Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH; PLA2G7)
Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH; PLA2G7). ..more
BioActive Compounds: 1675
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Brian J. Bahnson, University of Delaware
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: HL084366
Grant Proposal PI: Brian J. Bahnson, University of Delaware
External Assay ID: PPAFAH_INH_FP_1536_3X%INH CRUN
Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH; PLA2G7).
Atherosclerosis is a process in which plaques, deposits of low density lipoproteins (LDL), lipid-laden macrophages and other inflammatory cells, calcium, and cellular debris build up in the inner lining of an artery. These plaques interfere with blood flow, damage the arterial wall, and eventually rupture, causing debris to migrate downstream, leading to myocardial infarction or stroke (1-4). Human platelet activating factor acetylhydrolase (pPAFAH) is a Ca2+ independent phospholipase A2 (PLA2) identified in human plasma as the enzyme responsible for the hydrolysis/inactivation of platelet activating factor (PAF) (5, 6), a potent pro-inflammatory phospholipid signaling molecule (7). PAFAH catalyses the hydrolysis of the acetyl group at the sn-2 position of the glycerol backbone of PAF converting it to lyso-PAF (8-9). PAFAH has a catalytic cysteine residue and is consequently sensitive to broadly reactive thiol alkylating agents, including N-ethylmaleimide (10); however, selective inhibitors for PAFAH have not yet been identified. Inhibiting PAFAH may thus offer a new therapeutic strategy for cancer. Development of a selective inhibitor would also aid in the investigation into PAFAH involvement in the dysregulated biochemical pathways that support tumorigenesis. As a result, the identification of inhibitors of PAFAH would help to elucidate the physiological role of this enzyme and its contribution to atherosclerosis, cancer, and other inflammatory pathologies (11).
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.
PLA2G7, PAFAH, pPAFAH, PPAFAH, LP-LPA2, PAFAH2, PAFAH1b2, PAFAH1b3, hydrolase, plasma platelet activating factor acetylhydrolase, FP-Rh, atherosclerosis, heart disease, cancer, fluorescence polarization, FP, antagonist, inhibitor, inhibit, confirmation, triplicate, HTS, high throughput screening, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to confirm activity of compounds that act as inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH), which has a catalytic cysteine residue and is sensitive to thiol alkylating agents such as N-ethylmaleimide. Compounds selected for testing were identified as active in a previous set of assays entitled, Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH) (AID 463082). In this biochemical assay, recombinant pPAFAH protein is incubated with test compounds and a Rh-conjugated activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with pPAFAH for a defined period, followed by addition of the FP-rhodamine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as pPAFAH inhibitors will prevent pPAFAH-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in triplicate at a final nominal concentration of 3.39 uM.
Prior to the start of the assay, 4.0 uL of Assay Buffer (0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 12.5 nanomolar of pPAFAH protein were dispensed into 1536 microtiter plates. Next, 17 nL of test compound in DMSO or DMSO alone (0.34% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C.
The assay was started by dispensing 1.0 uL of 375 nM FP-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 15 minutes of incubation at 25 C, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as wells containing pPAFAH and DMSO.
Test_Compound is defined as wells containing pPAFAH in the presence of test compound.
High_Control is defined as wells containing no pPAFAH protein.
The average percent inhibition and standard deviation of each compound tested were calculated.
PubChem Activity Outcome and Score:
Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 463082) was declared active.
The reported PubChem Activity Score has been normalized to 100% of the highest observed inhibition value. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.
List of Reagents:
Recombinant pPAFAH protein (supplied by Assay Provider-Brian Bahnson)
FP-Rh probe (supplied by Assay Provider-Benjamin Cravatt)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic Acid (Invitrogen, part P6866)
DTT (Invitrogen, part 15508-013)
1536-well plates (Corning, part 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay. One compound (SID 49725089) was removed prior to confirmation because it produces a known fluorescence artifact.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)