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BioAssay: AID 504519

Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for PAFAH2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for PAFAH2. ..more
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Active(1)
 
 
AID: 504519
Data Source: The Scripps Research Institute Molecular Screening Center (PAFAH2_INH_LCMS_SILAC)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-21
Hold-until Date: 2011-10-06
Modify Date: 2011-10-06

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
492956Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Screening depositor-specified cross reference: Primary screen (PAFAH2 inhibitors in singlicate)
492969Summary of the probe development efforts to identify inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Summary1 depositor-specified cross reference: Summary (PAFAH2 inhibitors)
493030Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of human platelet activating factor acetylhydrolase 2 (PAFAH2)Screening depositor-specified cross reference: Primary screen (PAFAH2 inhibitors in triplicate)
463082Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the plasma platelet activating factor acetylhydrolase (pPAFAH)Screening same project related to Summary assay
504483Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) counterscreen assay to assess selectivity against anti-target pPAFAH in vitroOther same project related to Summary assay
504486Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS/MS assay to assess binding of compounds to active siteOther same project related to Summary assay
504491Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivity to assess carbamate vs. triazole urea scaffoldOther same project related to Summary assay
504494Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory same project related to Summary assay
504495Late 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) inhibitionOther same project related to Summary assay
504496Late 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) IC50Confirmatory same project related to Summary assay
504511Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory same project related to Summary assay
504513Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivityOther same project related to Summary assay
504527Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) general inhibition and selectivity of serine hydrolasesOther same project related to Summary assay
504531Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) assay to assess selectivity against anti-target pPAFAH in situOther same project related to Summary assay
Description:
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
External Assay ID: PAFAH2_INH_LCMS_SILAC

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAFAH2: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for PAFAH2.

Description:

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.

References:

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.

Keywords:

