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
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
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.
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 We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored.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.
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.
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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.
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
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)
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: mmoa characterization
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
Assay Format: Cell-based
Assay Test Type: In vivo