Late 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 compounds
Name: Late 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 compounds ..more
BioActive Compound: 1
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
Name: Late 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 compounds
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.
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.
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, inhibitor, active site, serine, trypsin, binding mode, covalent, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to assess the covalent nature of an inhibitor compound belonging to the carbamate scaffold and determine whether or not it labels the active site serine of pPAFAH. In this assay, purified enzyme is reacted with inhibitor compound, digested with trypsin, and the resulting peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting data are analyzed to identify sites of covalent labeling.
Two aliquots of purified, recombinant human pPAFAH (25 uL of 40 uM in DPBS; purified GST-fusion construct expressed in E. coli BL21 cells; protein contains residues 42-441 plus a non-native N-terminal extension (GSPNSRVD) and the following mutations in the hydrophobic patch region: I120A, L123A,L124A) were prepared. To one aliquot was added test compound (1 uL of 25x stock in DMSO, 100 uM final concentration). To the second (control) aliquot was added an equal volume of DMSO. Reactions were gently vortexed and incubated at 37 C for 60 minutes. To each reaction was added freshly prepared urea (75 uL of 6 M in DPBS), DDT (1.5 uL of 10 mM), and the reactions were incubated at 65 C for 25 minutes and then allowed to cool to room temperature. To each reaction was then added freshly prepared IAA (20 uL of 400 mM), and the reactions were rotated for 30 minutes at 25 C in the dark. Aqueous ammonium bicarbonate (150 uL of 50 mM) was added, followed by CaCl2 (1.5 uL of 100 mM), and sequencing grade modified trypsin (1 ug). Reactions were incubated at 37 C for 12 hours. The protein digests were acidified with formic acid (9 uL).
An Agilent 1200 series quaternary HPLC pump and Thermo Scientific LTQ-Orbitrap mass spectrometer were used for sample analysis. A fraction (20 uL) of the protein digest for each sample was pressure-loaded onto a 100 micron fused-silica column (with a 5 micron in-house pulled tip) packed with 10 cm of Aqua C18 reversed-phase packing material. Chromatography was carried out 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 (60 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). The MS2 spectra generated for each run were searched against the latest version of the IPI human protein database concatenated to a reversed decoy database using Sequest. Search parameters specified a static modification of +57.021 for cysteine due to alkylation, a variable modification of +253.12152 for serine to account for possible compound labeling, and no enzyme specificity. The resulting peptide identifications were assembled into protein identifications 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 achieve a user-defined false positive hit rate below 1% (as determined by number of hits against the reversed database) at the peptide level. No modified peptides were identified in the DMSO-treated sample.
PubChem Activity Outcome and Score:
Compounds observed to covalently modify the active site serine of pPAFAH were considered active. Compounds for which no covalent modification was observed 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 active compounds is 100-100. There are no inactive compounds.
List of Reagents:
Recombinant human pPAFAH (provided by assay provider)
DPBS (CellGro 21-030-CV)
Urea (Fisher U15-3)
Ammonium bicarbonate (Acros, AC37093-0010)
DTT (Sigma 43815)
IAA (iodoacetamide; Sigma I1149)
Trypsin (Promega V5111)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Formic acid (Fluka 06440)
This assay was performed by the assay provider with powder samples of test 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: mmoa characterization
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
Assay Format: Biochemical
Data Table (Concise)