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BioAssay: AID 1751

Confirmation Concentration-Response 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(120)
 
 
Active(90)
 
 
Inactive(23)
 
 
Inconclusive(8)
 
 
 Tested Substances
 Tested Substances
All(125)
 
 
Active(94)
 
 
Inactive(23)
 
 
Inconclusive(8)
 
 
AID: 1751
Data Source: NCGC (PYKH323a)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-05-13
Modify Date: 2010-01-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 90
Related Experiments
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AIDNameTypeProbeComment
954qHTS Assay for Activators of Human Muscle Pyruvate KinaseConfirmatory depositor-specified cross reference
1631qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate KinaseConfirmatory depositor-specified cross reference
1780Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 1 Pyruvate KinaseConfirmatory depositor-specified cross reference
1781Confirmation Concentration-Response Assay for Activators of Human Liver Pyruvate KinaseConfirmatory depositor-specified cross reference
1782Confirmation Concentration-Response Assay for Activators of Human Reticulocyte 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
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
2533Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: for Probe SARConfirmatory same project related to Summary assay
2534Secondary Concentration-Response Assay for Activators of Human Reticulocyte 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
2576Secondary LDH Assay for Activators of Human Pyruvate Kinase M2 isoform: 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
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 (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 (column 3) and AC100 inhibition (no enzyme in column 2). To monitor activation, the first column contained a titration of the activator, NCGC00031955-03 (16-point 1:2 dilutions in duplicate starting at 57 uM) and the first 16 rows of column 4 contained 57 uM of NCGC00031955-03.
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 0.0000034256 uM (3.42555e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000068511 uM (6.85111e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
20Activity at 0.0002192355 uM (0.000219235μM**)% Activity at given concentration.Float%
21Activity at 0.0003244270 uM (0.000324427μM**)% Activity at given concentration.Float%
22Activity at 0.0004384709 uM (0.000438471μM**)% Activity at given concentration.Float%
23Activity at 0.0008769419 uM (0.000876942μM**)% Activity at given concentration.Float%
24Activity at 0.0009732809 uM (0.000973281μM**)% Activity at given concentration.Float%
25Activity at 0.00175 uM (0.00175388μM**)% Activity at given concentration.Float%
26Activity at 0.00292 uM (0.00292μM**)% Activity at given concentration.Float%
27Activity at 0.00351 uM (0.00350777μM**)% Activity at given concentration.Float%
28Activity at 0.00702 uM (0.00701554μM**)% Activity at given concentration.Float%
29Activity at 0.00876 uM (0.00876μM**)% Activity at given concentration.Float%
30Activity at 0.014 uM (0.0140311μM**)% Activity at given concentration.Float%
31Activity at 0.026 uM (0.026μM**)% Activity at given concentration.Float%
32Activity at 0.028 uM (0.0280621μM**)% Activity at given concentration.Float%
33Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
34Activity at 0.079 uM (0.079μM**)% Activity at given concentration.Float%
35Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
36Activity at 0.224 uM (0.224497μM**)% Activity at given concentration.Float%
37Activity at 0.237 uM (0.237μM**)% Activity at given concentration.Float%
38Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
39Activity at 0.710 uM (0.71μM**)% Activity at given concentration.Float%
40Activity at 0.898 uM (0.897989μM**)% Activity at given concentration.Float%
41Activity at 1.796 uM (1.79598μM**)% Activity at given concentration.Float%
42Activity at 2.129 uM (2.129μM**)% Activity at given concentration.Float%
43Activity at 3.592 uM (3.59195μM**)% Activity at given concentration.Float%
44Activity at 6.386 uM (6.386μM**)% Activity at given concentration.Float%
45Activity at 7.184 uM (7.18391μM**)% Activity at given concentration.Float%
46Activity at 14.37 uM (14.3678μM**)% Activity at given concentration.Float%
47Activity at 19.16 uM (19.16μM**)% Activity at given concentration.Float%
48Activity at 28.74 uM (28.7356μM**)% Activity at given concentration.Float%
49Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
50Compound 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
Classification
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