pM).

%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as PAFAH2 in cells treated with test compound.
High_Control is defined as PAFAH2 in cells 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 four 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 an IC50 less than 0.1 uM were considered active. Compounds with an IC50 greater than or equal to 0.1 uM were considered inactive.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 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 100-100. There are no inactive compounds.

List of Reagents:

BW5147-derived murine T-cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
FP-Rh (provided by the Assay Provider)
Comment
This assay was performed by the assay provider with powder samples of compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: BW5147
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The value for concentration at which 50% inhibition is observed in situ; IC50 shown in uM.FloatμM
2Inhibition at 0.000075 uM [1] (7.5e-05μM**)Value of % Inhibition in situ at 0.000075 uM inhibitor concentration; replicate one.Integer%
3Inhibition at 0.000075 uM [2] (7.5e-05μM**)Value of % Inhibition in situ at 0.000075 uM inhibitor concentration; replicate two.Integer%
4Inhibition at 0.000075 uM [3] (7.5e-05μM**)Value of % Inhibition in situ at 0.000075 uM inhibitor concentration; replicate three.Integer%
5Inhibition at 0.00025 uM [1] (0.00025μM**)Value of % Inhibition in situ at 0.00025 uM inhibitor concentration; replicate one.Integer%
6Inhibition at 0.00025 uM [2] (0.00025μM**)Value of % Inhibition in situ at 0.00025 uM inhibitor concentration; replicate two.Integer%
7Inhibition at 0.00025 uM [3] (0.00025μM**)Value of % Inhibition in situ at 0.00025 uM inhibitor concentration; replicate three.Integer%
8Inhibition at 0.00075 uM [1] (0.00075μM**)Value of % Inhibition in situ at 0.00075 uM inhibitor concentration; replicate one.Integer%
9Inhibition at 0.00075 uM [2] (0.00075μM**)Value of % Inhibition in situ at 0.00075 uM inhibitor concentration; replicate two.Integer%
10Inhibition at 0.00075 uM [3] (0.00075μM**)Value of % Inhibition in situ at 0.00075 uM inhibitor concentration; replicate three.Integer%
11Inhibition at 0.0025 uM [1] (0.0025μM**)Value of % Inhibition in situ at 0.0025 uM inhibitor concentration; replicate one.Integer%
12Inhibition at 0.0025 uM [2] (0.0025μM**)Value of % Inhibition in situ at 0.0025 uM inhibitor concentration; replicate two.Integer%
13Inhibition at 0.0025 uM [3] (0.0025μM**)Value of % Inhibition in situ at 0.0025 uM inhibitor concentration; replicate three.Integer%

* 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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