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BioAssay: AID 651978

Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro

Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro. ..more
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AID: 651978
Data Source: The Scripps Research Institute Molecular Screening Center (LYPLA1_INH_LCMS_ABPP_SILAC_INVITRO)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-01-15
Hold-until Date: 2013-10-18
Modify Date: 2013-10-18

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Primary screen (LYPLA1 inhibitors in singlicate)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Primary screen (LYPLA2 inhibitors in singlicate)
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).Summary2 depositor-specified cross reference: Summary (LYPLA1 inhibitors)
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).Summary1 depositor-specified cross reference: Summary (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Confirmation screen (LYPLA2 inhibitors in triplicate)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Confirmation screen (LYPLA1 inhibitors in triplicate)
493105Assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition of recombinant and endogenous enzymeOther depositor-specified cross reference: Confirmation screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493108Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: fluorescence-based cell-based inhibitionOther depositor-specified cross reference: Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493109Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: LC-MS/MS assay to assess binding of compounds to active siteOther depositor-specified cross reference: Late stage LCMS assay (LYPLA1)
493110Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Gel-based Activity-Based Protein Profiling (ABPP) IC50 for LYPLA1 and LYPLA2Confirmatory depositor-specified cross reference: Late stage dose response (LYPLA1 and LYPLA2 inhibitors in triplicate)
493111Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther depositor-specified cross reference: Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493154Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Gel-based Activity-Based Protein Profiling (ABPP) IC50 for off-target ABHD11Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (ABHD11 inhibitors in triplicate)
493161Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (T-cell cytotoxicity in quadruplicate)
504482Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) IC50 for anti-target ABHD11Confirmatory depositor-specified cross reference: Late stage dose repsonse (ABHD11 inhibitors in triplicate)
504498Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: LC-MS/MS assay to assess binding of compounds to active site of anti-target ABHD11Other depositor-specified cross reference: Late stage MOA assay (ABDH11 LCMS)
504505Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) percent inhibition for anti-target ABHD11Other depositor-specified cross reference: Late stage screen (ABHD11 inhibitors in singlicate, in situ)
504507Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) IC50 for anti-target ABHD11 Set 2Confirmatory depositor-specified cross reference: Late stage dose response (ABHD11 inhibitors in triplicate)
504510Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds set 2Confirmatory depositor-specified cross reference: Late stage dose repsonse counterscreen (T-cell cytotoxicity in quadruplicate)
504520Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivityOther depositor-specified cross reference: Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
504522Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for anti-target ABHD11Other depositor-specified cross reference: Late stage panel screen (ABHD11 SILAC ratio)
504892Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11Other depositor-specified cross reference: Late stage counterscreen (ABHD11 inhibitors in singlicate)
651998Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: Substrate-based fluorescence-based biochemical determination of kinetic parametersConfirmatory depositor-specified cross reference
652001Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Substrate-based fluorescence-based biochemical determination of kinetic parametersConfirmatory depositor-specified cross reference
652003Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: fluorescence-based biochemical dose-response assayConfirmatory depositor-specified cross reference
652004Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: fluorescence-based biochemical dose-response assayConfirmatory depositor-specified cross reference
652018Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro, Set 2Other depositor-specified cross reference
652029Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibitionOther depositor-specified cross reference
652030Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther depositor-specified cross reference
743117Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition potency and selectivityOther depositor-specified cross reference
743118Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP in vitroConfirmatory depositor-specified cross reference
743119Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP in situConfirmatory depositor-specified cross reference
743127Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference
743132Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP in vitro in mouse brainConfirmatory depositor-specified cross reference
743133Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess inhibitor binding modeOther depositor-specified cross reference
743134Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP assay to assess in vivo activityOther depositor-specified cross reference
743137 On Hold
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 same project related to Summary assay
651979Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situOther same project related to Summary assay
651980Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situOther same project related to Summary assay
651981Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitroOther same project related to Summary assay
651985Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivoOther same project related to Summary assay
651986Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in situOther same project related to Summary assay
651987Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess binding modeOther same project related to Summary assay
651988Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther same project related to Summary assay
651990Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPPConfirmatory same project related to Summary assay
651991Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory same project related to Summary assay
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 same project related to Summary assay
651979Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situOther same project related to Summary assay
651980Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situOther same project related to Summary assay
651981Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitroOther same project related to Summary assay
651985Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivoOther same project related to Summary assay
651986Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in situOther same project related to Summary assay
651987Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess binding modeOther same project related to Summary assay
651988Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther same project related to Summary assay
651990Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPPConfirmatory same project related to Summary assay
651991Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory 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: 1 R01 CA132630
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: LYPLA1_INH_LCMS_ABPP_SILAC_INVITRO

Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro.

Description:

Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Numerous proteins have been identified as targets of palmitoylation, including cytoskeletal proteins, kinases, receptors, and other proteins involved in various aspects of cellular signaling and homeostasis (1). Using a global chemo-proteomic method for the metabolic incorporation and identification of palmitoylated proteins, we were able to identify hundreds of palmitoylated proteins, revealing palmitoylation as a widespread post-translational modification (PTM) (2). Palmitoylation involves an acyl-thioester linkage to specific cysteines (3,4). Given the labile properties of thioesters, palmitoylation is potentially reversible and may be regulated in a manner analogous to other PTMs (e.g., phosphorylation). As such, identification of proteins responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. For example, multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation (5), suggesting protein palmitoyl thioesterases may have tumor suppressor activity required to repress aberrant growth signaling. More than a decade ago, the cytosolic serine hydrolase acyl-protein thioesterase 1 (APT1) was identified as an in vitro HRAS palmitoyl thioesterase (6). Initially classified as lysophospholipase 1 (LYPLA1) (7), the enzyme has since been demonstrated to have several hundred-fold higher activity as a protein thioesterase. While the in vitro data (6,8) provided an intriguing clue to its possible role in vivo, prior to our studies, little was known about the in vivo thioesterase activity of LYPLA1. Upon retroviral shRNA knockdown of LYPLA1, we found that HRAS was robustly hyper-palmitoylated, providing the first evidence that the endogenous enzyme is a functional protein palmitoyl thioesterase capable of regulating HRAS palmitoylation in mammalian cells. However, shRNA resulted in only an 80% reduction in LYPLA1 expression (unpublished). LYPLA2 (a.k.a. APT2) is 65% identical to LYPLA1, and also exhibits lysophospholipase activity in vitro, but its potential role as a thioesterase is unknown (9). shRNA knockdown studies of LYPLA2 revealed only partial knockdown of the enzyme, making substrate identification inconclusive (unpublished). A principle goal of post-genomic research is the determination of the molecular and cellular role of uncharacterized enzymes like LYPLA1 and LYPLA2. As such, selective inhibitors of LYPLA1 or LYPLA2 would greatly aid investigations into the biological function of these enzymes. Several inhibitors of LYPLA1 have been described (10,11), but none of these agents have proven capable of inhibiting LYPLA1 activity in cells, and no selective inhibitors of LYPLA2 have been reported to date. To comprehensively identify LYPLA1 and LYPLA2 substrates and functionally test the role of these enzymes in dynamic de-palmitoylation and tumorigenesis, development of high affinity inhibitors, capable of achieving temporal and more complete control over activity, is critical.

References:

1. Dekker, F.J., et al., Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol., 2010. 6(6): p. 449-56.
2. Duncan, J.A. and A.G. Gilman, A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem., 1998. 273(25): p. 15830-7.
3. Sugimoto, H., H. Hayashi, and S. Yamashita, Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J. Biol. Chem., 1996. 271(13): p. 7705-11.
4. Toyoda, T., H. Sugimoto, and S. Yamashita, Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochim. Biophys. Acta, 1999. 1437(2): p. 182-93.
5. Biel, M., et al., Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chemistry, 2006. 12(15): p. 4121-43.
6. Deck, P., et al., Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. Engl., 2005. 44(31): p. 4975-80.
7. 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.
8. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
9. 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.
10. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19.
11. Schubert, C., The genomic basis of the Williams-Beuren syndrome. Cell. Mol. Life Sci., 2009. 66(7): p. 1178-97.

