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
Name: 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. ..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_LCMS_ABPP_SILAC
Name: 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.
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 We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored.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, liquid chromatography, LC, tandem mass spectrometry, MS/MS, activity-based protein profiling, ABPP, stable isotope labeling with amino acids in cell culture, SILAC, ABPP-SILAC, fluorophosphonate biotin, FP-Biotin, inhibitor, selectivity, anti-targets, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using activity-based protein profiling (ABPP) in combination with stable isotope labeling with amino acids in cell culture (SILAC). In this assay, cultured Neuro-2A cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with test compound and DMSO, respectively, in situ. Cells are lysed, proteomes are treated with the serine-hydrolase-specific activity-based fluorophosphonate-biotin (FP-biotin) affinity probe, and combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by multi-dimensional liquid chromatography tandem mass spectrometery LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, resulting in a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of 1.
Stable isotope labeling with amino acids in cell culture (SILAC). Neuro-2A murine neuroblastoma cells were initially grown for 10 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 2 mM L-glutamine. Light media was supplemented with 100 ug/mL L-arginine and 100 ug/mL L-lysine. Heavy media was supplemented with 100 ug/mL [13C615N4]-L-Arginine and 100 ug/mL [13C615N2]-L-Lysine. Light cells (in 10 mL media) were treated with 100 nM test compound (75 uL of a 200x stock in DMSO) and heavy cells were treated with DMSO (75 uL) for 2 hours at 37 C. Cells were washed 2 times with DPBS, harvested, and homogenized by sonication in DPBS (1mL). The soluble and membrane fractions were isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 2 mg/mL with DPBS in each fraction.
Sample preparation for ABPP-SILAC. The light and heavy proteomes were labeled with 10 uM of FP-biotin (500 uL total reaction volume) for 2 hours at 25 C. After incubation, light and heavy proteomes were mixed in 1:1 ratio, and the membrane proteomes were additionally solubilized with 1% Triton-X100. The proteomes were desalted over PD-10 desalting columns (GE Healthcare) and FP-labeled proteins were enriched with streptavidin beads. The beads were washed with 1% SDS in DPBS (1x), 6M urea (1x), and DPBS (2x), then resuspended in 6 M urea, reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 C in the dark. On-bead digestions were performed for 12 hours at 37 C with sequence-grade modified trypsin (Promega; 2 ug) in 2M urea in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid, pressure-loaded on to a biphasic (strong cation exchange/reversed phase) capillary column and analyzed as described below.
LC-MS/MS analysis. Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography (2D-LC) separation in combination with tandem mass spectrometry using an Agilent 1200-series quaternary pump and Thermo Scientific LTQ-Orbitrap Velos ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (20 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 30 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 126.96.36.199; publicly available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the ProLuCID algorithm (publicly available at http://fields.scripps.edu/downloads.php) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. ProLucid searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, 8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of Light/Heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were greater than 3 standard deviations from the median peptide value. Proteins with less than three peptides per protein ID were not included in the analysis.
Ratio = Average( AUC_light / AUC_heavy ) calculated for all unique peptides
AUC_light is the area-under-the-curve for the light peptide pair from cells treated with test compound.
AUC_heavy is the area-under-the-curve for the heavy peptide pair from cells treated with DMSO.
PubChem Activity Outcome and Score:
The following applies to each panel:
A compound with a light/heavy ratio of less than or equal to 0.5 for a particular target/anti-target was considered active. A compound with a light/heavy ratio of greater than 0.5 for a specified target/anti-target was considered inactive.
Overall Outcome and Score:
A compound was considered active if it was active for ABHD10 and inactive for all anti-target serine hydrolases tested.
The PubChem Activity Score is assigned a value of 100 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:
Neuro-2A murine cells (provided by Assay Provider)
SILAC DMEM media (Thermo 89985)
dialyzed FCS (Gemini 100-108)
1x Glutamine (CellGro 25-005-CI)
L-Arginine (SigmaAldrich A6969)
L-Lysine (SigmaAldrich L9037)
[13C615N4]-L-Arginine (SigmaAldrich 608033)
[13C615N2]-L-Lysine (SigmaAldrich 608041)
DPBS (Cellgro 20-031-CV)
FP-biotin (provided by Assay Provider)
PD-10 desalting columns (GE Healthcare 17-0851-01)
SDS (SigmaAldrich L6026)
Urea (Fisher U15-3)
TCEP (SigmaAldrich 75259)
Iodoacetamide (SigmaAldrich I1149)
Trypsin (Promega V5111)
CaCl2 (SigmaAldrich C1016)
Streptavidin beads (Pierce 20349)
Fused-silica (Agilent 160-2635-10)
Strong cation exchange (Phenomenex CH0-2257)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Formic acid (Fluka 06440)
Triton-X100 (Fisher AC21568-0010)
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: 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 Cell Type: Neuro-2A