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, stable isotope labeling with amino acids in cell culture, SILAC, liquid chromatography-tandem mass spectrometry, LC-MS/MS, 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
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PAFAH21platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus] [gi:225579137]
Taxonomy id: 10090
Gene id: 100163
2ABHD61Abhydrolase domain containing 6 [Mus musculus] [gi:20073260]
Taxonomy id: 10090
Gene id: 66082
3PLA2G61Pla2g6 protein [Mus musculus] [gi:34784362]
Taxonomy id: 10090
Gene id: 53357
4APEH1Apeh protein [Mus musculus] [gi:20072022]
Taxonomy id: 10090
Gene id: 235606
5ESD1Esterase D/formylglutathione hydrolase [Mus musculus] [gi:55777188]
Taxonomy id: 10090
Gene id: 13885
6CTSA1cathepsin A [Mus musculus] [gi:123240058]
Taxonomy id: 10090
Gene id: 19025
7SERHL1Serine hydrolase-like [Mus musculus] [gi:127797596]
Taxonomy id: 10090
Gene id: 68607
8FAM108A1Family with sequence similarity 108, member A [Mus musculus] [gi:52789434]
Taxonomy id: 10090
Gene id: 216169
9PPME11protein phosphatase methylesterase 1 [Mus musculus] [gi:30794138]
Taxonomy id: 10090
Gene id: 72590
10DPP71dipeptidylpeptidase 7 [Mus musculus] [gi:123227495]
Taxonomy id: 10090
Gene id: 83768
11DPP41dipeptidylpeptidase 4 [Mus musculus] [gi:123233284]
Taxonomy id: 10090
Gene id: 13482
12ACOT11Acyl-CoA thioesterase 1 [Mus musculus] [gi:20380528]
Taxonomy id: 10090
Gene id: 26897
13FAM108B11Fam108b protein [Mus musculus] [gi:61403362]
Taxonomy id: 10090
Gene id: 226016
14IAH11isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus] [gi:27754071]
Taxonomy id: 10090
Gene id: 67732
15ACOT21Acyl-CoA thioesterase 2 [Mus musculus] [gi:39992610]
Taxonomy id: 10090
Gene id: 171210
16ABHD131abhydrolase domain-containing protein 13 [Mus musculus] [gi:299473802]
Taxonomy id: 10090
Gene id: 68904
17PARL1presenilin associated, rhomboid-like [Mus musculus] [gi:148665146]
Taxonomy id: 10090
Gene id: 381038
18PAFAH1B21platelet-activating factor acetylhydrolase isoform Ib beta subunit [Mus musculus] [gi:1373363]
Taxonomy id: 10090
Gene id: 18475
19ACOT71acyl-CoA thioesterase 7 [Mus musculus] [gi:123857891]
Taxonomy id: 10090
Gene id: 70025
20LYPLA31lysophospholipase 3, isoform CRA_a [Mus musculus] [gi:148679401]
Taxonomy id: 10090
Gene id: 192654
21ABHD121abhydrolase domain containing 12 [Mus musculus] [gi:123241061]
Taxonomy id: 10090
Gene id: 76192
22FAM108C1Family with sequence similarity 108, member C [Mus musculus] [gi:17391206]
Taxonomy id: 10090
Gene id: 70178
23PREPL1Prepl protein [Mus musculus] [gi:13435484]
Taxonomy id: 10090
Gene id: 213760
24LYPLA11Lypla1 [Mus musculus] [gi:71059731]
Taxonomy id: 10090
Gene id: 18777
25FASN1fatty acid synthase [Mus musculus] [gi:123288587]
Taxonomy id: 10090
Gene id: 14104
26PREP1prolyl endopeptidase [Mus musculus] [gi:148673089]
Taxonomy id: 10090
Gene id: 19072
27BAT51abhydrolase domain-containing protein 16A [Mus musculus] [gi:30519896]
Taxonomy id: 10090
Gene id: 193742
28FAAH1fatty acid amide hydrolase [Mus musculus] [gi:123253900]
Taxonomy id: 10090
Gene id: 14073
29SIAE1sialic acid acetylesterase [Mus musculus] [gi:148693491]
Taxonomy id: 10090
Gene id: 22619
30PAFAH1B31platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus] [gi:6679201]
Taxonomy id: 10090
Gene id: 18476
31DPP81Dipeptidylpeptidase 8 [Mus musculus] [gi:37590654]
Taxonomy id: 10090
Gene id: 74388
32AADACL11Arylacetamide deacetylase-like 1 [Mus musculus] [gi:52139053]
Taxonomy id: 10090
Gene id: 320024
33LACTB1Lactamase, beta [Mus musculus] [gi:187955464]
Taxonomy id: 10090
Gene id: 80907
34DPP91dipeptidyl peptidase 9 [Mus musculus] [gi:255003757]
Taxonomy id: 10090
Gene id: 224897
35ABHD101Abhydrolase domain containing 10 [Mus musculus] [gi:37231537]
Taxonomy id: 10090
Gene id: 213012
36LYPLA21lysophospholipase 2 [Mus musculus] [gi:123122209]
Taxonomy id: 10090
Gene id: 26394
37PRCP1Prcp protein [Mus musculus] [gi:32967631]
Taxonomy id: 10090
Gene id: 72461
38ABHD111Abhydrolase domain containing 11 [Mus musculus] [gi:47682716]
Taxonomy id: 10090
Gene id: 68758
39DDHD11Ddhd1 protein [Mus musculus] [gi:27694042]
Taxonomy id: 10090
Gene id: 114874
40LIPA1lysosomal acid lipase 1 [Mus musculus] [gi:148709804]
Taxonomy id: 10090
Gene id: 16889
41PNPLA71patatin-like phospholipase domain-containing protein 7 [Mus musculus] [gi:225007615]
Taxonomy id: 10090
Gene id: 241274
42PNPLA61Pnpla6 protein [Mus musculus] [gi:34784201]
Taxonomy id: 10090
Gene id: 50767

