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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 ):           All
Tested Compound:
Depositor Specified Assays
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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 Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).summary3 Summary (PME-1 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).screening Counterscreen (LYPLA1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening Confirmation assay (PME-1 inhibitors)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen confirmation (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening 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 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 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 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 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 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 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 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 siteother1 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 Counterscreen (Cytotoxicity HeLa)
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 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 Late stage fluorescence-based biochemical gel-based ABPP inhibition and selectivity (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 Late stage fluorescence-base cell-based inhibition (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 Late stage gel-based ABPP dose response (PME-1 inhibitors)
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 Late stage dose response (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 Late stage results (Inhibition of PME-1 mediated demethylation of PP2A)
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 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 Late stage dose response (ABPP ABHD10 selectivity assay)
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 Late stage results (ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assay)
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 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 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 Late stage dose response assay (cytotoxicity in six replicates)
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 Late stage results (ABPP inhibition of ABHD10)
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 Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
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 Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
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
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
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: selectivity

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: protein complex format

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: meta target: molecular target: protein target: enzyme: generic hydrolase

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: detection technology: fluorescence: fluorescence intensity

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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