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_SEL_HTS

Name: Late 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 proteome.

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, selectivity, rhodamine-conjugated fluorophosphonate, FP-Rh, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Targets

Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		pPAFAH		12	Phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) [Mus musculus] [gi:14715116]		Taxonomy id: 10090 Gene id: 27226
2		FAAH	1	11	fatty acid amide hydrolase [Mus musculus] [gi:123253900]		Taxonomy id: 10090 Gene id: 14073
3		ABHD3		12	abhydrolase domain-containing protein 3 [Mus musculus] [gi:19527360]		Taxonomy id: 10090 Gene id: 106861
4		ABHD4		12	Abhydrolase domain containing 4 [Mus musculus] [gi:17028430]		Taxonomy id: 10090 Gene id: 105501
5		MAGL		12	RecName: Full=Monoglyceride lipase; Short=MGL; AltName: Full=Monoacylglycerol lipase; Short=MAGL [gi:47117040]		Taxonomy id: 10090 Gene id: 23945
6		ABHD6		12	Abhydrolase domain containing 6 [Mus musculus] [gi:20073260]		Taxonomy id: 10090 Gene id: 66082
7		MW 32 kDa		12
8		MW 30 kDa		12
9		LYPLA2		12	lysophospholipase 2 [Mus musculus] [gi:123122209]		Taxonomy id: 10090 Gene id: 26394
10		LYPLA1		12	Lypla1 [Mus musculus] [gi:71059731]		Taxonomy id: 10090 Gene id: 18777

---

§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine whether cherry picked HTS hit compounds active in a previous assay, AID 588474 [Late 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 pPAFAH], can inhibit pPAFAH in a gel-based competitive activity-based proteomic profiling (ABPP) assay and to assess compound selectivity in a complex proteome. Test compound is incubated with recombinantly-expressed target enzyme pPAFAH (pPAFAH assay) or serine hydrolase (potential anti-target) rich complex proteome (anti-target assay) 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 pPAFAH or anti-target 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:

pPAFAH Assay:

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 (10, 1, or 0.1 uM final concentration; 1 uL of a 50x stock in DMSO) or DMSO (1 uL) for 30 minutes at 37 C. FP-Rh (1 uL of a 50 uM solution in DMSO; 1 uM final concentration) was added, and 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 pPAFAH band relative to a DMSO-only (no compound) control.

Anti-target Assay:

Mouse brain membrane proteome (50 uL of 1 mg/mL) in Dulbecco's PBS (DPBS) was treated with test compound (10, 1, or 0.1 uM final concentration; 1 uL of a 50x stock in DMSO) or DMSO (1 uL) for 30 minutes at 37 C. FP-Rh (1 uL of a 25 uM solution in DMSO; 1 uM final concentration) was added, and 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 for each anti-target (fatty acid amide hydrolase [FAAH], monoacylglycerol lipase [MAGL], abhydrolase domain-containing proteins 3, 4, and 6 [ABHD3, ABHD4, ABHD6], lysophospholipases 1 and 2 [LYPLA1, LYPLA2], and unidentified proteins with molecular weight [MW] of 32kDa and 30kDa) was determined by measuring the integrated optical density of the individual protein 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 enzyme treated with test compound.
High_Control is defined as target or anti-target enzyme treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

Fold_selectivity > [Conc_<50%_INH_Anti-target] / [Conc_>=50%_INH_target]

Where:
[Conc_<50%_INH_Anti-target] is the test compound concentration at which less than 50% inhibition of the anti-target is observed.
[Conc_>=50%_INH_target] is the test compound concentration at which greater than or equal to 50% inhibition of target is observed.

If [Conc_>=50%_INH_target] is not determined, then Fold Selectivity is not determined.
If [Conc_<50%_INH_Anti-target] < [Conc_>=50%_INH_target], Fold Selectivity is 0.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with greater than or equal to 50% inhibition at 0.1 uM test compound concentration were considered active. Compounds with less than 50% inhibition at 0.1 uM test compound concentration were considered inactive.

The reported PubChem Activity Score has been normalized to 100% of the observed value at 0.1 uM.

pPaFAH Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

FAAH: Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 45-0.

ABHD3 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

ABHD4 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

MAGL Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

ABHD6 Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

MW 32 kDa Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

MW 30 kDa Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

LYPLA2 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

LYPLA1 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

Overall Outcome and Score:

Compounds with greater than or equal to 50% inhibition of pPAFAH at 0.1 uM test compound concentration and 10-fold or greater selectivity against all anti-targets were considered active. Compounds with less than 50% inhibition at 0.1 uM test compound concentration and/or less than 10-fold selectivity against all anti-targets were considered inactive.

The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.

The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.

List of Reagents:

Overexpressed pPAFAH 293T membrane proteome (Open Biosystems Accession BC010726, supplied by Assay Provider)
Mouse brain membrane proteome (supplied by Assay Provider)
FP-Rh (supplied by Assay Provider)
DPBS (Cellgro, part 21-030-CV)

This assay was performed by the assay provider with liquid cherry-picked compounds.

BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate confirmatory

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: single protein format

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: meta target: molecular target: protein target: enzyme: generic hydrolase

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: detection technology: fluorescence: fluorescence intensity

Grant Number: 1R01HL084366