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BioAssay: AID 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

Name: 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 ..more
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 Related BioAssays
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AID: 588835
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD10_INH_FLUO_GELBASEDABPP_3XIC50_INSITU_SEL)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-11-30
Hold-until Date: 2012-05-31
Modify Date: 2012-05-31

Data Table ( Complete ):           All
Tested Compound:
Depositor Specified Assays
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AIDNameTypeProbeComment
2130Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).summary3 Summary (PME-1 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).screening Counterscreen (LYPLA1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening Confirmation assay (PME-1 inhibitors)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen confirmation (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening Counterscreen confirmation (LYPLA1 inhibitors)
2291Fluorescence 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)
2363Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2ascreening MOA assay (Demethylation of PP2a)
2365Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Luminescence-based counterscreen assay to identify cytotoxic compoundsconfirmatory Counterscreen (Cytotoxicity HEC 293T)
2366Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50confirmatory ABPP dose response screen (PME-1 inhibitors)
2368Late 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 Assayscreening MOA assay (PME-1 inhibitors, gel filtration assay)
2369Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) Inhibitionscreening ABPP screen (PME-1 inhibitors)
2371Late 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 enzymeconfirmatory ABPP dose response screen (PME-1 inhibitors, purified enzyme)
463090Late 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 siteother1 MOA assay (PME-1 inhibitors, LC-MS assay)
463091Late 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 compoundsconfirmatory Counterscreen (Cytotoxicity HeLa)
463146Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Fluorescence-based biochemical gel-based ABPPother Activity-based protein profiling (PME1 inhibitors)
463149Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityother Activity-based protein profiling (PME1 inhibiton and selectivity)
463131Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based inhibitionscreening Activity-based protein profiling (PME1 inhibiton in singlicate)
463130Late 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 1confirmatory Activity-based protein profiling, dose response (PME1 inhibitors)
463124Late 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 2confirmatory Activity-based protein profiling, dose response (PME1 inhibitors)
463132Late 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 PP2Ascreening Mechanism of action (PP2a demethylation)
588801Late 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 ABHD10confirmatory Activity-based protein profiling (ABHD10 inhibitors)
588803Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assayother1 Activity-based protein profiling (PME-1 inhibitors, LC-MS/MS based SILAC )
588804Late 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 compoundsconfirmatory Cytotoxicity assay (PME-1 inhibitors)
588806Late 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 ABHD10other Activity-based protein profiling (ABHD10 inhibitors)
588807Late 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 ABHD10other Activity-based protein profiling (ABHD10 inhibiton and selectivity)
588802Late 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 1confirmatory Activity-based protein profiling, dose response (ABHD10 inhibitors)
588796Late 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 2confirmatory Activity-based protein profiling, dose response (ABHD10 inhibitors)
588805Late 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 assayother Activity-based protein profiling (ABHD10 inhibitors, LC-MS/MS based SILAC )
602468Late 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 proteinsother
602485Late 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 vivoother
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_3XIC50_INSITU_SEL

Name: 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

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, abhdyrolase domain containing protein 6, ABHD6, prolyl endopeptidase, PREP, uncharacterized, PME-1, protein phosphatase methylesterase 1, PPME-1, counterscreen, activity-based protein profiling, ABPP, inhibition, IC50, dose response, in situ, Neuro-2A, 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
In situ assays
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1ABHD6 in situ assay1monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]
Taxonomy id: 10090
Gene id: 66082
2PREP in situ assay1prolyl endopeptidase [Mus musculus] [gi:6755152]
Taxonomy id: 10090
Gene id: 19072

§ Panel component ID.
Protocol

Assay Overview:

The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD10 anti-target inhibition in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested, homogenized, and reacted 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 of the bands. As designed, test compounds that act as anti-target 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:

Cultured Neuro-2A murine neuroblastoma cells in serum-free DMEM medium (1 mL total volume) were treated with DMSO or test compound (10 uL of a 100x stock in DMSO) for 2 hours at 37 degrees Celsius. Cells were harvested, washed with DPBS (1 mL), and resuspended in DPBS (1 mL). Centrifugation (1000 x g, 5 minutes) provided a cell pellet which was resuspended in DPBS (200 uL) and homogenized by sonication. The protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 1 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, 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 the anti-target ABHD6 or PREP band relative to a DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (10 uM, 1 uM, 250 nM, 50 nM, 10 nM, 5 nM, and 1 nM).

%_Inhibition = ( 1 - ( IOD_Test_Compound-MedianIOD_Low_Control ) / ( MedianIOD_High_Control-MedianIOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as anti-target treated with test compound.
High_Control is defined as anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

For each test compound, percent inhibition was plotted against the log of the compound concentration. A four parameter variable slope equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.

ABHD6 and PREP Assay Outcomes:

Compounds with IC50 > 0.5 uM were considered inactive. There are no active compounds.

PubChem Activity Outcome:

Compounds that were "inactive" in both ABHD6 and PREP assays were considered "inactive". Compounds that were "active" in one or both ABHD6 and PREP assays were considered "active".

PubChem Activity Score:

The PubChem Activity Score is assigned a value of 50 for active compounds, and 0 for inactive compounds.

List of Reagents:

Neuro-2A murine cells (provided by Assay Provider)
FP-Rh (provided by the Assay Provider)
DMEM Medium (CellGro 10-017-CV)
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: selectivity

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
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Outcome [ABHD6]One of Active, Inactive, or Not Tested1monoacylglycerol lipase ABHD6 [Mus musculus]Outcome
2IC50 [ABHD6]*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in nanomolar.1FloatμM
3Inhibition of ABHD6 at 10 uM [1] (10μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 10 uM compound. Replicate 1.1Float%
4Inhibition of ABHD6 at 10 uM [2] (10μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 10 uM compound. Replicate 2.1Float%
5Inhibition of ABHD6 at 10 uM [3] (10μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 10 uM compound. Replicate 3.1Float%
6Inhibition of ABHD6 at 1 uM [1] (1μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 1 uM compound. Replicate 1.1Float%
7Inhibition of ABHD6 at 1 uM [2] (1μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 1 uM compound. Replicate 2.1Float%
8Inhibition of ABHD6 at 1 uM [3] (1μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 1 uM compound. Replicate 3.1Float%
9Inhibition of ABHD6 at 0.25 uM [1] (0.25μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.25 uM compound. Replicate 1.1Float%
10Inhibition of ABHD6 at 0.25 uM [2] (0.25μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.25 uM compound. Replicate 2.1Float%
11Inhibition of ABHD6 at 0.25 uM [3] (0.25μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.25 uM compound. Replicate 3.1Float%
12Inhibition of ABHD6 at 0.05 uM [1] (0.05μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.05 uM compound. Replicate 1.1Float%
13Inhibition of ABHD6 at 0.05 uM [2] (0.05μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.05 uM compound. Replicate 2.1Float%
14Inhibition of ABHD6 at 0.05 uM [3] (0.05μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.05 uM compound. Replicate 3.1Float%
15Inhibition of ABHD6 at 0.01 uM [1] (0.01μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.01 uM compound. Replicate 1.1Float%
16Inhibition of ABHD6 at 0.01 uM [2] (0.01μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.01 uM compound. Replicate 2.1Float%
17Inhibition of ABHD6 at 0.01 uM [3] (0.01μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.01 uM compound. Replicate 3.1Float%
18Inhibition of ABHD6 at 0.005 uM [1] (0.005μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.005 uM compound. Replicate 1.1Float%
19Inhibition of ABHD6 at 0.005 uM [2] (0.005μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.005 uM compound. Replicate 2.1Float%
20Inhibition of ABHD6 at 0.005 uM [3] (0.005μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.005 uM compound. Replicate 3.1Float%
21Inhibition of ABHD6 at 0.001 uM [1] (0.001μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.001 uM compound. Replicate 1.1Float%
22Inhibition of ABHD6 at 0.001 uM [2] (0.001μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.001 uM compound. Replicate 2.1Float%
23Inhibition of ABHD6 at 0.001 uM [3] (0.001μM**)The value for percent inhibition of endogenous mouse ABHD6 in situ at 0.001 uM compound. Replicate 3.1Float%
24Outcome [PREP]One of Active, Inactive, or Not Tested2prolyl endopeptidase [Mus musculus]Outcome
25IC50 [PREP]*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in nanomolar.2FloatμM
26Inhibition of PREP at 10 uM [1] (10μM**)The value for percent inhibition of endogenous mouse PREP in situ at 10 uM compound. Replicate 1.2Float%
27Inhibition of PREP at 10 uM [2] (10μM**)The value for percent inhibition of endogenous mouse PREP in situ at 10 uM compound. Replicate 2.2Float%
28Inhibition of PREP at 10 uM [3] (10μM**)The value for percent inhibition of endogenous mouse PREP in situ at 10 uM compound. Replicate 3.2Float%
29Inhibition of PREP at 1 uM [1] (1μM**)The value for percent inhibition of endogenous mouse PREP in situ at 1 uM compound. Replicate 1.2Float%
30Inhibition of PREP at 1 uM [2] (1μM**)The value for percent inhibition of endogenous mouse PREP in situ at 1 uM compound. Replicate 2.2Float%
31Inhibition of PREP at 1 uM [3] (1μM**)The value for percent inhibition of endogenous mouse PREP in situ at 1 uM compound. Replicate 3.2Float%
32Inhibition of PREP at 0.25 uM [1] (0.25μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.25 uM compound. Replicate 1.2Float%
33Inhibition of PREP at 0.25 uM [2] (0.25μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.25 uM compound. Replicate 2.2Float%
34Inhibition of PREP at 0.25 uM [3] (0.25μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.25 uM compound. Replicate 3.2Float%
35Inhibition of PREP at 0.05 uM [1] (0.05μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.05 uM compound. Replicate 1.2Float%
36Inhibition of PREP at 0.05 uM [2] (0.05μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.05 uM compound. Replicate 2.2Float%
37Inhibition of PREP at 0.05 uM [3] (0.05μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.05 uM compound. Replicate 3.2Float%
38Inhibition of PREP at 0.01 uM [1] (0.01μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.01 uM compound. Replicate 1.2Float%
39Inhibition of PREP at 0.01 uM [2] (0.01μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.01 uM compound. Replicate 2.2Float%
40Inhibition of PREP at 0.01 uM [3] (0.01μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.01 uM compound. Replicate 3.2Float%
41Inhibition of PREP at 0.005 uM [1] (0.005μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.005 uM compound. Replicate 1.2Float%
42Inhibition of PREP at 0.005 uM [2] (0.005μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.005 uM compound. Replicate 2.2Float%
43Inhibition of PREP at 0.005 uM [3] (0.005μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.005 uM compound. Replicate 3.2Float%
44Inhibition of PREP at 0.001 uM [1] (0.001μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.001 uM compound. Replicate 1.2Float%
45Inhibition of PREP at 0.001 uM [2] (0.001μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.001 uM compound. Replicate 2.2Float%
46Inhibition of PREP at 0.001 uM [3] (0.001μM**)The value for percent inhibition of endogenous mouse PREP in situ at 0.001 uM compound. Replicate 3.2Float%

* Activity Concentration. ** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R01 CA132630

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