Matthew G. Vander Heiden, M.D., Ph.D. [Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115] ..more
Target
| Sequence: pyruvate kinase isozymes R/L isoform 2 [Homo sapiens] Description ..   Protein Family: Pyruvate_Kinase Gene:PKLR Related Protein 3D Structures |
Tested Compounds:
Depositor Specified Assays
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| AID | Name | Type | Probe | Comment |
|---|---|---|---|---|
| 1540 | Secondary assay for Activators of Human Pyruvate Kinase M2 isoform | confirmatory | ||
| 1631 | qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase | confirmatory | ||
| 1751 | Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase | confirmatory | ||
| 2095 | qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: Summary | summary | 6 | |
| 2533 | Confirmation Concentration-Response Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: for Probe SAR | confirmatory | ||
| 2534 | Secondary Concentration-Response Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SAR | confirmatory | ||
| 2535 | Secondary Concentration-Response Assay for Activators of Human Liver Pyruvate Kinase: for Probe SAR | confirmatory | ||
| 2536 | Secondary Concentration-Response Assay for Activators of Human Muscle isoform 1 Pyruvate Kinase: for Probe SAR | confirmatory | ||
| 2562 | Secondary LDH Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SAR | confirmatory | ||
| 2576 | Secondary LDH Assay for Activators of Human Pyruvate Kinase M2 isoform: for Probe SAR | confirmatory | ||
| 2653 | Secondary Assay for Activators of Human Pyruvate Kinase M2 isoform - Cell Titer Glo Cytotoxicity for Probe SAR | confirmatory |
Description:
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-L)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.
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-L)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-L 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.
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-L 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.
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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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | 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. | | Float | μM | |
| 3 | Efficacy | Maximal 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 | % | |
| 4 | Analysis Comment | Annotation/notes on a particular compound's data or its analysis. | String | |||
| 5 | Rate-Curve_Description | A 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 | |||
| 6 | Rate-Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 7 | Rate-Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 8 | Rate-Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 9 | Rate-Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 10 | Rate-Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 11 | Rate-Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 12 | Rate-Excluded_Points | Which 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 | |||
| 13 | Rate-Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 14 | Rate-Activity at 0.0003224475 uM (0.000322447μM**) | % Activity at given concentration. | | Float | % | |
| 15 | Rate-Activity at 0.0004554583 uM (0.000455458μM**) | % Activity at given concentration. | | Float | % | |
| 16 | Rate-Activity at 0.0009673425 uM (0.000967342μM**) | % Activity at given concentration. | | Float | % | |
| 17 | Rate-Activity at 0.00137 uM (0.00136637μM**) | % Activity at given concentration. | | Float | % | |
| 18 | Rate-Activity at 0.00290 uM (0.00290203μM**) | % Activity at given concentration. | | Float | % | |
| 19 | Rate-Activity at 0.00410 uM (0.00409912μM**) | % Activity at given concentration. | | Float | % | |
| 20 | Rate-Activity at 0.00871 uM (0.00870608μM**) | % Activity at given concentration. | | Float | % | |
| 21 | Rate-Activity at 0.012 uM (0.0122974μM**) | % Activity at given concentration. | | Float | % | |
| 22 | Rate-Activity at 0.022 uM (0.0215858μM**) | % Activity at given concentration. | | Float | % | |
| 23 | Rate-Activity at 0.027 uM (0.0266725μM**) | % Activity at given concentration. | | Float | % | |
| 24 | Rate-Activity at 0.065 uM (0.0647574μM**) | % Activity at given concentration. | | Float | % | |
| 25 | Rate-Activity at 0.080 uM (0.0800175μM**) | % Activity at given concentration. | | Float | % | |
| 26 | Rate-Activity at 0.235 uM (0.235064μM**) | % Activity at given concentration. | | Float | % | |
| 27 | Rate-Activity at 0.332 uM (0.332029μM**) | % Activity at given concentration. | | Float | % | |
| 28 | Rate-Activity at 0.705 uM (0.705193μM**) | % Activity at given concentration. | | Float | % | |
| 29 | Rate-Activity at 0.996 uM (0.996087μM**) | % Activity at given concentration. | | Float | % | |
| 30 | Rate-Activity at 2.116 uM (2.11558μM**) | % Activity at given concentration. | | Float | % | |
| 31 | Rate-Activity at 2.988 uM (2.98826μM**) | % Activity at given concentration. | | Float | % | |
| 32 | Rate-Activity at 6.347 uM (6.34673μM**) | % Activity at given concentration. | | Float | % | |
| 33 | Rate-Activity at 8.965 uM (8.96478μM**) | % Activity at given concentration. | | Float | % | |
| 34 | Rate-Activity at 15.74 uM (15.736μM**) | % Activity at given concentration. | | Float | % | |
| 35 | Rate-Activity at 19.44 uM (19.4443μM**) | % Activity at given concentration. | | Float | % | |
| 36 | Rate-Activity at 47.21 uM (47.2081μM**) | % Activity at given concentration. | | Float | % | |
| 37 | Rate-Activity at 58.24 uM (58.243μM**) | % Activity at given concentration. | | Float | % | |
| 38 | Rate-Activity at 82.76 uM (82.7586μM**) | % Activity at given concentration. | | Float | % | |
| 39 | Compound QC | NCGC 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)
Classification
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