Late 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 vivo. ..more
BioActive Compound: 1
Depositor Specified Assays
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| AID | Name | Type | Probe | Comment |
|---|---|---|---|---|
| 2130 | Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). | screening | Primary screen (PME-1 inhibitors) | |
| 2143 | Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). | summary | 3 | Summary (PME-1 inhibitors) |
| 2174 | Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1). | screening | Counterscreen (LYPLA1 inhibitors) | |
| 2171 | Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). | screening | Confirmation assay (PME-1 inhibitors) | |
| 2177 | Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2). | screening | Counterscreen (LYPLA2 inhibitors) | |
| 2232 | Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2). | screening | Counterscreen confirmation (LYPLA2 inhibitors) | |
| 2233 | Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1). | screening | Counterscreen confirmation (LYPLA1 inhibitors) | |
| 2291 | Fluorescence 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) | |
| 2363 | Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2a | screening | MOA assay (Demethylation of PP2a) | |
| 2365 | Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Luminescence-based counterscreen assay to identify cytotoxic compounds | confirmatory | Counterscreen (Cytotoxicity HEC 293T) | |
| 2366 | Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50 | confirmatory | ABPP dose response screen (PME-1 inhibitors) | |
| 2368 | Late 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 Assay | screening | MOA assay (PME-1 inhibitors, gel filtration assay) | |
| 2369 | Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) Inhibition | screening | ABPP screen (PME-1 inhibitors) | |
| 2371 | Late 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 enzyme | confirmatory | ABPP dose response screen (PME-1 inhibitors, purified enzyme) | |
| 463090 | Late 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 site | other | 1 | MOA assay (PME-1 inhibitors, LC-MS assay) |
| 463091 | Late 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 compounds | confirmatory | Counterscreen (Cytotoxicity HeLa) | |
| 463124 | Late 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 2 | confirmatory | Late stage dose response (PME-1 inhibitors) | |
| 463130 | Late 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 1 | confirmatory | Late stage gel-based ABPP dose response (PME-1 inhibitors) | |
| 463131 | Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based inhibition | screening | Late stage fluorescence-base cell-based inhibition (PME-1 inhibitors) | |
| 463132 | Late 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 PP2A | screening | Late stage (Inhibition of PME-1 mediated demethylation of PP2A) | |
| 463146 | Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Fluorescence-based biochemical gel-based ABPP | other | Late stage fluorescence-based gel-based ABPP (PME-1 inhibitors) | |
| 463149 | Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity | other | Late stage fluorescence-based biochemical gel-based ABPP inhibition and selectivity (PME-1 inhibitors) | |
| 588796 | Late 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 2 | confirmatory | Late stage dose response (ABPP inhibition of ABHD10 in triplicate) | |
| 588801 | Late 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 ABHD10 | confirmatory | Late stage dose response(ABPP inhibition of ABHD10 in triplicate) | |
| 588802 | Late 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 1 | confirmatory | Late stage dose response (ABPP inhibition of ABHD10 in triplicate) | |
| 588803 | Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assay | other | 1 | Late stage results (LC-MS/MS-based cell-based ABPP-SILAC) |
| 588804 | Late 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 compounds | confirmatory | Late stage dose response assay (cytotoxicity in six replicates) | |
| 588805 | Late 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 assay | other | Late stage results (ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assay) | |
| 588806 | Late 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 | other | Late stage results(ABPP inhibition of ABHD10) | |
| 588807 | 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 in a complex proteome for ABHD10 | other | Late stage assay (ABPP inhibition and selectivity in a complex proteome for ABHD10) | |
| 588835 | Late 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 assay | other | Late stage dose response (ABPP ABHD10 selectivity assay) | |
| 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 | other | Late stage assay (cysteine-reactive proteins inhibition and selectivity) |
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_INVIVO
Late 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 vivo.
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. PMID: 11060018.
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. PMID: 19293187.
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, activity-based protein profiling, ABPP, inhibition, in vivo, fluorophosphonate rhodamine, FP-Rh, inhibitor, selectivity, anti-targets, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
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_INVIVO
Late 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 vivo.
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. PMID: 11060018.
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. PMID: 19293187.
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, activity-based protein profiling, ABPP, inhibition, in vivo, fluorophosphonate rhodamine, FP-Rh, inhibitor, selectivity, anti-targets, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Protocol
Assay Overview:
The purpose of this assay is to determine whether or not test compounds can inhibit ABHD10 in vivo and to assess selectivity using a gel-based activity-based protein profiling (ABPP) assay. In this assay, test compounds are administered to mice. Mice are sacrificed, and their heart tissue harvested, homogenized, and the soluble fraction isolated and reacted with the serine-hydrolase-specific activity-based probe FP-Rh. 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 of the bands. As designed, test compounds that act as ABHD10 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:
Purpose-bred C57BL/6 laboratory mice were administered test compound (6, 13, or 25 mg/kg in vehicle solution, i.p.) or vehicle only (n=1 per group). After six hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and heart tissues removed and snap frozen in liquid nitrogen. Tissues were homogenized and the soluble fraction isolated by centrifugation (45 min, 100K x g) and adjusted to 1 mg/mL in 50 mM Dulbecco's PBS (DPBS). For control, one aliquot (50 uL) of vehicle-treated proteome was reacted with test compound (1 uL of a 50x stock in DMSO, 1 uM final concentration) for 30 minutes at 25 C. All aliquots (50 uL) were treated with FP-Rh (1 uL of 50x stock in DMSO, 1 uM final concentration). The reaction was incubated for 30 minutes at 25 C, quenched with 4x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining of ABHD10 and anti-target bands was determined by measuring the integrated optical density of test compound bands relative to vehicle bands. Protein bands were counted as anti-targets if greater than or equal to 50% inhibition was observed at any test compound concentration.
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 ABHD10 or anti-target treated with test compound.
High_Control is defined as ABHD10 or anti-target treated with vehicle only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
ABHD10 Assay Outcome:
Compounds with greater than or equal to 50% inhibition at 25 mg/kg were considered active. Compounds with less than 50% inhibition at 25 mg/kg were considered inactive.
Selectivity Assay Outcome:
Compounds were considered active if one or more anti-targets were observed at any test compound concentration. Compounds were considered inactive if no anti-targets were observed at any test compound concentration.
Overall Outcome and Score:
Compounds active in the ABHD10 Assay in inactive in the Selectivity Assay were considered active. Compounds inactive in the ABHD10 Assay and/or active in the Selectivity Assay were considered inactive.
The PubChem Activity Score is assigned a value of 1000 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:
C57BL/6 laboratory mice (provided by
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
The purpose of this assay is to determine whether or not test compounds can inhibit ABHD10 in vivo and to assess selectivity using a gel-based activity-based protein profiling (ABPP) assay. In this assay, test compounds are administered to mice. Mice are sacrificed, and their heart tissue harvested, homogenized, and the soluble fraction isolated and reacted with the serine-hydrolase-specific activity-based probe FP-Rh. 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 of the bands. As designed, test compounds that act as ABHD10 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:
Purpose-bred C57BL/6 laboratory mice were administered test compound (6, 13, or 25 mg/kg in vehicle solution, i.p.) or vehicle only (n=1 per group). After six hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and heart tissues removed and snap frozen in liquid nitrogen. Tissues were homogenized and the soluble fraction isolated by centrifugation (45 min, 100K x g) and adjusted to 1 mg/mL in 50 mM Dulbecco's PBS (DPBS). For control, one aliquot (50 uL) of vehicle-treated proteome was reacted with test compound (1 uL of a 50x stock in DMSO, 1 uM final concentration) for 30 minutes at 25 C. All aliquots (50 uL) were treated with FP-Rh (1 uL of 50x stock in DMSO, 1 uM final concentration). The reaction was incubated for 30 minutes at 25 C, quenched with 4x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining of ABHD10 and anti-target bands was determined by measuring the integrated optical density of test compound bands relative to vehicle bands. Protein bands were counted as anti-targets if greater than or equal to 50% inhibition was observed at any test compound concentration.
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 ABHD10 or anti-target treated with test compound.
High_Control is defined as ABHD10 or anti-target treated with vehicle only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
ABHD10 Assay Outcome:
Compounds with greater than or equal to 50% inhibition at 25 mg/kg were considered active. Compounds with less than 50% inhibition at 25 mg/kg were considered inactive.
Selectivity Assay Outcome:
Compounds were considered active if one or more anti-targets were observed at any test compound concentration. Compounds were considered inactive if no anti-targets were observed at any test compound concentration.
Overall Outcome and Score:
Compounds active in the ABHD10 Assay in inactive in the Selectivity Assay were considered active. Compounds inactive in the ABHD10 Assay and/or active in the Selectivity Assay were considered inactive.
The PubChem Activity Score is assigned a value of 1000 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:
C57BL/6 laboratory mice (provided by
FP-Rh (provided by the 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: mmoa characterization
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
BAO: bioassay specification: assay stage: secondary: mmoa characterization
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
| TID | Name | Description | PID§ | Panel Targets | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||||
| Score | The BioAssay activity ranking score |  | Integer | |||||
| 1 | Outcome [ABHD10] | One of Active, Inactive, or Not Tested | 1 | abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus] | | Outcome | ||
| 2 | Inhibition at 6 mg/kg [ABHD10] | The value for percent inhibition of endogenous mouse ABHD10 in vivo at 6 mg/kg compound. | 1 | | Integer | % | ||
| 3 | Inhibition at 13 mg/kg [ABHD10] | The value for percent inhibition of endogenous mouse ABHD10 in vivo at 13 mg/kg compound. | 1 | | Integer | % | ||
| 4 | Inhibition at 25 mg/kg [ABHD10] | The value for percent inhibition of endogenous mouse ABHD10 in vivo at 25 mg/kg compound. | 1 | | Integer | % | ||
| 5 | Outcome [Selectivity] | One of Active, Inactive, or Not Tested | 2 | | Outcome | |||
| 6 | Count of Anti-targets [Selectivity] | The number of observed anti-targets at all tested compound concentrations | 2 | | Integer |
Additional Information
Grant Number: 1 R01 CA132630
Classification
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