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BioAssay: AID 602377

qHTS for Activators of Human Muscle Isoform Pyruvate Kinase: Extended Characterization Using A Lactate Dehydrogenase (LDH) Assay

Human pyruvate kinase muscle 2 (hPK-M2)enzyme was supplied as a highly purified (>95% pure) preparation from Harvard Medical School and a secondary assay was used to evaluate compounds. This assay couples the formation of pyruvate from hPK-M2 using lactate dehydrogenase (LDH) and NADH. The depletion of NADH is followed in a fluorescent-based kinetic assay that follows the formation of pyruvate to more ..
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(6)
 
 
AID: 602377
Data Source: NCGC (PYKH301)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 6
Related Experiments
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AIDNameTypeProbeComment
602359Extended Characterization of Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary depositor-specified cross reference: Extended Characterization Summary AID
2095qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: SummarySummary6 same project related to Summary assay
602379qHTS for Activators of Human Muscle Isoform Pyruvate Kinase: Extended Characterization against Liver Pyruvate KinaseConfirmatory same project related to Summary assay
602381qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase (PK): Extended Characterization using Reticulocyte PKConfirmatory same project related to Summary assay
602383qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase: Muscle Isoform 1 AssayConfirmatory same project related to Summary assay
602384qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase: Extended CharacterizationConfirmatory same project related to Summary assay
624387qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase: Extended Characterization using M1 for Probe 2Confirmatory same project related to Summary assay
624388qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase (PK): Extended Characterization using Reticulocyte PK Probe 2Confirmatory same project related to Summary assay
624389qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase: Extended Characterization Using A Lactate Dehydrogenase (LDH) Assay Probe 2Confirmatory same project related to Summary assay
624390qHTS for Activators of Human Muscle Isoform Pyruvate Kinase: Extended Characterization against Liver Pyruvate Kinase Probe 2Confirmatory same project related to Summary assay
624391qHTS for Activators of Human Muscle Isoform 2 Pyruvate Kinase: Extended Characterization for Probe 2Confirmatory same project related to Summary assay
Description:
Human pyruvate kinase muscle 2 (hPK-M2)enzyme was supplied as a highly purified (>95% pure) preparation from Harvard Medical School and a secondary assay was used to evaluate compounds. This assay couples the formation of pyruvate from hPK-M2 using lactate dehydrogenase (LDH) and NADH. The depletion of NADH is followed in a fluorescent-based kinetic assay that follows the formation of pyruvate to lactate, as catalyzed LDH (described by Hannaert et al., 2002)1 Pyruvate kinase substrates, PEP and ADP, were present in the assay at 0.1 mM and 4 mM respectively. In this kinetic assay initial rates were determined by following the fluorescence of NADH depletion.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH085679
Assay Submitter (PI): Matthew G. Vander Heiden, Harvard Medical School
Protocol
Three uL of substrate mix (final concentration, 50 mM Tris-Cl pH 8.0, 200 mM KCl, 15 mM MgCl2, 0.1 mM PEP, 4.0 mM ADP, and 0.2 mM NADH) was dispensed into black-solid 1536 well plates. 23 nL of compounds were delivered by a pin tool and 1 uL of enzyme mix (final concentrations, 10 nM hPK-M2 and 1 uM of LDH) was added. Plates were immediately placed in ViewLux (Perkin Elmer) and NADH fluorescence was determined at 30 second exposure intervals for between 3 and 6 minutes. Data were normalized to the uninhibited and EC100 activation using known activators such as fructose-bis-phosphate.
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.
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0003284687 uM (0.000328469μM**)% Activity at given concentration.Float%
16Activity at 0.0009854062 uM (0.000985406μM**)% Activity at given concentration.Float%
17Activity at 0.00292 uM (0.00291984μM**)% Activity at given concentration.Float%
18Activity at 0.00410 uM (0.00409912μM**)% Activity at given concentration.Float%
19Activity at 0.00876 uM (0.00875953μM**)% Activity at given concentration.Float%
20Activity at 0.012 uM (0.0122974μM**)% Activity at given concentration.Float%
21Activity at 0.026 uM (0.0262916μM**)% Activity at given concentration.Float%
22Activity at 0.037 uM (0.0368921μM**)% Activity at given concentration.Float%
23Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
24Activity at 0.080 uM (0.0800154μM**)% Activity at given concentration.Float%
25Activity at 0.239 uM (0.239342μM**)% Activity at given concentration.Float%
26Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
27Activity at 0.711 uM (0.71078μM**)% Activity at given concentration.Float%
28Activity at 0.996 uM (0.996087μM**)% Activity at given concentration.Float%
29Activity at 2.126 uM (2.12585μM**)% Activity at given concentration.Float%
30Activity at 3.081 uM (3.08132μM**)% Activity at given concentration.Float%
31Activity at 6.386 uM (6.3857μM**)% Activity at given concentration.Float%
32Activity at 8.640 uM (8.63993μM**)% Activity at given concentration.Float%
33Activity at 14.37 uM (14.3678μM**)% Activity at given concentration.Float%
34Activity at 19.45 uM (19.4511μM**)% Activity at given concentration.Float%
35Activity at 58.11 uM (58.1075μM**)% Activity at given concentration.Float%
36Activity at 82.76 uM (82.7586μM**)% Activity at given concentration.Float%
37Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH085679

Data Table (Concise)
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