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using stable isotope labeling with amino acids in cell culture (SILAC) ABPP. In this assay, cultured BW5147-derived murine T-cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with inhibitor and DMSO, respectively, in situ. Cells are lysed, proteomes are treated with FP-biotin, and combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, giving a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of 1.
Protocol Summary:
Stable isotope labeling with amino acids in cell culture (SILAC):
BW5147-derived murine T-cell hybridoma cells were initially grown for 6 passages in either light or heavy SILAC RPMI 1640 media supplemented with 10% dialyzed FCS and 1x PenStrep Glutamine. Light media was supplemented with 100 ug/mL L-arginine (Sigma) and 100 ug/mL L-lysine (Sigma). Heavy media was supplemented with 100 ug/mL [13C615N4]-L-Arginine (Isotek) and 100 ug/mL [13C615N2]-L-Lysine (Isotek). Cells were treated with 3 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 and membrane fractions were isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS.
Sample preparation for ABPP-SILAC:
The light and heavy proteomes were labeled with 7 muM of FP-biotin (500 muL total reaction volume) for 1.5 hours at 25 C. After incubation, light and heavy proteomes were mixed in 1:1 ratio, and the membrane proteomes were additionally solubilized with 1% Triton-X100. The proteomes were desalted over PD-10 desalting columns (GE Healthcare) and FP-labeled proteins were enriched with streptavidin beads. The beads were washed with 1% SDS in PBS (1x), PBS (3x), and H2O (3x), then resuspended in 6 M urea, reduced with DTT for 15 minutes at 60 C, and alkylated with iodoacetamide for 30 minutes at 25 C in the dark. On-bead digestions were performed for 12 hours at 37 C with trypsin (Promega; 4 muL of 0.5 mug/muL) in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% formic acid, pressure-loaded on to a biphasic (strong cation exchange/reverse phase) capillary column and analyzed as described below.
LC-MS/MS analysis:
Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography (2D-LC) separation in combination with tandem mass spectrometry using an Agilent 1100-series quaternary pump and Thermo Scientific LTQ Orbitrap ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (60 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publicly available at http://fields.scripps.edu/?q=content/download). MS2 spectra data were searched using the SEQUEST algorithm (Version 3.0) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. SEQUEST searches allowed for variable oxidation of methionine (+16), static modification of cysteine residues (+57 due to alkylation), and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0.41) using the --trypstat option, which applies different statistical models for the analysis of tryptic, half-tryptic, non-tryptic peptides. DTASelect 2.0 uses a quadratic discriminant analysis to achieve a user-defined maximum peptide false positive rate; the default parameters (maximum false positive rate of 2%) was used for the search; however, the actual false positive rate was much lower (1%). Ratios of Light/Heavy peaks were calculated using in-house software; reported ratios represent the mean of all unique, quantified peptides per protein.
Ratio = Average( AUClight / AUCheavy ) calculated for all unique peptides
Where:
AUClight is the area-under-the-curve for the light peptide pair from cells treated with test compound.
AUCheavy is the area-under-the-curve for the heavy peptide pair from cells treated with DMSO.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
A compound was considered active for a particular target/anti-target with a light/heavy ratio of less than or equal to 0.5. A compound was considered in active for a specified target/anti-target with a light/heavy ratio of greater than 0.5.
Overall Outcome and Score:
A compound was considered active if it was active for PAFAH2 and inactive for all anti-target serine hydrolases tested.
Active compounds were given a score of 100 and inactive compounds were given a score of 0.
