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BioAssay: AID 504491

Late 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 scaffold

Name: Late 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 scaffold. ..more
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 Tested Compounds
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Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
AID: 504491
Data Source: The Scripps Research Institute Molecular Screening Center (PAFAH2_INH_FLUO_GEL_1XINH_SEL_SET2)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-21
Hold-until Date: 2011-10-06
Modify Date: 2011-10-06

Data Table ( Complete ):           View Active Data    View All Data
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
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
504496Late 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) IC50Confirmatory 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: 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, Univ. of Delaware, Benjamin Cravatt, TSRI
External Assay ID: PAFAH2_INH_FLUO_GEL_1XINH_SEL_SET2

Name: Late 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 scaffold.

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, counterscreen, inhibitor, selectivity, anti-targets, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, BW5147, murine T cells, T cells, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PAFAH211platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus] [gi:225579137]
Taxonomy id: 10090
Gene id: 100163
2ABHD112Abhydrolase domain containing 11 [Mus musculus] [gi:47682716]
Taxonomy id: 10090
Gene id: 68758
3APEH11Apeh protein [Mus musculus] [gi:20072022]
Taxonomy id: 10090
Gene id: 235606
4ESD11Esterase D/formylglutathione hydrolase [Mus musculus] [gi:55777188]
Taxonomy id: 10090
Gene id: 13885
5LYPLA111Lypla1 [Mus musculus] [gi:71059731]
Taxonomy id: 10090
Gene id: 18777
6LYPLA211lysophospholipase 2 [Mus musculus] [gi:123122209]
Taxonomy id: 10090
Gene id: 26394

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to determine whether powder samples of test compounds of different chemotypes-carbamate and triazole urea-can inhibit PAFAH2 in a complex proteomic lysate and to estimate compound selectivity in an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction 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 (IOD) 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. Percent inhibition is calculated relative to a DMSO (no compound) control.
Protocol Summary:
Soluble proteome (1 mg/ml in DPBS) of BW5147-derived murine T cells was treated with 20 uM test compound (1 uL of a 50x stock in DMSO). Test compounds were incubated for 30 minutes at 25 C (50 uL reaction volume). FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM. 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 target (PAFAH2) and anti-target (N-acylaminoacyl-peptide hydrolase [APEH], alpha/beta hydrolase domain-containing protein 11 [ABHD11], esterase D/formylglutathione hydrolase [ESD], lysophospholipase 1 [LYPLA1], and lysophospholipase 2 [LYPLA2]) bands relative to a DMSO-only (no compound) control.
%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as target or anti-target in cells treated with test compound.
High_Control is defined as target or anti-target in cells treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with greater than or equal to 50% inhibition were considered active. Compounds with less than 50% inhibition were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
PAFAH2 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
ABHD11 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
APEH Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
ESD Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
LYPLA1 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
LYPLA2 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
Overall Outcome and Score:
Compounds that were active for inhibition of PAFAH2 were assigned a PubChem Activity Outcome of active, regardless of inhibition of anti-targets. Compounds that were inactive for PAFAH2 were assigned a PubChem Activity Outcome of inactive, regardless of inhibition of anti-targets.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
Soluble proteome of BW5147-derived murine T-cells
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [PAFAH2]One of Active, Inactive, or Not Tested for INH of PAFAH21platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus]Outcome
2Score [PAFAH2]The BioAssay activity ranking score1Integer
3Inhibition at 20 uM [PAFAH2] (20μM**)Inhibition of PAFAH2 in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.1Integer%
4Outcome [ABHD11]One of Active, Inactive, or Not Tested for INH of ABHD112Abhydrolase domain containing 11 [Mus musculus]Outcome
5Score [ABHD11]The BioAssay activity ranking score2Integer
6Inhibition at 20 uM [ABHD11] (20μM**)Percent inhibition of ABHD11 in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.2Integer%
7Outcome [APEH]One of Active, Inactive, or Not Tested for INH of APEH3Apeh protein [Mus musculus]Outcome
8Score [APEH]The BioAssay activity ranking score3Integer
9Inhibition at 20 uM [APEH] (20μM**)Percent inhibition of APEH in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.3Integer%
10Outcome [ESD]One of Active, Inactive, or Not Tested for INH of ESD4Esterase D/formylglutathione hydrolase [Mus musculus]Outcome
11Score [ESD]The BioAssay activity ranking score4Integer
12Inhibition at 20 uM [ESD] (20μM**)Percent inhibition of ESD in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.4Integer%
13Outcome [LYPLA1]One of Active, Inactive, or Not Tested for INH of LYPLA15Lypla1 [Mus musculus]Outcome
14Score [LYPLA1]The BioAssay activity ranking score5Integer
15Inhibition at 20 uM [LYPLA1] (20μM**)Percent inhibition of LYPLA1 in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.5Integer%
16Outcome [LYPLA2]One of Active, Inactive, or Not Tested for INH of LYPLA26lysophospholipase 2 [Mus musculus]Outcome
17Score [LYPLA2]The BioAssay activity ranking score6Integer
18Inhibition at 20 uM [LYPLA2] (20μM**)Percent inhibition of LYPLA2 in murine T-cell soluble proteome upon 20 uM compound treatment as assessed by competitive ABPP.6Integer%

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1R01HL084366

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