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BioAssay: AID 504495

Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) inhibition

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) inhibition ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 504495
Data Source: The Scripps Research Institute Molecular Screening Center (PAFAH2_INH_FLUO_ABPP_INSITU_1XINH)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-21
Hold-until Date: 2011-12-12
Modify Date: 2011-12-12

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
492969Summary of the probe development efforts to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)summarySummary (PAFAH2 inhibitors)
492956Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)screeningPrimary screen (PAFAH2 inhibitors in singlicate)
493030Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)screeningPrimary screen (PAFAH2 inhibitors in triplicate)
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R01HL084366
Grant Proposal PI: Brian Bahnson, Univ. of Delaware, Benjamin Cravatt, TSRI

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) inhibition


Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders (1-5). Phospholipids in particular are susceptible to oxidative damage, and (per)oxidized phospholipids can have deleterious effects, including disruption of membrane bilayers and production of toxic byproducts (4, 6-8). One hypothesized pathway for removal of oxidatively damaged species involves hydrolysis by phospholipase A2-type enzymes. Candidate hydrolytic enzymes include the platelet-activating factor acetylhydrolases (PAFAHs) (4,9). The initial role assigned to the PAFAHs was the hydrolysis of platelet activating factor (PAF) (10,11), a potent pro-inflammatory phospholipid signaling molecule (12), which plays a role in myriad physiological processes including inflammation, anaphylaxis, fetal development, and reproduction (4,13). The PAFAHs are subdivided into three classes: plasma (p)PAFAH, and intracellular types 1 and 2. In terms of sequence homology, pPAFAH and PAFAH2 are close homologs and show little similarity to type 1 enzymes. This project aims to develop specific inhibitors for pPAFAH and three counterscreen enzymes: PAFAH2, PAFAH1b2, and PAFAH1b3.

pPAFAH is associated with inflammatory pathways involved in atherosclerosis, asthma, anaphylactic shock, and other allergic reactions (14,15). Numerous studies have also linked increases in pPAFAH concentration and/or activity to an increased risk of various cardiovascular diseases (16,17); however, the biological function of pPAFAH in the development of coronary heart diseases is controversial, with both anti- and proinflammatory roles attributed to it (18,19). Dr. Bahnson and colleagues recently reported the first high-resolution crystal structure of pPAFAH (20), 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 (21), its properties are not suitable for the proposed studies.

PAFAH2 has also been shown to play a role in inflammatory processes via hydrolysis of oxidized phospholipids. Over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death (22,23) and to mediate repair of oxidative-stress induced tissue injury (4). The enzyme also undergoes N-terminal myristoylation and subsequent translocation to the membrane under conditions of oxidative stress (22,23). This evidence suggests that PAFAH2 functions as an important, and perhaps primary, antioxidant enzyme in certain tissues (4); however, its substrate specificity and pathway involvement are far from being fully elucidated. Currently no suitable inhibitors exist for co-crystallization or biochemical studies of PAFAH2.

Given the complex biology of the PAFAH enzymes, chemical tools capable of interrogating enzyme architecture and providing precise temporal control over PAFAH activity are necessary for complete characterization of patho/physiological roles of the PAFAHs in phospholipid metabolism and inflammatory disease processes. Towards that goal, we developed a HTS assay for inhibitor discovery for four PAFAH enzymes: pPAFAH, PAFAH2, PAFAH1b2, and PAFAH1b3, based on their reactivity with the serine-hydrolase-specific fluorophosphonate (FP) (24) activity-based protein profiling (ABPP) probe. This reactivity can be exploited for inhibitor discovery using a competitive-ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling (25). Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP (26). Following the HTS campaign, top inhibitors for each enzyme will be characterized and medchem optimized with the goal of delivering key reagents for elucidating the biology of the PAFAH enzymes.


