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BioAssay: AID 504892

Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11

Name: Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11. ..more
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
AID: 504892
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD11_INH_FLUO_GEL_1XINH_HUMAN)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-07-01
Hold-until Date: 2011-10-06
Modify Date: 2011-10-06

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
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AIDNameTypeProbeComment
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).summary2 Summary (LYPLA1 inhibitors)
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).summary1 Summary (LYPLA2 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).screening Primary screen (LYPLA1 inhibitors in singlicate)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening Confirmation screen (LYPLA1 inhibitors in triplicate)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Primary screen (LYPLA2 inhibitors in singlicate)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Confirmation screen (LYPLA2 inhibitors in triplicate)
493105Assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition of recombinant and endogenous enzymeother Confirmation screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493108Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: fluorescence-based cell-based inhibitionother Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493109Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: LC-MS/MS assay to assess binding of compounds to active siteother Late stage LCMS assay (LYPLA1)
493110Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Gel-based Activity-Based Protein Profiling (ABPP) IC50 for LYPLA1 and LYPLA2confirmatory Late stage dose response (LYPLA1 and LYPLA2 inhibitors in triplicate)
493111Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityother Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
493154Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Gel-based Activity-Based Protein Profiling (ABPP) IC50 for off-target ABHD11confirmatory Late stage dose response counterscreen (ABHD11 inhibitors in triplicate)
493161Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsconfirmatory Late stage dose response counterscreen (T-cell cytotoxicity in quadruplicate)
504482Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) IC50 for anti-target ABHD11confirmatory Late stage dose repsonse (ABHD11 inhibitors in triplicate)
504498Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: LC-MS/MS assay to assess binding of compounds to active site of anti-target ABHD11other Late stage MOA assay (ABDH11 LCMS)
504505Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based cell-based gel-based Activity-Based Protein Profiling (ABPP) percent inhibition for anti-target ABHD11other Late stage screen (ABHD11 inhibitors in singlicate, in situ)
504507Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) IC50 for anti-target ABHD11 Set 2confirmatory Late stage dose response (ABHD11 inhibitors in triplicate)
504510Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds set 2confirmatory Late stage dose repsonse counterscreen (T-cell cytotoxicity in quadruplicate)
504520Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition and selectivityother Late stage screen (LYPLA1 and LYPLA2 inhibitors in singlicate)
504522Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: LC-MS-based cell-based SILAC Activity-Based Protein Profiling (ABPP) for anti-target ABHD11other Late stage panel screen (ABHD11 SILAC ratio)
651978Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitroother
651979Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situother
651980Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situother
651981Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitroother
651985Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivoother
651986Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in situother
651987Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess binding modeother
651988Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityother
651990Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPPconfirmatory
651991Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsconfirmatory
651998Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: Substrate-based fluorescence-based biochemical determination of kinetic parametersconfirmatory
652001Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Substrate-based fluorescence-based biochemical determination of kinetic parametersconfirmatory
652003Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: fluorescence-based biochemical dose-response assayconfirmatory
652004Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: fluorescence-based biochemical dose-response assayconfirmatory
652018Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro, Set 2other
652029Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibitionother
652030Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityother
743117 On Hold
743118 On Hold
743119 On Hold
743127 On Hold
743132 On Hold
743133 On Hold
743134 On Hold
743137 On Hold
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: ABHD11_INH_FLUO_GEL_1XINH_HUMAN

Name: Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11.

Description:

Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling, and identification of proteins responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. For example, it has been suggested by Waldmann et al. that preventing depalmitoylation of the oncogene Ras by inhibiting thioesterase activity may disrupt targeted distribution, thereby downregulating oncogenic signaling (1). As such, inhibitors of protein palmitoyl thioesterases may act as tumor suppressors by preventing aberrant growth signaling. More than a decade ago, the cytosolic serine hydrolase acyl-protein thioesterase 1 (APT1) was identified as an in vitro HRAS palmitoyl thioesterase (2). Initially classified as lysophospholipase 1 (LYPLA1) (3), the enzyme has since been demonstrated to have several hundred-fold higher activity as a protein thioesterase. Upon retroviral shRNA knockdown of LYPLA1, we found that over-expressed HRAS was hyper-palmitoylated, providing evidence that endogenous LYPLA1 a functional protein palmitoyl thioesterase in mammalian cells. LYPLA2 (a.k.a. APT2) is a close homologue of LYPLA1 (65% identical) and exhibits lysophospholipase activity in vitro, but its potential role as a thioesterase is unknown (4). Several inhibitors of LYPLA1 have been described (1,5,6), but none of these agents have proven capable of selectively inhibiting LYPLA1 activity in cells. No selective inhibitors of LYPLA2 have been reported.

As serine hydrolases, the LYPLAs are readily labeled by activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group (7), which can be exploited for inhibitor discovery using a competitive-ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling (8). Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP (9). Seeking selective, in situ active chemical tools to investigate the potential roles of the LYPLAs in cancer pathogenesis, we conducted a FluoPol-ABPP HTS campaign to identify inhibitors of LYPLA1 (AIDs 2174 and 2233) and the structurally related LYPLA2 (AIDs 2177 and 2232). HTS identified a modestly potent triazole urea inhibitor (SID 7974398; IC50 795 nM vs. LYPLA1, 5200 nM vs. LYPLA2), which was successfully optimized via several rounds of SAR-by-synthesis as ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 (IC50 17 nM) and LYPLA2 (IC50 30 nM). Out of more than 20 serine hydrolases profiled by gel-based competitive ABPP, ML211 is 50-fold selective for all except for one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11; IC50 10 nM). However, during our SAR campaign, we fortuitously discovered a selective ABHD11 inhibitor from among the synthetic triazole urea library compounds. This compound (ML226, SID 99445332) was presented as a anti-probe in the ML211 Probe Report for use as a control.