Keywords:

late stage, late stage AID, assay provider, powders, LYPLA1, lysophospholipase 1, APT1, acyl-protein thioesterase 1, serine hydrolase, palmitoylation, protein palmitoylation, counterscreen, inhibitor, inhibition, selectivity, anti-targets, liquid chromatography, LC, tandem mass spectrometry, MS/MS, activity-based protein profiling, ABPP, stable isotope labeling with amino acids in cell culture, SILAC, ABPP-SILAC, inhibitor, inhibition, fluorophosphonate-peg-biotin, FP-PEG-biotin, BW5147-derived murine T-cell hybridoma cells, BW5147, murine T-cell hybridoma, 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
1LYPLA11acyl-protein thioesterase 1 [Mus musculus] [gi:6678760]
Taxonomy id: 10090
Gene id: 18777
2ABHD111alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Mus musculus] [gi:269784760]
Taxonomy id: 10090
Gene id: 213012
3ABHD101alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Mus musculus] [gi:269784760]
Taxonomy id: 10090
Gene id: 213012
4LYPLA21acyl-protein thioesterase 2 [Mus musculus] [gi:7242156]
Taxonomy id: 10090
Gene id: 26394
5DPP71dipeptidyl peptidase 2 precursor [Mus musculus] [gi:31981425]
Taxonomy id: 10090
Gene id: 83768
6PREPL1prolyl endopeptidase-like isoform a [Mus musculus] [gi:254939518]
Taxonomy id: 10090
Gene id: 213760
7PRCP1lysosomal Pro-X carboxypeptidase precursor [Mus musculus] [gi:33469015]
Taxonomy id: 10090
Gene id: 72461
8SIAE1sialic acid acetylesterase [Mus musculus] [gi:148693491]
Taxonomy id: 10090
Gene id: 22619
9PAFAH1B31platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus] [gi:6679201]
Taxonomy id: 10090
Gene id: 18476
10PAFAH1B21platelet-activating factor acetylhydrolase IB subunit beta [Mus musculus] [gi:40254624]
Taxonomy id: 10090
Gene id: 18475
11ESD1S-formylglutathione hydrolase [Mus musculus] [gi:13937355]
Taxonomy id: 10090
Gene id: 13885
12AADACL11neutral cholesterol ester hydrolase 1 [Mus musculus] [gi:30520239]
Taxonomy id: 10090
Gene id: 320024
13APEH1Acylpeptide hydrolase [Mus musculus] [gi:19343726]
Taxonomy id: 10090
Gene id: 235606
14DPP91dipeptidyl peptidase 9 [Mus musculus] [gi:255003757]
Taxonomy id: 10090
Gene id: 224897
15PREP1prolyl endopeptidase [Mus musculus] [gi:6755152]
Taxonomy id: 10090
Gene id: 19072
16DPP81dipeptidyl peptidase 8 [Mus musculus] [gi:31542571]
Taxonomy id: 10090
Gene id: 74388
17ACOT21acyl-coenzyme A thioesterase 2, mitochondrial precursor [Mus musculus] [gi:238624114]
Taxonomy id: 10090
Gene id: 171210
18FASN1fatty acid synthase [Mus musculus] [gi:93102409]
Taxonomy id: 10090
Gene id: 14104
19CTSA1lysosomal protective protein isoform a precursor [Mus musculus] [gi:84042525]
Taxonomy id: 10090
Gene id: 19025
20LIPA1lysosomal acid lipase/cholesteryl ester hydrolase precursor [Mus musculus] [gi:162287343]
Taxonomy id: 10090
Gene id: 16889
21ABHD61monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]
Taxonomy id: 10090
Gene id: 66082
22FAAH1fatty acid amide hydrolase [Mus musculus] [gi:123253900]
Taxonomy id: 10090
Gene id: 14073
23PNPLA61neuropathy target esterase isoform 1 [Mus musculus] [gi:390608665]
Taxonomy id: 10090
Gene id: 50767
24ACOT11acyl-coenzyme A thioesterase 1 [Mus musculus] [gi:6753550]
Taxonomy id: 10090
Gene id: 26897
25LACTB1serine beta-lactamase-like protein LACTB, mitochondrial precursor [Mus musculus] [gi:13507666]
Taxonomy id: 10090
Gene id: 80907
26DPP41dipeptidyl peptidase 4 isoform 1 [Mus musculus] [gi:6753674]
Taxonomy id: 10090
Gene id: 13482
27ABHD121monoacylglycerol lipase ABHD12 [Mus musculus] [gi:159110817]
Taxonomy id: 10090
Gene id: 76192
28PNPLA71patatin-like phospholipase domain-containing protein 7 [Mus musculus] [gi:225007615]
Taxonomy id: 10090
Gene id: 241274
29PAFAH21platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus] [gi:225579137]
Taxonomy id: 10090
Gene id: 100163

