Bookmark and Share
BioAssay: AID 504522

Late 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 ABHD11

Name: Late 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 ABHD11. ..more
_
   
 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 504522
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD11_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
Show more
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)
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
651978Late 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 vitroOther depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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 CA132630-01
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: ABHD11_INH_LCMS_SILAC

Name: Late 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 ABHD11.

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, a dual inhibitor selective for both LYPLA1 and LYPLA2 would greatly aid investigations into the biological function of these related 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 a high affinity inhibitor, capable of achieving temporal and more complete control over activity, is critical. Alpha/beta hydrolase domain-containing protein 11 (ABHD11) is a poorly characterized serine hydrolase; all that is known about its biology is that it is a mitochondrial enzyme (12) with broad tissue distribution, has little sequence homology to other proteins, and its gene is located in a region of chromosome 7 that is hemizygously deleted in Williams-Beuren syndrome, a rare genetic disease with symptoms that include vascular stenosis, mental retardation, and excessive sociability (13).

References:

1. Smotrys, J.E. and Linder, M.E. PALMITOYLATION OF INTRACELLULAR SIGNALING PROTEINS: Regulation and Function. Annual Review of Biochemistry, 2004. 73: 559-587.
2. Martin, B.R. and Cravatt, B.F. Large-scale profiling of protein palmitoylation in mammalian cells. Nat Methods, 2009. 6: 135-138.
3. Magee, A.I., Koyama, A.H., Malfer, C., Wen, D. and Schlesinger, M. J. Release of fatty acids from virus glycoproteins by hydroxylamine. Biochimica et Biophysica Acta (BBA) - General Subjects, 1984. 798: 156-166.
4. Rose, J.K., Adams, G.A. and Gallione, C.J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci USA, 1984. 81: 2050-2054.
5. Willumsen, B.M., Cox, A.D., Solski, P.A., Der, C.J. and Buss, J.E. Novel determinants of H-Ras plasma membrane localization and transformation. Oncogene, 1996. 13: 1901-1909.
6. Duncan, J.A. and Gilman, A.G. A Cytoplasmic Acyl-Protein Thioesterase That Removes Palmitate from G Protein alpha Subunits and p21RAS. J Biol Chem, 1998. 273: 15830-15837.
7. Sugimoto, H., Hayashi, H. & Yamashita, S. Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J Biol Chem, 1996. 271: 7705-7711.
8. Hirano, T. et al. Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2009. 1791: 797-805.
9. Toyoda, T., Sugimoto, H. and Yamashita, S. Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1999. 1437: 182-193.
10. Biel, M., Deck, P., Giannis, A. and Waldmann, H. Synthesis and Evaluation of Acyl Protein Thioesterase 1 (APT1) Inhibitors. Chemistry - A European Journal, 2006. 12: 4121-4143.
11. Deck, P. et al. Development and Biological Evaluation of Acyl Protein Thioesterase 1 (APT1) Inhibitors. Angewandte Chemie International Edition, 2005. 44: 4975-4980.
12. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19.
13. 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, LYPLA2, lysophospholipase 2, APT1, acyl-protein thioesterase 1, APT2, acyl-protein thioesterase 2, palmitoylation, alpha/beta hydrolase domain-containing protein 11, abhydrolase domain-containing protein 11, ABHD11, oncogene, tumor suppressor, serine hydrolase, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, 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