ABHD11 is a poorly characterized serine hydrolase; all that is known about its biology is that it is a mitochondrial enzyme (10) with broad tissue distribution, has little sequence homology to other proteins, and its gene is located in a region of chromosome 7 that is hemizygously deleted in Williams-Beuren syndrome, a rare genetic disease with symptoms that include vascular stenosis, mental retardation, and excessive sociability (11). However, the underlying biology and contribution of ABHD11 to the disease is unknown, as its biochemical function, substrates, and products have not been identified. A principle goal of post-genomic research is the determination of the molecular and cellular roles of uncharacterized enzymes like ABHD11. As such, potent and selective inhibitors of ABHD11 not only serve as key controls for ML211, but important chemical tools in their own right for investigation of ABHD11 biology. This AID provides ABHD11-specific characterization of the triazole urea library members, including ML226.

References:

1. Dekker, F.J., et al., Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol., 2010. 6(6): p. 449-56.
2. Duncan, J.A. and A.G. Gilman, A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem., 1998. 273(25): p. 15830-7.
3. Sugimoto, H., H. Hayashi, and S. Yamashita, Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J. Biol. Chem., 1996. 271(13): p. 7705-11.
4. Toyoda, T., H. Sugimoto, and S. Yamashita, Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochim. Biophys. Acta, 1999. 1437(2): p. 182-93.
5. Biel, M., et al., Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chemistry, 2006. 12(15): p. 4121-43.
6. Deck, P., et al., Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. Engl., 2005. 44(31): p. 4975-80.
7. Jessani, N., et al., Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A., 2002. 99(16): p. 10335-40.
8. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
9. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol., 2009. 27(4): p. 387-94.
10. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19.
11. Schubert, C., The genomic basis of the Williams-Beuren syndrome. Cell. Mol. Life Sci., 2009. 66(7): p. 1178-97.

Keywords:

late stage, late stage AID, assay provider, powders, LYPLA1, lysophospholipase 1, LYPLA2, lysophospholipase 2, APT1, acyl-protein thioesterase 1, APT2, acyl-protein thioesterase 2, palmitoylation, alpha/beta hydrolase domain-containing protein 11, abhydrolase domain-containing protein 11, ABHD11, oncogene, tumor suppressor, serine hydrolase, counterscreen, inhibitor, selectivity, anti-targets, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of test compounds can inhibit the human isoform of ABHD11 in a complex proteomic lysate. In this assay, a complex proteome containing recombinant human ABHD11 is incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-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 ABHD11 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:

Recombinant human ABHD11 (50 ug/mL) was spiked into mouse brain membrane proteome (1 mg/mL in DPBS, 50 uL reaction volume) and treated with 0 (DMSO only), 3, 10, 30, or 150 nM or 20 uM test compound (1 uL of a 50x stock in DMSO). (Note: mouse membrane proteome alone is used as a control for visualization and quantification of the human ABHD11 band). Test compounds were incubated for 30 minutes at 25 C. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM. The reaction was incubated for 30 minutes at 25 C, quenched with 2x 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 the human ABHD11 band.

%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as target treated with test compound.
High_Control is defined as target treated with DMSO only (no compound).
Low_Control is defined as background in the "no enzyme" control (mouse membrane proteome only, no added ABHD11).

PubChem Activity Outcome and Score:

Compounds with greater than or equal to 50% inhibition at 0.030 uM were considered active. Compounds with less than 50% inhibition at 30 nM were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed inhibition at 0.030 uM. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.

List of Reagents:

Mouse brain membrane proteome (provided by the Assay Provider)
Recombinant human ABHD11 (provided by the Assay Provider)
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 0.003 uM (0.003μM**)Inhibition of recombinant, human ABHD11 spiked into mouse brain membrane proteome upon 3 nM compound treatment as assessed by competitive ABPP.Float%
2Inhibition at 0.010 uM (0.01μM**)Inhibition of recombinant, human ABHD11 spiked into mouse brain membrane proteome upon 10 nM compound treatment as assessed by competitive ABPP.Float%
3Inhibition at 0.030 uM (0.03μM**)Inhibition of recombinant, human ABHD11 spiked into mouse brain membrane proteome upon 30 nM compound treatment as assessed by competitive ABPP.Float%
4Inhibition at 0.150 uM (0.15μM**)Inhibition of recombinant, human ABHD11 spiked into mouse brain membrane proteome upon 150 nM compound treatment as assessed by competitive ABPP.Float%
5Inhibition at 20 uM (20μM**)Inhibition of recombinant, human ABHD11 spiked into mouse brain membrane proteome upon 20000 nM compound treatment as assessed by competitive ABPP.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R01 CA132630

Data Table (Concise)
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