§ 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) activity-based protein profiling (ABPP) in vitro. In this assay, cultured BW5147-derived murine T-cells are metabolically labeled with light or heavy amino acids. Cells are harvested, homogenized, and proteome fractions isolated. Light and heavy samples are treated with inhibitor and DMSO, respectively, followed by the serine-hydrolase-specific ABPP affinity probe fluorophosphonate-peg-biotin (FP-PEG-biotin). FP-PEG-biotin is similar to FP-biotin, but shows slower reaction kinetics, facilitating analysis of target inhibition by reversible compounds. Light and heavy samples are combined in a 1:1 (w/w) ratio, and 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-PEG-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, resulting in a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio close to 1.
Protocol Summary:
Sample Preparation:
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 [(isotope-13)C6(isotope-15)N4]-L-Arginine (Isotek) and 100 ug/mL [(isotope-13)C6(isotope-15)N2]-L-Lysine (Isotek). Cells were harvested 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. Membrane and soluble fractions of light cells were treated with test compound (1 uM) and membrane and soluble fractions of heavy cells were treated with DMSO for 30 minutes at 37 C. All samples were reacted with the activity-based affinity probe FP-PEG-Biotin (5 uM) for 30 minutes at 25 C. Light and heavy proteomes were mixed in 1:1 (w/w) ratio to generate one membrane and one soluble sample, which were desalted over PD-10 columns (GE Healthcare). SDS was added to a final concentration of 0.5% in 3 mL total reaction volume and biotinylated proteins were enriched on streptavidin beads (50 uL, 1 hour, 25 C). The beads were washed with 1% SDS in DPBS (1x), 6M urea (1x), and DPBS (2x), then resuspended in 6 M urea (150 uL), reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 C in the dark, and urea concentration was reduced to 2 M with 2x volume DPBS. On-bead digestions were performed for 12 hours at 37 C with sequence-grade modified trypsin (Promega; 2 ug) in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid and stored at -80 C prior to analysis.
LC-MS/MS analysis:
Samples were analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT) 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/downloads.php). MS2 spectra data were searched using the Sequest algorithm against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. Sequest searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, +8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of light/heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were >3 standard deviations from the median peptide value. Proteins with less than three peptides per protein ID were not included in the analysis.
Ratio = Average( AUC_light / AUC_heavy) calculated for all unique peptides
Where:
AUC_light is the area-under-the-curve for the light peptide pair from cells treated with test compound.
AUC_heavy 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 inactive 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 LYPLA1 and inactive for all other serine hydrolases tested, otherwise inactive.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