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)
SILAC RPMI 1640 media (Thermo 89984)
dialyzed FCS (Gemini 100-108)
1x PenStrep Glutamine (CellGro 30-002-CI)
L-Arginine (Sigma A6969)
L-Lysine (Sigma L9037)
[13C615N4]-L-Arginine (Sigma 608033)
[13C615N2]-L-Lysine (Sigma 608041)
DPBS (Cellgro 20-031-CV)
FP-biotin (provided by Assay Provider)
PD-10 desalting columns (GE Healthcare 17-0851-01)
SDS (Sigma L6026)
Urea (Fisher U15-3)
DTT (Sigma 43815)
Iodoacetamide(Sigma I1149)
Trypsin (Promega V5111)
CaCl2 (Sigma C1016)
streptavidin beads (Pierce 20349)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Millipore-filtered Water
Formic acid (Fluka 06440)
Triton-X100 (Fisher AC21568-0010)
Comment
This assay was performed by the assay provider with powder samples of compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: BW5147
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [PAFAH2]One of Active, Inactive, or Not Tested1platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus]Outcome
2Average Silac Ratio [PAFAH2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 1Floatratio
3Standard Error [PAFAH2]Calculated standard error for mean1Float
4Outcome [ABHD6]One of Active, Inactive, or Not Tested2Abhydrolase domain containing 6 [Mus musculus]Outcome
5Average Silac Ratio [ABHD6]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 2Floatratio
6Standard Error [ABHD6]Calculated standard error for mean2Float
7Outcome [PLA2G6]One of Active, Inactive, or Not Tested3Pla2g6 protein [Mus musculus]Outcome
8Average Silac Ratio [PLA2G6]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 3Floatratio
9Standard Error [PLA2G6]Calculated standard error for mean3Float
10Outcome [APEH]One of Active, Inactive, or Not Tested4Apeh protein [Mus musculus]Outcome
11Average Silac Ratio [APEH]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 4Floatratio
12Standard Error [APEH]Calculated standard error for mean4Float
13Outcome [ESD]One of Active, Inactive, or Not Tested5Esterase D/formylglutathione hydrolase [Mus musculus]Outcome
14Average Silac Ratio [ESD]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 5Floatratio
15Standard Error [ESD]Calculated standard error for mean5Float
16Outcome [CTSA]One of Active, Inactive, or Not Tested6cathepsin A [Mus musculus]Outcome
17Average Silac Ratio [CTSA]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 6Floatratio
18Standard Error [CTSA]Calculated standard error for mean6Float
19Outcome [SERHL]One of Active, Inactive, or Not Tested7Serine hydrolase-like [Mus musculus]Outcome
20Average Silac Ratio [SERHL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 7Floatratio
21Standard Error [SERHL]Calculated standard error for mean7Float
22Outcome [FAM108A]One of Active, Inactive, or Not Tested8Family with sequence similarity 108, member A [Mus musculus]Outcome
23Average Silac Ratio [FAM108A]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 8Floatratio
24Standard Error [FAM108A]Calculated standard error for mean8Float
25Outcome [PPME1]One of Active, Inactive, or Not Tested9protein phosphatase methylesterase 1 [Mus musculus]Outcome
26Average Silac Ratio [PPME1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 9Floatratio
27Standard Error [PPME1]Calculated standard error for mean9Float
28Outcome [DPP7]One of Active, Inactive, or Not Tested10dipeptidylpeptidase 7 [Mus musculus]Outcome
29Average Silac Ratio [DPP7]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 10Floatratio
30Standard Error [DPP7]Calculated standard error for mean10Float
31Outcome [DPP4]One of Active, Inactive, or Not Tested11dipeptidylpeptidase 4 [Mus musculus]Outcome
32Average Silac Ratio [DPP4]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 11Floatratio
33Standard Error [DPP4]Calculated standard error for mean11Float
34Outcome [ACOT1]One of Active, Inactive, or Not Tested12Acyl-CoA thioesterase 1 [Mus musculus]Outcome
35Average Silac Ratio [ACOT1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 12Floatratio
36Standard Error [ACOT1]Calculated standard error for mean12Float
