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BioAssay: AID 1542

Secondary assay for Activators of Human Pyruvate Kinase M1 isoform

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)
 
 
Inactive(45)
 
 
Inconclusive(79)
 
 
 Tested Substances
 Tested Substances
All(131)
 
 
Inactive(45)
 
 
Inconclusive(86)
 
 
 Related BioAssays
 Related BioAssays
AID: 1542
Data Source: NCGC (PYKHM1Confa)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-03-10
Modify Date: 2010-01-19

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
954qHTS Assay for Activators of Human Muscle Pyruvate KinaseConfirmatorydepositor-specified cross reference: Counter assay for activators of Human Pyruvate Kinase M2 isoform
Description:
Confirmatory assay
NIH Chemical Genomics Center [NCGC]
Matthew G. Vander Heiden, M.D., Ph.D. [Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115]

NCGC Assay Overview:

Human pyruvate kinase liver (hPK-M1)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-R 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. 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.
Protocol
NCGC Assay Protocol Summary:
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-M1 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 controls.
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
5Rate-Curve_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 signficant, but below 80% of control.String
6Rate-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Rate-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Rate-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Rate-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Rate-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Rate-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Rate-Excluded_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
13Rate-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Rate-Activity at 0.0000034256 uM (3.42555e-06μM**)% Activity at given concentration.Float%
15Rate-Activity at 0.0000068511 uM (6.85111e-06μM**)% Activity at given concentration.Float%
16Rate-Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
17Rate-Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
18Rate-Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
19Rate-Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
20Rate-Activity at 0.0002192355 uM (0.000219235μM**)% Activity at given concentration.Float%
21Rate-Activity at 0.0004384709 uM (0.000438471μM**)% Activity at given concentration.Float%
22Rate-Activity at 0.0008769419 uM (0.000876942μM**)% Activity at given concentration.Float%
23Rate-Activity at 0.00175 uM (0.00175388μM**)% Activity at given concentration.Float%
24Rate-Activity at 0.00351 uM (0.00350777μM**)% Activity at given concentration.Float%
25Rate-Activity at 0.00702 uM (0.00701554μM**)% Activity at given concentration.Float%
26Rate-Activity at 0.014 uM (0.0140311μM**)% Activity at given concentration.Float%
27Rate-Activity at 0.028 uM (0.0280621μM**)% Activity at given concentration.Float%
28Rate-Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
29Rate-Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
30Rate-Activity at 0.224 uM (0.224497μM**)% Activity at given concentration.Float%
31Rate-Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
32Rate-Activity at 0.898 uM (0.897989μM**)% Activity at given concentration.Float%
33Rate-Activity at 1.796 uM (1.79598μM**)% Activity at given concentration.Float%
34Rate-Activity at 3.592 uM (3.59195μM**)% Activity at given concentration.Float%
35Rate-Activity at 7.184 uM (7.18391μM**)% Activity at given concentration.Float%
36Rate-Activity at 14.37 uM (14.3678μM**)% Activity at given concentration.Float%
37Rate-Activity at 28.74 uM (28.7356μM**)% Activity at given concentration.Float%
38Rate-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
39Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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