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BioAssay: AID 493108

Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: fluorescence-based cell-based inhibition

Name: Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: fluorescence-based cell-based inhibition. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(2)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(2)
 
 
Inactive(1)
 
 
AID: 493108
Data Source: The Scripps Research Institute Molecular Screening Center (LYPLA1_INH_FLUO_ABPP_INSITU_1XINH)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-01-28
Hold-until Date: 2011-09-01
Modify Date: 2011-09-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
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: Counterscreen (LYPLA1 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
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Confirmation counterscreen (LYPLA1 inhibitors in triplicate)
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
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
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
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
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
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
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
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
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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening same project related to Summary assay
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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_FLUO_ABPP_INSITU_1XINH

Name: Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: fluorescence-based cell-based inhibition.

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.

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.

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, serine hydrolase, palmitoylation, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, inhibitor, in situ, cell-based assay, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Assays (see Protocol for details)
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1LYPLA121Lypla1 [Mus musculus] [gi:71059731]
Taxonomy id: 10090
Gene id: 18777
2LYPLA221lysophospholipase 2 [Mus musculus] [gi:123122209]
Taxonomy id: 10090
Gene id: 26394
3ABHD113Abhydrolase domain containing 11 [Mus musculus] [gi:47682716]
Taxonomy id: 10090
Gene id: 68758

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit LYPLA1, LYPLA2 and anti-target ABHD11 activity in situ. In this assay, cultured BW5147-derived murine T-cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as LYPLA1, LYPLA2 and/or ABHD11 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Protocol Summary:
BW5147-derived murine T-cells (5 mL total volume; supplemented with FCS) were treated with 30 nM test compound (5 uL of a 1000x stock in DMSO) for 2 hours at 37 C. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 C, quenched with 2x SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the LYPLA1 band, the LYPLA2 band, and the ABHD11 band relative to a DMSO-only (no compound) control.
% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as LYPLA1, LYPLA2, or ABHD11 in cells treated with test compound.
High_Control is defined as LYPLA1, LYPLA2, or ABHD11 in cells treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with greater than or equal to 90% inhibition were considered active. Compounds with inhibition less than 90% inhibition were considered inactive.
The score has been normalized to 100% of the observed percent inhibition.
LYPLA1 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
LYPLA2 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
ABHD11 Score: The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
Overall Outcome and Score:
Compounds that were active in both LYPLA1 and LYPLA2 assays were considered active. Compounds that were inactive in either LYPLA1 and/or LYPLA2 assay were considered inactive.
The overall score is 0 if the compound was inactive and 100 if the compound was active.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
BW5147-derived murine T-cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
FP-Rh (provided by the Assay Provider)
Comment
This assay was performed by the assay provider with powder samples of compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: BW5147
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [LYPLA1]One of Active, Inactive, or Not Tested1Lypla1 [Mus musculus]Outcome
2Score [LYPLA1]The BioAssay activity ranking score1Integer
3Inhibition at 0.03 uM [LYPLA1] (0.03μM**)In situ inhibition of LYPLA1 upon 30 nM compound treatment as assessed by competitive ABPP.1Float%
4Outcome [LYPLA2]One of Active, Inactive, or Not Tested.2lysophospholipase 2 [Mus musculus]Outcome
5Score [LYPLA2]The BioAssay activity ranking score.2Integer
6Inhibition at 0.03 uM [LYPLA2] (0.03μM**)In situ inhibition of LYPLA2 upon 30 nM compound treatment as assessed by competitive ABPP.2Float%
7Outcome [ABHD11]One of Active, Inactive, or Not Tested.3Abhydrolase domain containing 11 [Mus musculus]Outcome
8Score [ABHD11]The BioAssay activity ranking score.3Integer
9Inhibition at 0.03 uM [ABHD11] (0.03μM**)In situ inhibition of ABHD11 upon 30 nM compound treatment as assessed by competitive ABPP.3Float%

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

Classification
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