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BioAssay: AID 2534

Secondary Concentration-Response Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SAR

Wael M. Rabeh, Lyudmila Nedyalkova and Hee-Wan Park [Structural Genomics Consortium, 100 College St. Toronto, Ontario, Canada] ..more
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 Tested Compounds
 Tested Compounds
All(94)
 
 
Inactive(91)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(98)
 
 
Inactive(95)
 
 
Inconclusive(3)
 
 
AID: 2534
Data Source: NCGC (PYKHRh)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-12
Hold-until Date: 2010-06-15
Modify Date: 2010-10-19

Data Table ( Complete ):           View All Data
Target
Sequence: pyruvate kinase isozymes R/L isoform 1 [Homo sapiens]
Description ..   
Protein Family: Pyruvate_Kinase

Gene:PKLR     Related Protein 3D Structures     More BioActivity Data..
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
1631qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate KinaseConfirmatory depositor-specified cross reference
2095qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary6 depositor-specified cross reference
2266qHTS Assay for Inhibitors of Leishmania Mexicana Pyruvate Kinase (LmPK): SummarySummary 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
1540Secondary assay for Activators of Human Pyruvate Kinase M2 isoformConfirmatory same project related to Summary assay
1751Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate KinaseConfirmatory same project related to Summary assay
2533Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
2535Secondary Concentration-Response Assay for Activators of Human Liver Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
2536Secondary Concentration-Response Assay for Activators of Human Muscle isoform 1 Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
2562Secondary LDH Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
602359Extended Characterization of Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary same project related to Summary assay
945qHTS Validation Assay for Inhibitors of Leishmania Mexicana Pyruvate KinaseConfirmatory same project related to Summary assay
959qHTS Validation Assay for Activators of Leishmania Mexicana Pyruvate KinaseConfirmatory same project related to Summary assay
1721qHTS Assay for Inhibitors of Leishmania Mexicana Pyruvate Kinase (LmPK)Confirmatory same project related to Summary assay
1722qHTS Assay for Activators of Leishmania Mexicana Pyruvate Kinase (LmPK)Confirmatory same project related to Summary assay
2530Secondary Assay for Luciferase (Kinase-Glo TM) Inhibition CounterscreenConfirmatory same project related to Summary assay
2533Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
2559Confirmation Assay for Inhibitors of Leishmania Mexicana Pyruvate Kinase (LmPK)Confirmatory same project related to Summary assay
2561Confirmation Assay for Inhibitors of Leishmania Mexicana Pyruvate Kinase (LmPK): for Probe SARConfirmatory same project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
Wael M. Rabeh, Lyudmila Nedyalkova and Hee-Wan Park [Structural Genomics Consortium, 100 College St. Toronto, Ontario, Canada]

NCGC Assay Overview:

Human pyruvate kinase reticulocyte (hPK-R) enzyme was supplied as a highly purified (>95% pure) preparation from Structural Genomics Consortium in Toronto (Ontario, Canada) 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 for R-isoform

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 (final concentration of 0.1 nM) 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 1 second exposure/plate. Data were normalized to the uninhibited (row 31). Additionally, a no enzyme and 5-fold enzyme control was dispensed in row 32, split equally between the two conditions.
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". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
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.
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 0.0003244270 uM (0.000324427μM**)% Activity at given concentration.Float%
15Activity at 0.0009732809 uM (0.000973281μM**)% Activity at given concentration.Float%
16Activity at 0.00292 uM (0.00291984μM**)% Activity at given concentration.Float%
17Activity at 0.00876 uM (0.00875953μM**)% Activity at given concentration.Float%
18Activity at 0.026 uM (0.0262786μM**)% Activity at given concentration.Float%
19Activity at 0.079 uM (0.0788358μM**)% Activity at given concentration.Float%
20Activity at 0.237 uM (0.236507μM**)% Activity at given concentration.Float%
21Activity at 0.710 uM (0.709522μM**)% Activity at given concentration.Float%
22Activity at 2.129 uM (2.12857μM**)% Activity at given concentration.Float%
23Activity at 6.386 uM (6.3857μM**)% Activity at given concentration.Float%
24Activity at 19.16 uM (19.1571μM**)% Activity at given concentration.Float%
25Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
26Compound 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 All Data
Classification
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