BW5147-derived murine T-cell proteome (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)
[(isotope-13)C6(isotope-15)N4]-L-Arginine (Sigma 608033)
[(isotope-13)C6(isotope-15)N2]-L-Lysine (Sigma 608041)
DPBS (Cellgro 20-031-CV)
FP-PEG-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 synthetic compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Test Type: In vitro
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [LYPLA1]One of Active, Inactive, or Not Tested1acyl-protein thioesterase 1 [Mus musculus]Outcome
2Mean Ratio [LYPLA1] (1μM**)Calculated inhibitor-treated/DMSO ratio for LYPLA1 as mean for all unique peptides quantified1Floatratio
3Standard Deviation [LYPLA1]Calculated standard deviation for mean1Float
4Outcome [ABHD11]One of Active, Inactive, or Not Tested2alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Mus musculus]Outcome
5Mean Ratio [ABHD11] (1μM**)Calculated inhibitor-treated/DMSO ratio for ABHD11 as mean for all unique peptides quantified2Floatratio
6Standard Deviation [ABHD11]Calculated standard deviation for mean2Float
7Outcome [ABHD10]One of Active, Inactive, or Not Tested3alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Mus musculus]Outcome
8Mean Ratio [ABHD10] (1μM**)Calculated inhibitor-treated/DMSO ratio for ABHD10 as mean for all unique peptides quantified3Floatratio
9Standard Deviation [ABHD10]Calculated standard deviation for mean3Float
10Outcome [LYPLA2]One of Active, Inactive, or Not Tested4acyl-protein thioesterase 2 [Mus musculus]Outcome
11Mean Ratio [LYPLA2] (1μM**)Calculated inhibitor-treated/DMSO ratio for LYPLA2 as mean for all unique peptides quantified4Floatratio
12Standard Deviation [LYPLA2]Calculated standard deviation for mean4Float
13Outcome [DPP7]One of Active, Inactive, or Not Tested5dipeptidyl peptidase 2 precursor [Mus musculus]Outcome
14Mean Ratio [DPP7] (1μM**)Calculated inhibitor-treated/DMSO ratio for DPP7 as mean for all unique peptides quantified5Floatratio
15Standard Deviation [DPP7]Calculated standard deviation for mean5Float
16Outcome [PREPL]One of Active, Inactive, or Not Tested6prolyl endopeptidase-like isoform a [Mus musculus]Outcome
17Mean Ratio [PREPL] (1μM**)Calculated inhibitor-treated/DMSO ratio for PREPL as mean for all unique peptides quantified6Floatratio
18Standard Deviation [PREPL]Calculated standard deviation for mean6Float
19Outcome [PRCP]One of Active, Inactive, or Not Tested7lysosomal Pro-X carboxypeptidase precursor [Mus musculus]Outcome
20Mean Ratio [PRCP] (1μM**)Calculated inhibitor-treated/DMSO ratio for PRCP as mean for all unique peptides quantified7Floatratio
21Standard Deviation [PRCP]Calculated standard deviation for mean7Float
22Outcome [SIAE]One of Active, Inactive, or Not Tested8sialic acid acetylesterase [Mus musculus]Outcome
23Mean Ratio [SIAE] (1μM**)Calculated inhibitor-treated/DMSO ratio for SIAE as mean for all unique peptides quantified8Floatratio
24Standard Deviation [SIAE]Calculated standard deviation for mean8Float
25Outcome [PAFAH1B3]One of Active, Inactive, or Not Tested9platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus]Outcome
26Mean Ratio [PAFAH1B3] (1μM**)Calculated inhibitor-treated/DMSO ratio for PAFAH1B3 as mean for all unique peptides quantified9Floatratio
27Standard Deviation [PAFAH1B3]Calculated standard deviation for mean9Float