37Outcome [FAM108B1]One of Active, Inactive, or Not Tested13Fam108b protein [Mus musculus]Outcome
38Average Silac Ratio [FAM108B1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 13Floatratio
39Standard Error [FAM108B1]Calculated standard error for mean13Float
40Outcome [IAH1]One of Active, Inactive, or Not Tested14isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus]Outcome
41Average Silac Ratio [IAH1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 14Floatratio
42Standard Error [IAH1]Calculated standard error for mean14Float
43Outcome [ACOT2]One of Active, Inactive, or Not Tested15Acyl-CoA thioesterase 2 [Mus musculus]Outcome
44Average Silac Ratio [ACOT2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 15Floatratio
45Standard Error [ACOT2]Calculated standard error for mean15Float
46Outcome [ABHD13]One of Active, Inactive, or Not Tested16abhydrolase domain-containing protein 13 [Mus musculus]Outcome
47Average Silac Ratio [ABHD13]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 16Floatratio
48Standard Error [ABHD13]Calculated standard error for mean16Float
49Outcome [PARL]One of Active, Inactive, or Not Tested17presenilin associated, rhomboid-like [Mus musculus]Outcome
50Average Silac Ratio [PARL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 17Floatratio
51Standard Error [PARL]Calculated standard error for mean17Float
52Outcome [PAFAH1B2]One of Active, Inactive, or Not Tested18platelet-activating factor acetylhydrolase isoform Ib beta subunit [Mus musculus]Outcome
53Average Silac Ratio [PAFAH1B2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 18Floatratio
54Standard Error [PAFAH1B2]Calculated standard error for mean18Float
55Outcome [ACOT7]One of Active, Inactive, or Not Tested19acyl-CoA thioesterase 7 [Mus musculus]Outcome
56Average Silac Ratio [ACOT7]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 19Floatratio
57Standard Error [ACOT7]Calculated standard error for mean19Float
58Outcome [LYPLA3]One of Active, Inactive, or Not Tested20lysophospholipase 3, isoform CRA_a [Mus musculus]Outcome
59Average Silac Ratio [LYPLA3]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 20Floatratio
60Standard Error [LYPLA3]Calculated standard error for mean20Float
61Outcome [ABHD12]One of Active, Inactive, or Not Tested21abhydrolase domain containing 12 [Mus musculus]Outcome
62Average Silac Ratio [ABHD12]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 21Floatratio
63Standard Error [ABHD12]Calculated standard error for mean21Float
64Outcome [FAM108C]One of Active, Inactive, or Not Tested22Family with sequence similarity 108, member C [Mus musculus]Outcome
65Average Silac Ratio [FAM108C]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 22Floatratio
66Standard Error [FAM108C]Calculated standard error for mean22Float
67Outcome [PREPL]One of Active, Inactive, or Not Tested23Prepl protein [Mus musculus]Outcome
68Average Silac Ratio [PREPL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 23Floatratio
69Standard Error [PREPL]Calculated standard error for mean23Float
70Outcome [LYPLA1]One of Active, Inactive, or Not Tested24Lypla1 [Mus musculus]Outcome
71Average Silac Ratio [LYPLA1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 24Floatratio
72Standard Error [LYPLA1]Calculated standard error for mean24Float
73Outcome [FASN]One of Active, Inactive, or Not Tested25fatty acid synthase [Mus musculus]Outcome
74Average Silac Ratio [FASN]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 25Floatratio
75Standard Error [FASN]Calculated standard error for mean25Float
76Outcome [PREP]One of Active, Inactive, or Not Tested26prolyl endopeptidase [Mus musculus]Outcome
77Average Silac Ratio [PREP]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 26Floatratio
78Standard Error [PREP]Calculated standard error for mean26Float
79Outcome [BAT5]One of Active, Inactive, or Not Tested27abhydrolase domain-containing protein 16A [Mus musculus]Outcome
80Average Silac Ratio [BAT5]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 27Floatratio
81Standard Error [BAT5]Calculated standard error for mean27Float
