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BioAssay: AID 652029

Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition

Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition. ..more
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 Tested Compounds
 Tested Compounds
All(154)
 
 
Active(39)
 
 
Inactive(115)
 
 
 Tested Substances
 Tested Substances
All(154)
 
 
Active(39)
 
 
Inactive(115)
 
 
AID: 652029
Data Source: The Scripps Research Institute Molecular Screening Center (LYPLA2_INH_FLUO_ABPP_1XINH_HTS)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-02-12
Hold-until Date: 2013-02-15
Modify Date: 2013-02-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 39
Depositor Specified Assays
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AIDNameTypeProbeComment
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).summary2 Summary (LYPLA1 inhibitors)
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).summary1 Summary (LYPLA2 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).screening Primary screen (LYPLA1 inhibitors in singlicate)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening Confirmation screen (LYPLA1 inhibitors in triplicate)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Primary screen (LYPLA2 inhibitors in singlicate)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Confirmation screen (LYPLA2 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 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 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 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 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 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 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 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 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 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 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 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 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 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 Late stage (LC-MS-based cell-based SILAC ABPP for anti-target ABHD11)
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 Late stage assay (human isoform of ABHD11 inhibitors)
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 Late stage ABPP in vitro SILAC assay (LYPLA 1)
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 Late stage ABPP in situ SILAC assay (LYPLA 1)
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 Late stage assay (SILAC selectivity analysis in situ)
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 Late stage assay (SILAC selectivity analysis in vitro)
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 Late stage assay (LYPLA1 and LYPLA2 inhibitors activity in vivo)
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 Late stage assay (LYPLA1 and LYPLA2 inhibitors activity in situ)
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 Late stage assay (binding mode)
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 Late stage assay (inhibition and selectivity)
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 Late stage dose-response (LYPLA1 and LYPLA2 inhibitors in triplicate)
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 Late stage dose-response (cytotoxicity 6 replicates)
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 Late stage ABPP biochemical kinetics assay (LYPLA 1)
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 Late stage assay (LYPLA2 kinetic parameters in quadruplicate)
652003Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: fluorescence-based biochemical dose-response assayconfirmatory Late stage ABPP biochemical dose response assay (LYPLA 1) (4XIC50)
652004Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: fluorescence-based biochemical dose-response assayconfirmatory Late stage dose response (LYPLA2 inhibitors in quadruplicate)
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 Late stage assay (LYPLA1 and LYPLA2 inhibition and selectivity in singlicate)
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_FLUO_ABPP_1XINH_HTS

Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP 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, 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, cherry picks, LYPLA2, lysophospholipase 2, APT1, acyl-protein thioesterase 1, APT2, acyl-protein thioesterase 2, serine hydrolase, palmitoylation, proteome, soluble proteome, inhibitor, inhibition, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:

The purpose of this assay is to determine whether liquid samples of cherry picked test compounds can inhibit LYPLA2 in a complex proteomic lysate using a competitive activity-based proteomic profiling (ABPP) assay. In this assay, a mouse brain soluble proteome, containing spiked-in recombinant human LYPLA2 is incubated with test compounds followed by reaction with a serine-hydrolase-specific 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 LYPLA2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.

Protocol Summary:

To soluble proteome prepared from mouse brain (1 mg/ml in DPBS) is added 20 nM purified recombinant human (rh) LYPLA2. Proteome was treated with 20 uM test compound (1 uL of a 50x stock in DMSO) for 30 minutes at 25 C (50 uL reaction volume). FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM. 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 target rhLYPLA2 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 target or anti-target treated with test compound.
High_Control is defined as target or anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

PubChem Activity Outcome and Score:

Compounds with greater than or equal to 50% inhibition were considered active. Compounds with less than 50% inhibition were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 50-0.

List of Reagents:

Soluble proteome prepared from mouse brain (provided by the Assay Provider)
rhLYPLA2 (provided by Assay Provider)
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
Comment
This assay was performed by the assay provider with liquid samples of test compounds.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 20 uM (20μM**)Inhibition of 20 nM recombinant human LYPLA2 in soluble mousse brain proteome upon 20 uM compound treatment as assessed by gel-based competitive ABPP.Integer%

** Test Concentration.
Additional Information
Grant Number: 1 R01 CA132630

Data Table (Concise)
Classification
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