28Outcome [PAFAH1B2]One of Active, Inactive, or Not Tested10platelet-activating factor acetylhydrolase IB subunit beta [Mus musculus]Outcome
29Mean Ratio [PAFAH1B2] (1μM**)Calculated inhibitor-treated/DMSO ratio for PAFAH1B2 as mean for all unique peptides quantified10Floatratio
30Standard Deviation [PAFAH1B2]Calculated standard deviation for mean10Float
31Outcome [ESD]One of Active, Inactive, or Not Tested11S-formylglutathione hydrolase [Mus musculus]Outcome
32Mean Ratio [ESD] (1μM**)Calculated inhibitor-treated/DMSO ratio for ESD as mean for all unique peptides quantified11Floatratio
33Standard Deviation [ESD]Calculated standard deviation for mean11Float
34Outcome [AADACL1]One of Active, Inactive, or Not Tested12neutral cholesterol ester hydrolase 1 [Mus musculus]Outcome
35Mean Ratio [AADACL1] (1μM**)Calculated inhibitor-treated/DMSO ratio for AADACL1 as mean for all unique peptides quantified12Floatratio
36Standard Deviation [AADACL1]Calculated standard deviation for mean12Float
37Outcome [APEH]One of Active, Inactive, or Not Tested13Acylpeptide hydrolase [Mus musculus]Outcome
38Mean Ratio [APEH] (1μM**)Calculated inhibitor-treated/DMSO ratio for APEH as mean for all unique peptides quantified13Floatratio
39Standard Deviation [APEH]Calculated standard deviation for mean13Float
40Outcome [DPP9]One of Active, Inactive, or Not Tested14dipeptidyl peptidase 9 [Mus musculus]Outcome
41Mean Ratio [DPP9] (1μM**)Calculated inhibitor-treated/DMSO ratio for DPP9 as mean for all unique peptides quantified14Floatratio
42Standard Deviation [DPP9]Calculated standard deviation for mean14Float
43Outcome [PREP]One of Active, Inactive, or Not Tested15prolyl endopeptidase [Mus musculus]Outcome
44Mean Ratio [PREP] (1μM**)Calculated inhibitor-treated/DMSO ratio for PREP as mean for all unique peptides quantified15Floatratio
45Standard Deviation [PREP]Calculated standard deviation for mean15Float
46Outcome [DPP8]One of Active, Inactive, or Not Tested16dipeptidyl peptidase 8 [Mus musculus]Outcome
47Mean Ratio [DPP8] (1μM**)Calculated inhibitor-treated/DMSO ratio for DPP8 as mean for all unique peptides quantified16Floatratio
48Standard Deviation [DPP8]Calculated standard deviation for mean16Float
49Outcome [ACOT2]One of Active, Inactive, or Not Tested17acyl-coenzyme A thioesterase 2, mitochondrial precursor [Mus musculus]Outcome
50Mean Ratio [ACOT2] (1μM**)Calculated inhibitor-treated/DMSO ratio for ACOT2 as mean for all unique peptides quantified17Floatratio
51Standard Deviation [ACOT2]Calculated standard deviation for mean17Float
52Outcome [FASN]One of Active, Inactive, or Not Tested18fatty acid synthase [Mus musculus]Outcome
53Mean Ratio [FASN] (1μM**)Calculated inhibitor-treated/DMSO ratio for FASN as mean for all unique peptides quantified18Floatratio
54Standard Deviation [FASN]Calculated standard deviation for mean18Float
55Outcome [CTSA]One of Active, Inactive, or Not Tested19lysosomal protective protein isoform a precursor [Mus musculus]Outcome
56Mean Ratio [CTSA] (1μM**)Calculated inhibitor-treated/DMSO ratio for CTSA as mean for all unique peptides quantified19Floatratio
57Standard Deviation [CTSA]Calculated standard deviation for mean19Float
58Outcome [LIPA]One of Active, Inactive, or Not Tested20lysosomal acid lipase/cholesteryl ester hydrolase precursor [Mus musculus]Outcome