82Outcome [FAAH]One of Active, Inactive, or Not Tested28fatty acid amide hydrolase [Mus musculus]Outcome
83Average Silac Ratio [FAAH]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 28Floatratio
84Standard Error [FAAH]Calculated standard error for mean28Float
85Outcome [SIAE]One of Active, Inactive, or Not Tested29sialic acid acetylesterase [Mus musculus]Outcome
86Average Silac Ratio [SIAE]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 29Floatratio
87Standard Error [SIAE]Calculated standard error for mean29Float
88Outcome [PAFAH1B3]One of Active, Inactive, or Not Tested30platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus]Outcome
89Average Silac Ratio [PAFAH1B3]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 30Floatratio
90Standard Error [PAFAH1B3]Calculated standard error for mean30Float
91Outcome [DPP8]One of Active, Inactive, or Not Tested31Dipeptidylpeptidase 8 [Mus musculus]Outcome
92Average Silac Ratio [DPP8]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 31Floatratio
93Standard Error [DPP8]Calculated standard error for mean31Float
94Outcome [AADACL1]One of Active, Inactive, or Not Tested32Arylacetamide deacetylase-like 1 [Mus musculus]Outcome
95Average Silac Ratio [AADACL1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 32Floatratio
96Standard Error [AADACL1]Calculated standard error for mean32Float
97Outcome [LACTB]One of Active, Inactive, or Not Tested33Lactamase, beta [Mus musculus]Outcome
98Average Silac Ratio [LACTB]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 33Floatratio
99Standard Error [LACTB]Calculated standard error for mean33Float
100Outcome [DPP9]One of Active, Inactive, or Not Tested34dipeptidyl peptidase 9 [Mus musculus]Outcome
101Average Silac Ratio [DPP9]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 34Floatratio
102Standard Error [DPP9]Calculated standard error for mean34Float
103Outcome [ABHD10]One of Active, Inactive, or Not Tested35Abhydrolase domain containing 10 [Mus musculus]Outcome
104Average Silac Ratio [ABHD10]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 35Floatratio
105Standard Error [ABHD10]Calculated standard error for mean35Float
106Outcome [LYPLA2]One of Active, Inactive, or Not Tested36lysophospholipase 2 [Mus musculus]Outcome
107Average Silac Ratio [LYPLA2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 36Floatratio
108Standard Error [LYPLA2]Calculated standard error for mean36Float
109Outcome [PRCP]One of Active, Inactive, or Not Tested37Prcp protein [Mus musculus]Outcome
110Average Silac Ratio [PRCP]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 37Floatratio
111Standard Error [PRCP]Calculated standard error for mean37Float
112Outcome [ABHD11]One of Active, Inactive, or Not Tested38Abhydrolase domain containing 11 [Mus musculus]Outcome
113Average Silac Ratio [ABHD11]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 38Floatratio
114Standard Error [ABHD11]Calculated standard error for mean38Float
115Outcome [DDHD1]One of Active, Inactive, or Not Tested39Ddhd1 protein [Mus musculus]Outcome
116Average Silac Ratio [DDHD1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 39Floatratio
117Standard Error [DDHD1]Calculated standard error for mean39Float
118Outcome [LIPA]One of Active, Inactive, or Not Tested40lysosomal acid lipase 1 [Mus musculus]Outcome
119Average Silac Ratio [LIPA]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 40Floatratio
120Standard Error [LIPA]Calculated standard error for mean40Float
121Outcome [PNPLA7]One of Active, Inactive, or Not Tested41patatin-like phospholipase domain-containing protein 7 [Mus musculus]Outcome
122Average Silac Ratio [PNPLA7]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 41Floatratio
123Standard Error [PNPLA7]Calculated standard error for mean41Float
124Outcome [PNPLA6]One of Active, Inactive, or Not Tested42Pnpla6 protein [Mus musculus]Outcome
125Average Silac Ratio [PNPLA6]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified 42Floatratio
126Standard Error [PNPLA6]Calculated standard error for mean42Float
Additional Information
Grant Number: 1R01HL084366

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