1ABHD111Abhydrolase domain containing 11 [Mus musculus] [gi:47682716]
Taxonomy id: 10090
Gene id: 68758
2ABHD61Abhydrolase domain containing 6 [Mus musculus] [gi:20073260]
Taxonomy id: 10090
Gene id: 66082
3ACOT11Acyl-CoA thioesterase 1 [Mus musculus] [gi:20380528]
Taxonomy id: 10090
Gene id: 26897
4DPP41dipeptidylpeptidase 4 [Mus musculus] [gi:123233284]
Taxonomy id: 10090
Gene id: 13482
5ACOT21Acyl-CoA thioesterase 2 [Mus musculus] [gi:39992610]
Taxonomy id: 10090
Gene id: 171210
6PREPL1Prepl protein [Mus musculus] [gi:13435484]
Taxonomy id: 10090
Gene id: 213760
7FAM108B11Fam108b protein [Mus musculus] [gi:61403362]
Taxonomy id: 10090
Gene id: 226016
8PAFAH21platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus] [gi:225579137]
Taxonomy id: 10090
Gene id: 100163
9CTSA1cathepsin A [Mus musculus] [gi:123240058]
Taxonomy id: 10090
Gene id: 19025
10ABHD121abhydrolase domain containing 12 [Mus musculus] [gi:123241061]
Taxonomy id: 10090
Gene id: 76192
11PNPLA81Patatin-like phospholipase domain containing 8 [Mus musculus] [gi:117558667]
Taxonomy id: 10090
Gene id: 67452
12AADACL11Arylacetamide deacetylase-like 1 [Mus musculus] [gi:52139053]
Taxonomy id: 10090
Gene id: 320024
13DPP71dipeptidylpeptidase 7 [Mus musculus] [gi:123227495]
Taxonomy id: 10090
Gene id: 83768
14PAFAH1B31platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus] [gi:6679201]
Taxonomy id: 10090
Gene id: 18476
15ABHD101Abhydrolase domain containing 10 [Mus musculus] [gi:37231537]
Taxonomy id: 10090
Gene id: 213012
16ESD1Esterase D/formylglutathione hydrolase [Mus musculus] [gi:55777188]
Taxonomy id: 10090
Gene id: 13885
17IAH11isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus] [gi:27754071]
Taxonomy id: 10090
Gene id: 67732
18APEH1Apeh protein [Mus musculus] [gi:20072022]
Taxonomy id: 10090
Gene id: 235606
19LIPA1lysosomal acid lipase 1 [Mus musculus] [gi:148709804]
Taxonomy id: 10090
Gene id: 16889
20PNPLA61Pnpla6 protein [Mus musculus] [gi:34784201]
Taxonomy id: 10090
Gene id: 50767
21PLA2G61Pla2g6 protein [Mus musculus] [gi:34784362]
Taxonomy id: 10090
Gene id: 53357
22PREP1prolyl endopeptidase [Mus musculus] [gi:148673089]
Taxonomy id: 10090
Gene id: 19072
23PRCP1Prcp protein [Mus musculus] [gi:32967631]
Taxonomy id: 10090
Gene id: 72461
24PPME11protein phosphatase methylesterase 1 [Mus musculus] [gi:30794138]
Taxonomy id: 10090
Gene id: 72590
25ACOT71acyl-CoA thioesterase 7 [Mus musculus] [gi:123857891]
Taxonomy id: 10090
Gene id: 70025
26LACTB1Lactamase, beta [Mus musculus] [gi:187955464]
Taxonomy id: 10090
Gene id: 80907
27LYPLA31lysophospholipase 3, isoform CRA_a [Mus musculus] [gi:148679401]
Taxonomy id: 10090
Gene id: 192654
28SERHL1Serine hydrolase-like [Mus musculus] [gi:127797596]
Taxonomy id: 10090
Gene id: 68607
29PARL1presenilin associated, rhomboid-like [Mus musculus] [gi:148665146]
Taxonomy id: 10090
Gene id: 381038
30DPP81Dipeptidylpeptidase 8 [Mus musculus] [gi:37590654]
Taxonomy id: 10090
Gene id: 74388
31BAT51abhydrolase domain-containing protein 16A [Mus musculus] [gi:30519896]
Taxonomy id: 10090
Gene id: 193742
32LYPLA11Lypla1 [Mus musculus] [gi:71059731]
Taxonomy id: 10090
Gene id: 18777
33SIAE1sialic acid acetylesterase [Mus musculus] [gi:148693491]
Taxonomy id: 10090
Gene id: 22619
34FAAH1fatty acid amide hydrolase [Mus musculus] [gi:123253900]
Taxonomy id: 10090
Gene id: 14073
35DPP91dipeptidyl peptidase 9 [Mus musculus] [gi:255003757]
Taxonomy id: 10090
Gene id: 224897
36FASN1fatty acid synthase [Mus musculus] [gi:123288587]
Taxonomy id: 10090
Gene id: 14104
37LYPLA21lysophospholipase 2 [Mus musculus] [gi:123122209]
Taxonomy id: 10090
Gene id: 26394
38PAFAH1B21platelet-activating factor acetylhydrolase isoform Ib beta subunit [Mus musculus] [gi:1373363]
Taxonomy id: 10090
Gene id: 18475
39DDHD11Ddhd1 protein [Mus musculus] [gi:27694042]
Taxonomy id: 10090
Gene id: 114874