59Mean Ratio [LIPA] (1μM**)Calculated inhibitor-treated/DMSO ratio for LIPA as mean for all unique peptides quantified20Floatratio
60Standard Deviation [LIPA]Calculated standard deviation for mean20Float
61Outcome [ABHD6]One of Active, Inactive, or Not Tested21monoacylglycerol lipase ABHD6 [Mus musculus]Outcome
62Mean Ratio [ABHD6] (1μM**)Calculated inhibitor-treated/DMSO ratio for ABHD6 as mean for all unique peptides quantified21Floatratio
63Standard Deviation [ABHD6]Calculated standard deviation for mean21Float
64Outcome [FAAH]One of Active, Inactive, or Not Tested22fatty acid amide hydrolase [Mus musculus]Outcome
65Mean Ratio [FAAH] (1μM**)Calculated inhibitor-treated/DMSO ratio for FAAH as mean for all unique peptides quantified22Floatratio
66Standard Deviation [FAAH]Calculated standard deviation for mean22Float
67Outcome [PNPLA6]One of Active, Inactive, or Not Tested23neuropathy target esterase isoform 1 [Mus musculus]Outcome
68Mean Ratio [PNPLA6] (1μM**)Calculated inhibitor-treated/DMSO ratio for PNPLA6 as mean for all unique peptides quantified23Floatratio
69Standard Deviation [PNPLA6]Calculated standard deviation for mean23Float
70Outcome [ACOT1]One of Active, Inactive, or Not Tested24acyl-coenzyme A thioesterase 1 [Mus musculus]Outcome
71Mean Ratio [ACOT1] (1μM**)Calculated inhibitor-treated/DMSO ratio for ACOT1 as mean for all unique peptides quantified24Floatratio
72Standard Deviation [ACOT1]Calculated standard deviation for mean24Float
73Outcome [LACTB]One of Active, Inactive, or Not Tested25serine beta-lactamase-like protein LACTB, mitochondrial precursor [Mus musculus]Outcome
74Mean Ratio [LACTB] (1μM**)Calculated inhibitor-treated/DMSO ratio for LACTB as mean for all unique peptides quantified25Floatratio
75Standard Deviation [LACTB]Calculated standard deviation for mean25Float
76Outcome [DPP4]One of Active, Inactive, or Not Tested26dipeptidyl peptidase 4 isoform 1 [Mus musculus]Outcome
77Mean Ratio [DPP4] (1μM**)Calculated inhibitor-treated/DMSO ratio for DPP4 as mean for all unique peptides quantified26Floatratio
78Standard Deviation [DPP4]Calculated standard deviation for mean26Float
79Outcome [ABHD12]One of Active, Inactive, or Not Tested27monoacylglycerol lipase ABHD12 [Mus musculus]Outcome
80Mean Ratio [ABHD12] (1μM**)Calculated inhibitor-treated/DMSO ratio for ABHD12 as mean for all unique peptides quantified27Floatratio
81Standard Deviation [ABHD12]Calculated standard deviation for mean27Float
82Outcome [PNPLA7]One of Active, Inactive, or Not Tested28patatin-like phospholipase domain-containing protein 7 [Mus musculus]Outcome
83Mean Ratio [PNPLA7] (1μM**)Calculated inhibitor-treated/DMSO ratio for PNPLA7 as mean for all unique peptides quantified28Floatratio
84Standard Deviation [PNPLA7]Calculated standard deviation for mean28Float
85Outcome [PAFAH2]One of Active, Inactive, or Not Tested29platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus]Outcome
86Mean Ratio [PAFAH2] (1μM**)Calculated inhibitor-treated/DMSO ratio for PAFAH2 as mean for all unique peptides quantified29Floatratio
87Standard Deviation [PAFAH2]Calculated standard deviation for mean29Float

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R01 CA132630

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