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_LCMS_SOL

Name: Late 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 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, powders, assay provider, low throughput, secondary, PLA2G7, pPAFAH, serine hydrolase, platelet activating factor acetylhydrolase, inflammation, atherosclerosis, fluorescence, competitive activity-based protein profiling, ABPP, liquid chromatography, LC, tandem mass spectrometry, MS/MS, multidimensional protein identification technology, MudPIT, ABPP-MudPIT, inhibitor, selectivity, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Targets

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		Pla2g7 (pPAFAH)	1		platelet-activating factor acetylhydrolase [Mus musculus] [gi:31980752]		Taxonomy id: 10090 Gene id: 27226
2		Esd		1	S-formylglutathione hydrolase [Mus musculus] [gi:13937355]		Taxonomy id: 10090 Gene id: 13885
3		Prep		1	prolyl endopeptidase [Mus musculus] [gi:6755152]		Taxonomy id: 10090 Gene id: 19072
4		Prepl		1	prolyl endopeptidase-like isoform a [Mus musculus] [gi:254939518]		Taxonomy id: 10090 Gene id: 213760
5		Lypla2		1	acyl-protein thioesterase 2 [Mus musculus] [gi:7242156]		Taxonomy id: 10090 Gene id: 26394
6		Fasn		1	fatty acid synthase [Mus musculus] [gi:93102409]		Taxonomy id: 10090 Gene id: 14104
7		Acot2		1	acyl-coenzyme A thioesterase 2, mitochondrial precursor [Mus musculus] [gi:238624114]		Taxonomy id: 10090 Gene id: 171210
8		Acot1		1	acyl-coenzyme A thioesterase 1 [Mus musculus] [gi:6753550]		Taxonomy id: 10090 Gene id: 26897
9		Pafah1b2		1	platelet-activating factor acetylhydrolase IB subunit beta [Mus musculus] [gi:40254624]		Taxonomy id: 10090 Gene id: 18475
10		Apeh		1	Acylpeptide hydrolase [Mus musculus] [gi:19343726]		Taxonomy id: 10090 Gene id: 235606
11		Lypla1		1	acyl-protein thioesterase 1 [Mus musculus] [gi:6678760]		Taxonomy id: 10090 Gene id: 18777
12		Pme1		1	protein phosphatase methylesterase 1 [Mus musculus] [gi:30794138]		Taxonomy id: 10090 Gene id: 72590
13		Abhd10		1	abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus] [gi:269784760]		Taxonomy id: 10090 Gene id: 213012
14		Rbbp9		1	putative hydrolase RBBP9 [Mus musculus] [gi:86439977]		Taxonomy id: 10090 Gene id: 26450
15		Es1	1		liver carboxylesterase N precursor [Mus musculus] [gi:247269929]		Taxonomy id: 10090 Gene id: 13884
16		Acot7		1	cytosolic acyl coenzyme A thioester hydrolase isoform 1 [Mus musculus] [gi:225690616]		Taxonomy id: 10090 Gene id: 70025
17		Dpp9		1	dipeptidyl peptidase 9 [Mus musculus] [gi:255003757]		Taxonomy id: 10090 Gene id: 224897
18		Dpp8		1	dipeptidyl peptidase 8 [Mus musculus] [gi:31542571]		Taxonomy id: 10090 Gene id: 74388
19		Pafah1b3		1	platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus] [gi:6679201]		Taxonomy id: 10090 Gene id: 18476
20		Magl		1	monoglyceride lipase isoform d [Mus musculus] [gi:261878511]		Taxonomy id: 10090 Gene id: 23945
21		Abhd4		1	abhydrolase domain-containing protein 4 isoform 1 [Mus musculus] [gi:326937491]		Taxonomy id: 10090 Gene id: 105501
22		Abhd11		1	abhydrolase domain-containing protein 11 isoform 1 [Mus musculus] [gi:21644577]		Taxonomy id: 10090 Gene id: 68758
23		Pla2g15		1	group XV phospholipase A2 precursor [Mus musculus] [gi:19527008]		Taxonomy id: 10090 Gene id: 192654
24		Lyplal1		1	lysophospholipase-like protein 1 [Mus musculus] [gi:227496223]		Taxonomy id: 10090 Gene id: 226791
25		Lipe		1	hormone-sensitive lipase isoform 2 [Mus musculus] [gi:87239972]		Taxonomy id: 10090 Gene id: 16890
26		Tpp2		1	Tpp2 protein [Mus musculus] [gi:37194903]		Taxonomy id: 10090 Gene id: 22019

---

§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine the selectivity profile of powder samples of test compounds in mouse brain soluble proteome by activity-based protein profiling (ABPP) in combination with the multidimensional protein identification technology (MudPIT) liquid chromatography tandem mass spectrometry (LC-M/MS) analysis platform. In this assay, paired complex proteome samples are treated with test compound or DMSO, followed by reaction with the serine hydrolase specific activity-based fluorophosphonate-biotin (FP-biotin) affinity probe. Biotinylated proteins are enriched, trypsinized, and analyzed by MudPIT. Inhibition of target and anti-target activity is quantified by comparing spectra counts between the test compound- and DMSO-treated samples. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated sample relative to the DMSO-treated sample, thus giving a smaller spectra count ratio (test compound/DMSO) for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of 1.

