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BioAssay: AID 602468

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity among cysteine-reactive proteins

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity among cysteine-reactive proteins. ..more
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AID: 602468
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD10_INH_FLUO_GELBASEDABPP_SEL_SET2)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-03-22
Hold-until Date: 2012-05-31
Modify Date: 2012-05-31

Data Table ( Complete ):           View All Data
Tested Compound:
Related Experiments
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AIDNameTypeProbeComment
2130Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 depositor-specified cross reference: Summary (PME-1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Confirmation assay (PME-1 inhibitors)
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)
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: Counterscreen (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: Counterscreen confirmation (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Counterscreen confirmation (LYPLA1 inhibitors)
2291Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors, Maybridge Library)
2363Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2aScreening depositor-specified cross reference: MOA assay (Demethylation of PP2a)
2365Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Luminescence-based counterscreen assay to identify cytotoxic compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HEC 293T)
2366Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors)
2368Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) Gel Filtration AssayScreening depositor-specified cross reference: MOA assay (PME-1 inhibitors, gel filtration assay)
2369Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) InhibitionScreening depositor-specified cross reference: ABPP screen (PME-1 inhibitors)
2371Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50: Purified enzymeConfirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors, purified enzyme)
463090Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): LC-MS/MS assay to assess binding of compounds to active siteOther depositor-specified cross reference: MOA assay (PME-1 inhibitors, LC-MS assay)
463091Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HeLa)
463124Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 2Confirmatory depositor-specified cross reference: Late stage dose response (PME-1 inhibitors)
463130Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 1Confirmatory depositor-specified cross reference: Late stage gel-based ABPP dose response (PME-1 inhibitors)
463131Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based inhibitionScreening depositor-specified cross reference: Late stage fluorescence-base cell-based inhibition (PME-1 inhibitors)
463132Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Inhibition of PME-1-mediated demethylation of PP2AScreening depositor-specified cross reference: Late stage results (Inhibition of PME-1 mediated demethylation of PP2A)
463146Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Fluorescence-based biochemical gel-based ABPPOther depositor-specified cross reference: Late stage fluorescence-based gel-based ABPP (PME-1 inhibitors)
463149Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther depositor-specified cross reference: Late stage fluorescence-based biochemical gel-based ABPP inhibition and selectivity (PME-1 inhibitor
588796Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 2Confirmatory depositor-specified cross reference: Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
588801Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10Confirmatory depositor-specified cross reference: Late stage dose response(ABPP inhibition of ABHD10 in triplicate)
588802Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 1Confirmatory depositor-specified cross reference: Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
588803Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assayOther1 depositor-specified cross reference: Late stage results (LC-MS/MS-based cell-based ABPP-SILAC)
588804Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Late stage dose response assay (cytotoxicity in six replicates)
588805Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assayOther depositor-specified cross reference: Late stage results (ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assay)
588806Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10Other depositor-specified cross reference: Late stage results (ABPP inhibition of ABHD10)
588807Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity in a complex proteome for ABHD10Other depositor-specified cross reference: Late stage results (ABPP inhibition and selectivity in a complex proteome for ABHD10)
588835Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) ABHD10 selectivity assayOther depositor-specified cross reference: Late stage dose response (ABPP ABHD10 selectivity assay)
602485Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 in vivoOther depositor-specified cross reference
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).Summary2 same project related to Summary assay
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).Summary1 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: ABHD10_INH_FLUO_GELBASEDABPP_SEL_SET2

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity among cysteine-reactive proteins.

Description:

Protein phosphatase methylesterase-1 (PME-1)-mediated methylesterification is thought to control the binding of different subunits to protein phosphatase 2A (PP2A) (1), which, along with protein phosphatase 1 (PP1), is responsible for > 90% of all serine/threonine phosphatase activity (2). PME-1 has also been identified as a protector of sustained ERK pathway activity in malignant gliomas (3), suggesting a link between cancer progression and PME-1-regulated methylesterification. A fluorescence-polarization activity-based protein profiling (fluopol-ABPP) HTS assay for PME-1 inhibitor discovery (AIDs 2130 and 2171) unveiled a phenomenal class of potent and selective inhibitors, the aza-beta lactams (ABLs). During medicinal chemistry campaign to refine ABL inhibitors for PME-1 (See Probe Report for ML174), we observed that one of the common anti-targets of several ABL members was the uncharacterized serine hydrolase abhydrolase domain containing protein 10 (ABHD10). We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored. A principle goal of post-genomic research is to elucidate the molecular and cellular roles of uncharacterized enzymes like ABHD10, work that requires selective chemical tools to inactivate enzyme activity in a controlled manner.