1. Ames, B.N., Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science, 1983. 221(4617): p. 1256-64.
2. Halliwell, B. and J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol., 1990. 186: p. 1-85.
3. Harman, D., The aging process. Proc. Natl. Acad. Sci. U. S. A., 1981. 78(11): p. 7124-8.
4. Kono, N., et al., Protection against oxidative stress-induced hepatic injury by intracellular type II platelet-activating factor acetylhydrolase by metabolism of oxidized phospholipids in vivo. J. Biol. Chem., 2008. 283(3): p. 1628-36.
5. Southorn, P.A. and G. Powis, Free radicals in medicine. II. Involvement in human disease. Mayo. Clin. Proc., 1988. 63(4): p. 390-408.
6. Kinnunen, P.K., On the principles of functional ordering in biological membranes. Chem. Phys. Lipids, 1991. 57(2-3): p. 375-99.
7. Uchida, K., 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res., 2003. 42(4): p. 318-43.
8. Fruhwirth, G.O., A. Loidl, and A. Hermetter, Oxidized phospholipids: from molecular properties to disease. Biochim. Et Biophys. Acta, 2007. 1772(7): p. 718-36.
9. Nigam, S. and T. Schewe, Phospholipase A(2)s and lipid peroxidation. Biochim. Et Biophys. Acta, 2000. 1488(1-2): p. 167-81.
10. Blank, M.L., et al., A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid). J. Biol. Chem., 1981. 256(1): p. 175-8.
11. Farr, R.S., et al., Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF). Clin. Immunol. Immunopathol., 1980. 15(3): p. 318-330.
12. Zimmerman, G.A., et al., The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis. Crit. Care Med., 2002. 30(5 Suppl): p. S294-301.
13. Prescott, S.M., et al., Platelet-activating factor and related lipid mediators. Annu. Rev. Biochem., 2000. 69: p. 419-45.
14. Karasawa, K., et al., Plasma platelet activating factor-acetylhydrolase (PAF-AH). Prog. Lipid Res., 2003. 42(2): p. 93-114.
15. Leitinger, N., Oxidized phospholipids as triggers of inflammation in atherosclerosis. Mol. Nutr. Food Res., 2005. 49(11): p. 1063-71.
16. Anderson, J.L., Lipoprotein-associated phospholipase A2: an independent predictor of coronary artery disease events in primary and secondary prevention. Am. J. Cardiol., 2008. 101(12A): p. 23F-33F.
17. Sudhir, K., Clinical review: Lipoprotein-associated phospholipase A2, a novel inflammatory biomarker and independent risk predictor for cardiovascular disease. J. Clin. Endocrinol. Metab., 2005. 90(5): p. 3100-5.
18. Wilensky, R.L. and C.H. Macphee, Lipoprotein-associated phospholipase A(2) and atherosclerosis. Curr. Opin. Lipidol., 2009. 20(5): p. 415-20.
19. Karabina, S.A. and E. Ninio, Plasma PAF-acetylhydrolase: an unfulfilled promise? Biochim. Et Biophys. Acta, 2006. 1761(11): p. 1351-8.
20. Samanta, U. and B.J. Bahnson, Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis. J. Biol. Chem., 2008. 283(46): p. 31617-24.
21. Blackie, J.A., et al., The identification of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospholipase A2. Bioorg. Med. Chem. Lett., 2003. 13(6): p. 1067-70.
22. Matsuzawa, A., et al., Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II. J. Biol. Chem., 1997. 272(51): p. 32315-20.
23. Marques, M., et al., Identification of platelet-activating factor acetylhydrolase II in human skin. J. Invest. Dermatol., 2002. 119(4): p. 913-9.
24. Jessani, N., et al., Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A., 2002. 99(16): p. 10335-40.
25. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
26. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol., 2009. 27(4): p. 387-94.


late stage, late stage AID, assay provider, powders, platelet-activating factor acetylhydrolase, PAFAH, PAF-AH, plasma platelet-activating factor acetylhydrolase, pPAFAH, platelet-activating factor acetylhydrolase type II, PAFAH2, PAFAHII, cancer, inflammation, atherosclerosis, serine hydrolase, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, inhibitor, in situ, cell-based assay, BW5147, murine T cells, T cells, Scripps, 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 or not powder samples of test compounds can inhibit PAFAH2 activity in situ. In this assay, cultured BW5147-derived murine T-cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated 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 (IOD) of the bands. As designed, test compounds that act as PAFAH2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.

Protocol Summary:

BW5147-derived murine T cells (5 mL total volume; supplemented with FCS) were treated with 30 nM test compound (5 uL of a 1000x stock in DMSO) for 4 hours at 37 C. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 C, quenched with 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 PAFAH2 band relative to a DMSO-only (no compound) control.

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


Test_Compound is defined as PAFAH2 in cells treated with test compound.
High_Control is defined as PAFAH2 in cells 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 90% inhibition of PAFAH2 were considered active. Compounds with inhibition less than 90% inhibition of PAFAH2 were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. 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:

BW5147-derived murine T-cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
FP-Rh (provided by the Assay Provider)
This assay was performed by the assay provider with powder samples of compounds.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 0.03 uM (0.03μM**)In situ inhibition of PAFAH2 upon 30 nM compound treatment as assessed by competitive ABPP.Integer%

** Test Concentration.
Additional Information
Grant Number: 1R01HL084366

Data Table (Concise)