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_1X%INH

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 of pPAFAH.

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

Assay Overview:

The purpose of this assay is to determine whether cherry picked HTS hit compounds can inhibit pPAFAH in a gel-based competitive activity-based proteomic profiling (ABPP) assay. In this assay, the target enzyme pPAFAH is incubated with test compound 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 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:

Recombinant mouse pPAFAH (25 uL of 0.25 mg/mL) in Dulbecco's PBS (DPBS) was treated with test compound (1 uL of a 125 uM solution in DMSO; 5 uM final concentration) 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 room temperature, 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.

%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as target enzyme treated with test compound.
High_Control is defined as target enzyme 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 75% inhibition were considered "active". Compounds with less than 75% inhibition were considered "inactive".

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-14, and for inactive compounds 14-0.

List of Reagents:

Recombinant pPAFAH (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.

Grant Number: 1R01HL084366