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 in situ
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 in situ. ..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_FLUO_GELBASEDABPP_INSITU
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 in situ.
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 situ, 293T Hek, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to determine whether or not powders samples of test compounds can inhibit pPAFAH in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested, homogenized, and reacted 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 of the bands. As designed, test compounds that act as pPAFAH inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Transiently transfected 293T Hek cells overexpressing mouse pPAFAH (Open Biosystems Accession BC010726) were cultured in a 6-well dish for 48 hours in DMEM medium containing 10% FCS. Cells were washed with DBPS and fresh, serum-free DMEM medium (1 mL) was added. Cells were treated with test compound (10 uM, 1 uM, 0.1 uM, and 0.01 uM final concentration; 10 uL of 100x stock in DMSO) or DMSO only and incubated for 4 hours at 37 C. Cells were washed with DPBS (4x), harvested, and homogenized by sonication. The soluble and membrane fractions were separated by centrifugation (100K x g, 45 min) and the protein concentration in each fraction was adjusted to 1mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 1 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 37 C, quenched with an equal volume of 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 pPAFAH band relative to a DMSO-only (no compound) control.
The % inhibition was then calculated as follows:
%_Inhibition = ( 1 - ( IOD_Test_Compound - Median_IOD_Low_Control ) / ( Median_IOD_High_Control - Median_IOD_Low_Control ) ) * 100
Test_Compound is defined as pPAFAH treated with test compound.
High_Control is defined as pPAFAH treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
Compounds with greater than or equal to 50% inhibition at 0.1 uM compound concentration were considered active. Compounds with less than 50% inhibition at 0.1 uM compound concentration were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition at 0.1 uM. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
293T Hek cells overexpressing pPAFAH (supplied by Assay Provider)
FP-Rh (supplied by the Assay Provider)
DMEM Medium (CellGro 10-017-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
This assay was performed by the assay provider with powder samples of test compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)