§ 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 uM of FP-biotin (500 uL 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 uL of 0.5 ug/uL) 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( 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 ABHD11 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.
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [ABHD11]One of Active or Inactive1Abhydrolase domain containing 11 [Mus musculus]Outcome
2Average Silac Ratio [ABHD11]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified1Floatratio
3Standard Error [ABHD11]Calculated standard error for mean1Float
4Outcome [ABHD6]One of Active or Inactive2Abhydrolase 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 quantified2Floatratio
6Standard Error [ABHD6]Calculated standard error for mean2Float
7Outcome [ACOT1]One of Active or Inactive3Acyl-CoA thioesterase 1 [Mus musculus]Outcome
8Average Silac Ratio [ACOT1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified3Floatratio
9Standard Error [ACOT1]Calculated standard error for mean3Float
10Outcome [DPP4]One of Active or Inactive4dipeptidylpeptidase 4 [Mus musculus]Outcome
11Average Silac Ratio [DPP4]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified4Floatratio
12Standard Error [DPP4]Calculated standard error for mean4Float
13Outcome [ACOT2]One of Active or Inactive5Acyl-CoA thioesterase 2 [Mus musculus]Outcome
14Average Silac Ratio [ACOT2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified5Floatratio
15Standard Error [ACOT2]Calculated standard error for mean5Float
16Outcome [PREPL]One of Active or Inactive6Prepl protein [Mus musculus]Outcome
17Average Silac Ratio [PREPL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified6Floatratio
18Standard Error [PREPL]Calculated standard error for mean6Float
19Outcome [FAM108B1]One of Active or Inactive7Fam108b protein [Mus musculus]Outcome
20Average Silac Ratio [FAM108B1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified7Floatratio
21Standard Error [FAM108B1]Calculated standard error for mean7Float
22Outcome [PAFAH2]One of Active or Inactive8platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus]Outcome
23Average Silac Ratio [PAFAH2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified8Floatratio
24Standard Error [PAFAH2]Calculated standard error for mean8Float
25Outcome [CTSA]One of Active or Inactive9cathepsin A [Mus musculus]Outcome
26Average Silac Ratio [CTSA]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified9Floatratio
27Standard Error [CTSA]Calculated standard error for mean9Float
28Outcome [ABHD12]One of Active or Inactive10abhydrolase domain containing 12 [Mus musculus]Outcome
29Average Silac Ratio [ABHD12]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified10Floatratio
30Standard Error [ABHD12]Calculated standard error for mean10Float
31Outcome [PNPLA8]One of Active or Inactive11Patatin-like phospholipase domain containing 8 [Mus musculus]Outcome
32Average Silac Ratio [PNPLA8]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified11Floatratio
33Standard Error [PNPLA8]Calculated standard error for mean11Float
34Outcome [AADACL1]One of Active or Inactive12Arylacetamide deacetylase-like 1 [Mus musculus]Outcome
35Average Silac Ratio [AADACL1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified12Floatratio
36Standard Error [AADACL1]Calculated standard error for mean12Float
37Outcome [DPP7]One of Active or Inactive13dipeptidylpeptidase 7 [Mus musculus]Outcome
38Average Silac Ratio [DPP7]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified13Floatratio
39Standard Error [DPP7]Calculated standard error for mean13Float
40Outcome [PAFAH1B3]One of Active or Inactive14platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus]Outcome
41Average Silac Ratio [PAFAH1B3]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified14Floatratio
42Standard Error [PAFAH1B3]Calculated standard error for mean14Float
43Outcome [ABHD10]One of Active or Inactive15Abhydrolase domain containing 10 [Mus musculus]Outcome
44Average Silac Ratio [ABHD10]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified15Floatratio
45Standard Error [ABHD10]Calculated standard error for mean15Float
46Outcome [ESD]One of Active or Inactive16Esterase D/formylglutathione hydrolase [Mus musculus]Outcome
47Average Silac Ratio [ESD]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified16Floatratio
48Standard Error [ESD]Calculated standard error for mean16Float
49Outcome [IAH1]One of Active or Inactive17isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus]Outcome
50Average Silac Ratio [IAH1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified17Floatratio
51Standard Error [IAH1]Calculated standard error for mean17Float
52Outcome [APEH]One of Active or Inactive18Apeh protein [Mus musculus]Outcome
53Average Silac Ratio [APEH]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified18Floatratio
54Standard Error [APEH]Calculated standard error for mean18Float
55Outcome [LIPA]One of Active or Inactive19lysosomal acid lipase 1 [Mus musculus]Outcome
56Average Silac Ratio [LIPA]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified19Floatratio
57Standard Error [LIPA]Calculated standard error for mean19Float