References:

1. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J. B., Broach, J. R. (2000). Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 19, 5672-5681.
2. Oliver, C. J., Shenolikar, S. (1998). Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 3, D961-972.
3. Puustinen, P., Junttila, M. R., Vanhatupa, S., Sablina, A. A., Hector, M. E., Teittinen, K., Raheem, O., Ketola, K., Lin, S., Kast, J., Haapasalo, H., Hahn, W. C., Westermarck, J. (2009). PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 69, 2870-2877.

Keywords:

late stage, late stage AID, assay provider, powders, abhdyrolase domain containing protein 10, ABHD10, uncharacterized, PME-1, protein phosphatase methylesterase 1, PPME-1, counterscreen, anti-targets, activity-based protein profiling, ABPP, gel-based, chloroacetamide-rhodamine, CA-Rh, in vitro, in situ, inhibitor, selectivity, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Assays
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1In vitro MBM1
2In situ Neuro-2A1

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to assess selectivity among cysteine-containing proteins of powder samples of test compounds both in vitro (complex proteomic lysates) and in situ (cultured cells) using an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a rhodamine-conjugated chloroacetamide (CA-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 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:
In vitro MBM Assay: Mouse brain membrane (MBM) and soluble proteome (1 mg/mL in DPBS; 50 uL reaction volume) was treated with 20, 10, 1, or 0.1 uM test compound (1 uL of a 50x stock in DMSO). Test compounds were incubated for 30 minutes at 37 C, and CA-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 5 uM. The reaction was incubated for 30 minutes at 25 C, quenched with 4x 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 anti-target bands relative to a DMSO-only (no compound) control. Only proteins for which at least 50% inhibition is observed at any test compound concentration are counted as anti-targets.
In situ Neuro-2A Assay: Cultured Neuro-2A murine neuroblastoma cells in serum-free DMEM medium (1 mL total volume) were treated with DMSO or test compound (5 uL of a 200x stock in DMSO, 100 nM or 250 nM final concentration) for 2 hours at 37 C. Cells were washed with DPBS, harvested, and resuspended in DPBS (1 mL). Centrifugation (1000 x g, 5 minutes) provided a cell pellet that was resuspended in DPBS (200 uL) and homogenized by sonication. The membrane and soluble fractions were separated by centrifugation (100,000 x g, 45 minutes), and the protein concentrations were adjusted to 1 mg/mL with DPBS. CA-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 5 uM. The reaction was incubated for 30 minutes at 25 C, quenched with 4x 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 anti-target bands relative to a DMSO-only (no compound) control. Only proteins for which at least 50% inhibition is observed at any test compound concentration are counted as anti-targets.
The % inhibition was then calculated as follows:
%_Inhibition = ( 1 - ( IOD_Test_Compound - Median_IOD_Low_Control ) / ( Median_IOD_High_Control - Median_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:
The following applies to each panel:
Compounds with anti-targets at any test compound concentration were considered active. Compounds with no observed anti-targets at all test compound concentrations were considered inactive.
Overall Outcome and Score:
Compounds active in either one or both assays were considered active. Compounds inactive in both assays were considered 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 inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Mouse brain membrane proteome (provided by Assay Provider)
Neuro-2A cells (provided by Assay Provider)
DMEM Medium (CellGro 10-017-CV)
CA-Rh (provided by Assay Provider)
DPBS (Cellgro 20-031-CV)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: selectivity
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Outcome [MBM]One of Active, Inactive, or Not Tested1Outcome
2Anti-targets at 10 uM [MBM] (10μM**)Number of observed anti-targets in mouse brain proteome upon 10 uM compound treatment as assessed by gel-based competitive ABPP with the thiol-reactive ABPP probe CA-Rh.1Integer
3Anti-targets at 1 uM [MBM] (1μM**)Number of observed anti-targets in mouse brain proteome upon 1 uM compound treatment as assessed by gel-based competitive ABPP with the thiol-reactive ABPP probe CA-Rh.1Integer
4Anti-targets at 0.1 uM [MBM] (0.1μM**)Number of observed anti-targets in mouse brain proteome upon 0.1 uM compound treatment as assessed by gel-based competitive ABPP with the thiol-reactive ABPP probe CA-Rh.1Integer
5Outcome [Neuro-2A]One of Active, Inactive, or Not Tested2Outcome
6Anti-targets at 0.250 uM [Neuro-2A] (0.25μM**)Number of observed anti-targets in cultured Neuro-2A murine neuroblastoma cells upon 0.250 uM compound treatment as assessed by gel-based competitive ABPP with the thiol-reactive ABPP probe CA-Rh.2Integer
7Anti-targets at 0.100 uM [Neuro-2A] (0.1μM**)Number of observed anti-targets in cultured Neuro-2A murine neuroblastoma cells upon 0.100 uM compound treatment as assessed by gel-based competitive ABPP with the thiol-reactive ABPP probe CA-Rh.2Integer

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

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