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
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.
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 We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored.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.
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.
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
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.
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
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)
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
Assay Format: Biochemical
** Test Concentration. § Panel component ID.