58Outcome [PNPLA6]One of Active or Inactive20Pnpla6 protein [Mus musculus]Outcome
59Average Silac Ratio [PNPLA6]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified20Floatratio
60Standard Error [PNPLA6]Calculated standard error for mean20Float
61Outcome [PLA2G6]One of Active or Inactive21Pla2g6 protein [Mus musculus]Outcome
62Average Silac Ratio [PLA2G6]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified21Floatratio
63Standard Error [PLA2G6]Calculated standard error for mean21Float
64Outcome [PREP]One of Active or Inactive22prolyl endopeptidase [Mus musculus]Outcome
65Average Silac Ratio [PREP]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified22Floatratio
66Standard Error [PREP]Calculated standard error for mean22Float
67Outcome [PRCP]One of Active or Inactive23Prcp protein [Mus musculus]Outcome
68Average Silac Ratio [PRCP]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified23Floatratio
69Standard Error [PRCP]Calculated standard error for mean23Float
70Outcome [PPME1]One of Active or Inactive24protein phosphatase methylesterase 1 [Mus musculus]Outcome
71Average Silac Ratio [PPME1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified24Floatratio
72Standard Error [PPME1]Calculated standard error for mean24Float
73Outcome [ACOT7]One of Active or Inactive25acyl-CoA thioesterase 7 [Mus musculus]Outcome
74Average Silac Ratio [ACOT7]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified25Floatratio
75Standard Error [ACOT7]Calculated standard error for mean25Float
76Outcome [LACTB]One of Active or Inactive26Lactamase, beta [Mus musculus]Outcome
77Average Silac Ratio [LACTB]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified26Floatratio
78Standard Error [LACTB]Calculated standard error for mean26Float
79Outcome [LYPLA3]One of Active or Inactive27lysophospholipase 3, isoform CRA_a [Mus musculus]Outcome
80Average Silac Ratio [LYPLA3]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified27Floatratio
81Standard Error [LYPLA3]Calculated standard error for mean27Float
82Outcome [SERHL]One of Active or Inactive28Serine hydrolase-like [Mus musculus]Outcome
83Average Silac Ratio [SERHL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified28Floatratio
84Standard Error [SERHL]Calculated standard error for mean28Float
85Outcome [PARL]One of Active or Inactive29presenilin associated, rhomboid-like [Mus musculus]Outcome
86Average Silac Ratio [PARL]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified29Floatratio
87Standard Error [PARL]Calculated standard error for mean29Float
88Outcome [DPP8]One of Active or Inactive30Dipeptidylpeptidase 8 [Mus musculus]Outcome
89Average Silac Ratio [DPP8]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified30Floatratio
90Standard Error [DPP8]Calculated standard error for mean30Float
91Outcome [BAT5]One of Active or Inactive31abhydrolase domain-containing protein 16A [Mus musculus]Outcome
92Average Silac Ratio [BAT5]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified31Floatratio
93Standard Error [BAT5]Calculated standard error for mean31Float
94Outcome [LYPLA1]One of Active or Inactive32Lypla1 [Mus musculus]Outcome
95Average Silac Ratio [LYPLA1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified32Floatratio
96Standard Error [LYPLA1]Calculated standard error for mean32Float
97Outcome [SIAE]One of Active or Inactive33sialic acid acetylesterase [Mus musculus]Outcome
98Average Silac Ratio [SIAE]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified33Floatratio
99Standard Error [SIAE]Calculated standard error for mean33Float
100Outcome [FAAH]One of Active or Inactive34fatty acid amide hydrolase [Mus musculus]Outcome
101Average Silac Ratio [FAAH]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified34Floatratio
102Standard Error [FAAH]Calculated standard error for mean34Float
103Outcome [DPP9]One of Active or Inactive35dipeptidyl peptidase 9 [Mus musculus]Outcome
104Average Silac Ratio [DPP9]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified35Floatratio
105Standard Error [DPP9]Calculated standard error for mean35Float
106Outcome [FASN]One of Active or Inactive36fatty acid synthase [Mus musculus]Outcome
107Average Silac Ratio [FASN]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified36Floatratio
108Standard Error [FASN]Calculated standard error for mean36Float
109Outcome [LYPLA2]One of Active or Inactive37lysophospholipase 2 [Mus musculus]Outcome
110Average Silac Ratio [LYPLA2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified37Floatratio
111Standard Error [LYPLA2]Calculated standard error for mean37Float
112Outcome [PAFAH1B2]One of Active or Inactive38platelet-activating factor acetylhydrolase isoform Ib beta subunit [Mus musculus]Outcome
113Average Silac Ratio [PAFAH1B2]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified38Floatratio
114Standard Error [PAFAH1B2]Calculated standard error for mean38Float
115Outcome [DDHD1]One of Active or Inactive39Ddhd1 protein [Mus musculus]Outcome
116Average Silac Ratio [DDHD1]Calculated light/heavy (inhibitor-treated/DMSO) ratio for each protein as mean for all unique peptides quantified39Floatratio
117Standard Error [DDHD1]Calculated standard error for mean39Float
Additional Information
Grant Number: 1 R01 CA132630-01

Classification
PageFrom: