Bookmark and Share
BioAssay: AID 651980

Late 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 situ

Name: Late 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 situ. ..more
_
   
 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 651980
Data Source: The Scripps Research Institute Molecular Screening Center (LYPLA2_INH_LCMS_ABPP_SILAC_INSITU)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-01-15
Hold-until Date: 2013-10-29
Modify Date: 2013-10-29

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)
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
743137Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysisOther depositor-specified cross reference
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 same project related to Summary assay
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 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
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
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 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
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: LYPLA2_INH_LCMS_ABPP_SILAC_INSITU

Name: Late 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 situ.

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, HEK293T cells, in situ, 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
1LYPLA21acyl-protein thioesterase 2 [Homo sapiens] [gi:9966764]
Taxonomy id: 9606
Gene id: 11313
2MGLL1lysophospholipase-like protein 1 [Homo sapiens] [gi:20270341]
Taxonomy id: 9606
Gene id: 127018
3PREP1prolyl endopeptidase [Homo sapiens] [gi:41349456]
Taxonomy id: 9606
Gene id: 5550
4TPP21tripeptidyl peptidase II [Homo sapiens] [gi:119629464]
Taxonomy id: 9606
Gene id: 7174
5LYPLA11acyl-protein thioesterase 1 [Homo sapiens] [gi:5453722]
Taxonomy id: 9606
Gene id: 10434
6PPME11protein phosphatase methylesterase 1 isoform a [Homo sapiens] [gi:7706645]
Taxonomy id: 9606
Gene id: 51400
7AADACL11neutral cholesterol ester hydrolase 1 isoform b [Homo sapiens] [gi:68051721]
Taxonomy id: 9606
Gene id: 57552
8FASN1fatty acid synthase [Homo sapiens] [gi:41872631]
Taxonomy id: 9606
Gene id: 2194
9ESD1S-formylglutathione hydrolase [Homo sapiens] [gi:33413400]
Taxonomy id: 9606
Gene id: 2098
10DPP91RecName: Full=Dipeptidyl peptidase 9; Short=DP9; AltName: Full=Dipeptidyl peptidase IV-related protein 2; Short=DPRP-2; AltName: Full=Dipeptidyl peptidase IX; Short=DPP IX; AltName: Full=Dipeptidyl peptidase-like protein 9; Short=DPLP9 [gi:67460390]
Taxonomy id: 9606
Gene id: 91039
11ABHD101alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Homo sapiens] [gi:8923001]
Taxonomy id: 9606
Gene id: 55347
12LYPLAL11lysophospholipase-like protein 1 [Homo sapiens] [gi:20270341]
Taxonomy id: 9606
Gene id: 127018
13APEH1acylamino-acid-releasing enzyme [Homo sapiens] [gi:23510451]
Taxonomy id: 9606
Gene id: 327
14ABHD121monoacylglycerol lipase ABHD12 isoform a [Homo sapiens] [gi:109689718]
Taxonomy id: 9606
Gene id: 26090
15PNPLA61neuropathy target esterase isoform a [Homo sapiens] [gi:260656037]
Taxonomy id: 9606
Gene id: 10908
16CTSA1lysosomal protective protein isoform a precursor [Homo sapiens] [gi:119395729]
Taxonomy id: 9606
Gene id: 5476
17ABHD61monoacylglycerol lipase ABHD6 [Homo sapiens] [gi:189027141]
Taxonomy id: 9606
Gene id: 57406
18PLA2G4A1cytosolic phospholipase A2 [Homo sapiens] [gi:23943920]
Taxonomy id: 9606
Gene id: 5321

§ 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 HEK293T cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with inhibitor and DMSO, respectively, in situ, followed by the cell-permeable alkyne-functionalized triazole urea ABPP probe NHU5. NHU5 covalently reacts with a subset of serine hydrolases, including LYPLA1 and LYPLA2, and shows slower reaction kinetics than other cell-permeable serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Cells are harvested, proteome fractions isolated and reacted with a biotin-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition ("click chemistry") reaction conditions for enrichment of probe-tagged proteins. Light and heavy samples are 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 NHU5 probe 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:
HEK293T cells were initially grown for 6 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 1x PenStrep Glutamine in a humidified incubator (37 C, 5% CO2). Light medium was supplemented with 100 ug/mL L-arginine (Sigma) and 100 ug/mL L-lysine (Sigma). Heavy medium 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). Light cells were treated with test compound (5 uM) and heavy cells were treated with DMSO for 3 hours at 37 C. Activity-based alkyne probe NHU5 (50 uM) was added, and cells incubated for 1 hour at 37 C. After 1 hour, cells were washed (4x DPBS), harvested, isolated by centrifugation at 1,400 x g for 3 minutes, re-suspended in methanol/chloroform/dH2O (4:1:3 v/v), and vortexed. After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and the solution vortexed. Protein was pelleted by centrifugation at 13,000 x g for 6 minutes. The supernatant was removed and the pellet was air-dried and re-suspended in PBS by sonication. Light and heavy proteomes were mixed in 1:1 (w/w) ratio to generate one membrane and one soluble sample. For affinity purification of probe-labeled proteins, click chemistry with a biotin-azide tag (biotin-N3; 100 uM) was carried out under standard conditions (1 mM TCEP, 100 uM TBTA ligand, 1 mM Cu(II)sulfate, 1 hour, 25 C), and excess reagents removed by washing the precipitated protein with cold methanol (3 x 750 uL). The protein pellets were resolubilized in 2.5% SDS/DPBS (750 uL) by sonication and heating (60 C, 5 minutes). The concentration of SDS was reduced to 0.5% with DPBS (3 mL), and biotinylated proteins enriched with streptavidin beads (50 uL, 1 hour, 25 C). The beads were washed with 1% SDS in DPBS (1x), 6 M urea (1x), and DPBS (2x), then resuspended in 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 LYPLA2 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:
HEK293T cells (provided by Assay Provider)
SILAC DMEM Medium (Thermo 89985)
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 Format: Cell-based
Assay Cell Type: HEK 293T
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [LYPLA2]One of Active, Inactive, or Not Tested1acyl-protein thioesterase 2 [Homo sapiens]Outcome
2Mean Ratio [LYPLA2] (5μM**)Calculated inhibitor-treated/DMSO ratio for LYPLA2 as mean for all unique peptides quantified1Floatratio
3Standard Deviation [LYPLA2]Calculated standard deviation for mean1Float
4Outcome [MGLL]One of Active, Inactive, or Not Tested2lysophospholipase-like protein 1 [Homo sapiens]Outcome
5Mean Ratio [MGLL] (5μM**)Calculated inhibitor-treated/DMSO ratio for MGLL as mean for all unique peptides quantified2Floatratio
6Standard Deviation [MGLL]Calculated standard deviation for mean2Float
7Outcome [PREP]One of Active, Inactive, or Not Tested3prolyl endopeptidase [Homo sapiens]Outcome
8Mean Ratio [PREP] (5μM**)Calculated inhibitor-treated/DMSO ratio for PREP as mean for all unique peptides quantified3Floatratio
9Standard Deviation [PREP]Calculated standard deviation for mean3Float
10Outcome [TPP2]One of Active, Inactive, or Not Tested4tripeptidyl peptidase II [Homo sapiens]Outcome
11Mean Ratio [TPP2] (5μM**)Calculated inhibitor-treated/DMSO ratio for TPP2 as mean for all unique peptides quantified4Floatratio
12Standard Deviation [TPP2]Calculated standard deviation for mean4Float
13Outcome [LYPLA1]One of Active, Inactive, or Not Tested5acyl-protein thioesterase 1 [Homo sapiens]Outcome
14Mean Ratio [LYPLA1] (5μM**)Calculated inhibitor-treated/DMSO ratio for LYPLA1 as mean for all unique peptides quantified5Floatratio
15Standard Deviation [LYPLA1]Calculated standard deviation for mean5Float
