Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH and selectivity analysis in vivo
Name: Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH and selectivity analysis in vivo. ..more
BioActive Compound: 1
Depositor Specified Assays
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_INVIVO_SEL
Name: Late stage assay provider results from the probe development effort to identify inhibitors of pPAFAH: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of pPAFAH and selectivity analysis in vivo.
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, gel-based, inhibitor, in vivo, selectivity, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit pPAFAH in vivo and to assess selectivity. In this assay, test compounds are orally administered to mice. Mice are sacrificed, and their brain tissue harvested, homogenized, and the membrane fraction isolated and reacted with the activity-based probes HT-01 and fluorophosphonate-rhodamine (FP-Rh). HT-01 bears a BODIPY fluorophore and urea triazole reactive group that selectively labels several serine hydrolases, including pPAFAH. This reagent is used in addition to the standard serine hydrolase activity-based probe FP-Rh to enhance visualization of pPAFAH, which is otherwise obscured by other serine hydrolases upon SDS-PAGE separation/visualization. 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 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.
Purpose-bred WT laboratory mice were orally administered test compound (50 mg/kg in PEG300 vehicle solution) or vehicle only (n=2 per group). After four hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and brain tissues removed and snap frozen in liquid nitrogen. Tissues were homogenized and the membrane fraction isolated by centrifugation (45 min, 100K x g) and adjusted to 1 mg/mL in 50 mM Dulbecco's PBS (DPBS). Aliquots (50 uL) were treated with HT-01 (1uL of 50x stock in DMSO, 1uM final concentration) or FP-Rh (1uL of 50x stock in DMSO, 1uM final concentration). The reaction was incubated for 30 minutes at 37 degrees Celsius, 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 test compound bands relative to vehicle bands.
The % inhibition was then calculated as follows:
%_Inhibition = ( 1 - ( IOD_Test_Compound-MedianIOD_Low_Control ) / ( MedianIOD_High_Control-MedianIOD_Low_Control ) ) * 100
Test_Compound is defined as target or anti-target band from mice treated with test compound.
High_Control is defined as target or anti-target band from mice treated with vehicle only (no compound).
Low_Control is defined as background in a blank region of the gel.
Assay Outcome (%INH pPAFAH and %INH carboxylesterase 1 [CES1]):
Compounds with ?50% inhibition were considered active. Compounds with <50% inhibition were considered inactive.
Assay Outcome (FP-Rh Anti-targets):
Compounds with 2 or more observed anti-targets were considered active. Compounds with fewer than 2 observed anti-targets were considered inactive.
Compounds active for pPAFAH inhibition and inactive for FP-Rh anti-targets were considered active, regardless of the outcome of the CES1 assay. Compounds inactive for pPAFAH inhibition and/or active for FP-Rh anti-targets were considered inactive, regardless of the outcome of the CES1 assay.
PubChem Activity Score:
The PubChem Activity Score is assigned a value of 50 for active compounds, and 0 for inactive compounds.
List of Reagents:
Purpose-bred WT laboratory mice (supplied by Assay Provider)
HT-01 (supplied by Assay Provider)
FP-Rh (supplied by Assay Provider)
PEG (SigmaAldrich 202371)
DPBS (Cellgro, part 21-030-CV)
This assay was performed by the assay provider with powder samples of test compounds.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: selectivity
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