Protocol Summary:

Sample Preparation: paired aliquots of mouse brain soluble proteome (1 mL of 1 mg/mL in 50 mM Tris pH 8.0 [Tris] buffer) were treated with either 1 uM test compound (20 uL of a 50x stock in DMSO) or DMSO for 30 minutes at 37 C. Each reaction was labeled with 5 uM of FP-biotin (5 uL of a 50x stock in DMSO) for 2 hours at 25 C. TritonX100 was added to 1% final concentration (v/v), and the proteomes were desalted over PD-10 desalting columns (GE Healthcare). SDS was added to the eluate to give 0.5% final concentration, and the samples heated to 90 C for 8 minutes. FP-labeled proteins were enriched with streptavidin beads (100 uL of 50% slurry; 25 C, 1 hour, 8.5 mL total volume Tris). Following enrichment, the beads were washed with 1% SDS in Tris (2x), 6 M Urea in Tris (2x), and Tris (2x), and then resuspended in 8 M urea, reduced with TCEP (10 mM) for 25 minutes at 25 C, and alkylated with iodoacetamide (12.5 mM) for 25 minutes at 25 C in the dark. The urea concentration was reduced to 2 M with Tris, and on-bead digestions were performed for 12 hours at 37 C with sequence-grade modified trypsin (Promega; 2 ug) in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid, pressure-loaded on to a biphasic (strong cation exchange/reversed phase) MudPIT capillary column and analyzed as described below. A total of 4 biological replicates were prepared and analyzed for each experiment.

LC-MS/MS analysis: digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (MudPIT) using an Agilent 1200-series quaternary pump and Thermo Scientific LTQ ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate, with chromatographic elution following each salt pulse accomplished using an increasing gradient of aqueous acetonitrile containing 0.1% formic acid over 125 minutes. Data were collected in data-dependent acquisition mode with dynamic exclusion turned on (90 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publically available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the PROLUCID algorithm (publically available at http://fields.scripps.edu/downloads.php) against the latest version of the mouse UniProt database concatenated with the reversed database for assessment of false-discovery rates. PROLUCID searches allowed for variable oxidation of methionine (+16), static modification of cysteine (+57 due to alkylation), and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0.41) using the -trypstat and --modstat options, which apply different statistical models for the analysis of tryptic, half-tryptic, non-tryptic, and modified peptides. DTASelect 2.0 uses a quadratic discriminant analysis to maintain a user-defined false positive hit rate (as determined by number of hits against the reversed database) below 1% at the peptide level. Spectra count abundance ratios (test_compound/DMSO) were calculated for all serine hydrolases with an average of 10 or more spectra counts in the DMSO control samples.

Ratio = Average_Spectra_Counts_Test_Compound / Average_Spectra_Counts_DMSO_Control

Where:

Average_Spectra_Counts_Test_Compound is the average number of spectra counts for a given serine hydrolase across all test compound-treated samples.
Average_Spectra_Counts_DMSO_Control is the average number of spectra counts for a given serine hydrolase across all DMSO control samples.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

A compound was considered active for a particular target/anti-target with an abundance ratio of less than or equal to 0.5. A compound was considered inactive for a specified target/anti-target with an abundance ratio of greater than 0.5.

Overall Outcome and Score:

A compound was considered active if it was active for pPAFAH (aka Pla2g7) and active for fewer than 2 serine hydrolase anti-targets. A compound was considered inactive if it was inactive for pPAFAH (aka Pla2g7) and/or active for 2 or more serine hydrolase 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. There are no inactive compounds.

List of Reagents:

Mouse brain soluble proteome (provided by assay provider)
FP-biotin (provided by Assay Provider)
Tris-HCl (SigmaAldrich T5941)
Triton-X100 (Fisher AC21568-0010)
PD-10 desalting columns (GE Healthcare 17-0851-01)
SDS (SigmaAldrich L6026)
Urea (Fisher U15-3)
TCEP (SigmaAldrich 75259)
Iodoacetamide (iodoacetamide; SigmaAldrich I1149)
Trypsin (Promega V5111)
CaCl2 (SigmaAldrich C1016)
Streptavidin beads (Pierce 20349)
Formic acid (Fluka 06440)
Fused-silica (Agilent 160-2635-10)
Strong cation exchange (Phenomenex CH0-2257)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Millipore-filtered Water
Ammonium bicarbonate (Acros, AC37093-0010)

This assay was performed by the assay provider with powder samples of test compounds.

BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: mmoa characterization

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

Grant Number: 1R01HL084366