16Outcome [PPME1]One of Active, Inactive, or Not Tested6protein phosphatase methylesterase 1 isoform a [Homo sapiens]Outcome
17Mean Ratio [PPME1] (5μM**)Calculated inhibitor-treated/DMSO ratio for PPME1 as mean for all unique peptides quantified6Floatratio
18Standard Deviation [PPME1]Calculated standard deviation for mean6Float
19Outcome [AADACL1]One of Active, Inactive, or Not Tested7neutral cholesterol ester hydrolase 1 isoform b [Homo sapiens]Outcome
20Mean Ratio [AADACL1] (5μM**)Calculated inhibitor-treated/DMSO ratio for AADACL1 as mean for all unique peptides quantified7Floatratio
21Standard Deviation [AADACL1]Calculated standard deviation for mean7Float
22Outcome [FASN]One of Active, Inactive, or Not Tested8fatty acid synthase [Homo sapiens]Outcome
23Mean Ratio [FASN] (5μM**)Calculated inhibitor-treated/DMSO ratio for FASN as mean for all unique peptides quantified8Floatratio
24Standard Deviation [FASN]Calculated standard deviation for mean8Float
25Outcome [ESD]One of Active, Inactive, or Not Tested9S-formylglutathione hydrolase [Homo sapiens]Outcome
26Mean Ratio [ESD] (5μM**)Calculated inhibitor-treated/DMSO ratio for ESD as mean for all unique peptides quantified9Floatratio
27Standard Deviation [ESD]Calculated standard deviation for mean9Float
28Outcome [DPP9]One of Active, Inactive, or Not Tested10RecName: Full=Dipeptidyl peptidase 9; Short=DP9; AltName: Full=Dipeptidyl peptidase IV-related protein 2; Short=DPRP-2; AltName: Full=Dipeptidyl peptidase IX; Short=DPP IX; AltName: Full=Dipeptidyl peptidase-like protein 9; Short=DPLP9Outcome
29Mean Ratio [DPP9] (5μM**)Calculated inhibitor-treated/DMSO ratio for DPP9 as mean for all unique peptides quantified10Floatratio
30Standard Deviation [DPP9]Calculated standard deviation for mean10Float
31Outcome [ABHD10]One of Active, Inactive, or Not Tested11alpha/beta hydrolase domain-containing protein 10, mitochondrial isoform 1 precursor [Homo sapiens]Outcome
32Mean Ratio [ABHD10] (5μM**)Calculated inhibitor-treated/DMSO ratio for ABHD10 as mean for all unique peptides quantified11Floatratio
33Standard Deviation [ABHD10]Calculated standard deviation for mean11Float
34Outcome [LYPLAL1]One of Active, Inactive, or Not Tested12lysophospholipase-like protein 1 [Homo sapiens]Outcome
35Mean Ratio [LYPLAL1] (5μM**)Calculated inhibitor-treated/DMSO ratio for LYPLAL1 as mean for all unique peptides quantified12Floatratio
36Standard Deviation [LYPLAL1]Calculated standard deviation for mean12Float
37Outcome [APEH]One of Active, Inactive, or Not Tested13acylamino-acid-releasing enzyme [Homo sapiens]Outcome
38Mean Ratio [APEH] (5μM**)Calculated inhibitor-treated/DMSO ratio for APEH as mean for all unique peptides quantified13Floatratio
39Standard Deviation [APEH]Calculated standard deviation for mean13Float
40Outcome [ABHD12]One of Active, Inactive, or Not Tested14monoacylglycerol lipase ABHD12 isoform a [Homo sapiens]Outcome
41Mean Ratio [ABHD12] (5μM**)Calculated inhibitor-treated/DMSO ratio for ABHD12 as mean for all unique peptides quantified14Floatratio
42Standard Deviation [ABHD12]Calculated standard deviation for mean14Float
43Outcome [PNPLA6]One of Active, Inactive, or Not Tested15neuropathy target esterase isoform a [Homo sapiens]Outcome
44Mean Ratio [PNPLA6] (5μM**)Calculated inhibitor-treated/DMSO ratio for PNPLA6 as mean for all unique peptides quantified15Floatratio
45Standard Deviation [PNPLA6]Calculated standard deviation for mean15Float
46Outcome [CTSA]One of Active, Inactive, or Not Tested16lysosomal protective protein isoform a precursor [Homo sapiens]Outcome
47Mean Ratio [CTSA] (5μM**)Calculated inhibitor-treated/DMSO ratio for CTSA as mean for all unique peptides quantified16Floatratio
48Standard Deviation [CTSA]Calculated standard deviation for mean16Float
49Outcome [ABHD6]One of Active, Inactive, or Not Tested17monoacylglycerol lipase ABHD6 [Homo sapiens]Outcome
50Mean Ratio [ABHD6] (5μM**)Calculated inhibitor-treated/DMSO ratio for ABHD6 as mean for all unique peptides quantified17Floatratio
51Standard Deviation [ABHD6]Calculated standard deviation for mean17Float
52Outcome [PLA2G4A]One of Active, Inactive, or Not Tested18cytosolic phospholipase A2 [Homo sapiens]Outcome
53Mean Ratio [PLA2G4A] (5μM**)Calculated inhibitor-treated/DMSO ratio for PLA2G4A as mean for all unique peptides quantified18Floatratio
54Standard Deviation [PLA2G4A]Calculated standard deviation for mean18Float

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

Classification
PageFrom: