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BioAssay: AID 602379

qHTS for Activators of Human Muscle Isoform Pyruvate Kinase: Extended Characterization against Liver Pyruvate Kinase

Human pyruvate kinase liver (hPK-L) 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. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Inactive(6)
 
 
AID: 602379
Data Source: NCGC (PYHK300)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-13

Data Table ( Complete ):           View All Data
Target
Sequence: pyruvate kinase isozymes R/L isoform 2 [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
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
602377qHTS for Activators of Human Muscle Isoform Pyruvate Kinase: Extended Characterization Using A Lactate Dehydrogenase (LDH) AssayConfirmatory 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 liver (hPK-L) 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.


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 (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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
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 0.0000034256 uM (3.42555e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000068511 uM (6.85111e-06μM**)% Activity at given concentration.Float%
17Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
20Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
21Activity at 0.0002966441 uM (0.000296644μM**)% Activity at given concentration.Float%
22Activity at 0.0004388382 uM (0.000438838μM**)% Activity at given concentration.Float%
23Activity at 0.0009492488 uM (0.000949249μM**)% Activity at given concentration.Float%
24Activity at 0.00260 uM (0.00260209μM**)% Activity at given concentration.Float%
25Activity at 0.00352 uM (0.00351983μM**)% Activity at given concentration.Float%
26Activity at 0.00831 uM (0.00831131μM**)% Activity at given concentration.Float%
27Activity at 0.015 uM (0.0145137μM**)% Activity at given concentration.Float%
28Activity at 0.027 uM (0.0267091μM**)% Activity at given concentration.Float%
29Activity at 0.073 uM (0.0729086μM**)% Activity at given concentration.Float%
30Activity at 0.112 uM (0.112214μM**)% Activity at given concentration.Float%
31Activity at 0.233 uM (0.233292μM**)% Activity at given concentration.Float%
32Activity at 0.459 uM (0.459231μM**)% Activity at given concentration.Float%
33Activity at 0.749 uM (0.748803μM**)% Activity at given concentration.Float%
34Activity at 2.043 uM (2.04284μM**)% Activity at given concentration.Float%
35Activity at 3.695 uM (3.69525μM**)% Activity at given concentration.Float%
36Activity at 6.566 uM (6.56563μM**)% Activity at given concentration.Float%
37Activity at 17.92 uM (17.9193μM**)% Activity at given concentration.Float%
38Activity at 28.69 uM (28.6937μM**)% Activity at given concentration.Float%
39Activity at 57.33 uM (57.3344μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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