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BioAssay: AID 1631

qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase

Matthew G. Vander Heiden, M.D., Ph.D. [Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115] ..more
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 Tested Compounds
 Tested Compounds
All(263668)
 
 
Active(892)
 
 
Inactive(259044)
 
 
Inconclusive(3753)
 
 
 Tested Substances
 Tested Substances
All(264516)
 
 
Active(892)
 
 
Inactive(259866)
 
 
Inconclusive(3758)
 
 
AID: 1631
Data Source: NCGC (PYKH221a)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-03-27
Modify Date: 2009-06-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 892
Related Experiments
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AIDNameTypeProbeComment
1540Secondary assay for Activators of Human Pyruvate Kinase M2 isoformConfirmatory depositor-specified cross reference
1751Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate KinaseConfirmatory depositor-specified cross reference
1898Secondary assay for Activators of Human Pyruvate Kinase M2 isoform - Cell Titer Glo cytotoxicity assayConfirmatory depositor-specified cross reference
2095qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary6 depositor-specified cross reference
2263Confirmation Concentration-Response Assay for Inhibitors of Human Muscle isoform 2 Pyruvate KinaseConfirmatory depositor-specified cross reference
2265Secondary assay for Inhibitors of Human Pyruvate Kinase M2 isoform: FLuc CounterscreenConfirmatory depositor-specified cross reference
2267Secondary assay for Inhibitors of Human Pyruvate Kinase M2 isoformConfirmatory depositor-specified cross reference
2533Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2534Secondary Concentration-Response Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2535Secondary Concentration-Response Assay for Activators of Human Liver Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2536Secondary Concentration-Response Assay for Activators of Human Muscle isoform 1 Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2562Secondary LDH Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2576Secondary LDH Assay for Activators of Human Pyruvate Kinase M2 isoform: for Probe SARConfirmatory depositor-specified cross reference
2620Secondary LDH Assay for Activators of Human Pyruvate Kinase M1 Isoform: for Probe SARConfirmatory depositor-specified cross reference
2625Secondary LDH Assay for Activators of Human Liver Pyruvate Kinase: for Probe SARConfirmatory depositor-specified cross reference
2653Secondary Assay for Activators of Human Pyruvate Kinase M2 isoform - Cell Titer Glo Cytotoxicity for Probe SARConfirmatory depositor-specified cross reference
602359Extended Characterization of Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary same project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
Matthew G. Vander Heiden, M.D., Ph.D. [Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115]
MLPCN Grant: 1 R03 MH085679-01

NCGC Assay Overview:
Human pyruvate kinase muscle 2 (hPK-M2) enzyme was supplied as a highly purified (>95% pure) preparation from Harvard Medical School and assayed for its ability to generate ATP from ADP using phosphoenolpyruvate (PEP) as a substrate. ATP generation was detected in a coupled reaction by luciferase-mediated luminescence, an ATP-dependent process. Pyruvate kinase substrates, PEP and ADP, were present in the assay at Km and approximately 10-fold below Km respectively. The enzyme was assayed at an intermediate level of activity to screen for both inhibitors and activators.
Protocol
NCGC Assay Protocol Summary:
Three uL of substrate mix (at r.t.) in assay buffer (50 mM Imidazole pH7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween 20, 0.05% BSA) was dispensed into white solid bottom 1,536 well microtiter plates so that the final concentrations of substrates in the assay were 0.1 mM ADP and 0.5 mM PEP. 23 nL of compound were delivered by a pin tool and 1 uL of enzyme mix in assay buffer (4 degree Celsius) was added. Plates were incubated at room temperature for 1 hour. Two uL of detection mix (Kinase-Glo, Promega; at 4 degree Celsius protected from light) was added and luminescence read by a ViewLux (Perkin Elmer) at 5 second exposure/plate. Data were normalized to the uninhibited and AC100 inhibition (no enzyme). To monitor activation, the first column contained a titration of the activator, NCGC00031955-01 (16-point 1:2 dilutions in duplicate starting at 57 uM) and the first 16 rows of column 4 contained 57 uM of NCGC00031955-01.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007358821 uM (0.000735882μM**)% Activity at given concentration.Float%
15Activity at 0.00368 uM (0.00367905μM**)% Activity at given concentration.Float%
16Activity at 0.018 uM (0.0183957μM**)% Activity at given concentration.Float%
17Activity at 0.092 uM (0.0919691μM**)% Activity at given concentration.Float%
18Activity at 0.460 uM (0.459827μM**)% Activity at given concentration.Float%
19Activity at 1.030 uM (1.03μM**)% Activity at given concentration.Float%
20Activity at 2.299 uM (2.29904μM**)% Activity at given concentration.Float%
21Activity at 5.140 uM (5.14μM**)% Activity at given concentration.Float%
22Activity at 11.50 uM (11.496μM**)% Activity at given concentration.Float%
23Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
24Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
25Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH085679-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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