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BioAssay: AID 602179

qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): qHTS

Unbiased genomic sequencing for 22 glioma genomes found recurrent mutation of isocitrate dehydrogenase 1 (IDH1) on chromosome 2q33-a gene encoding the cytosolic isoform of IIDH1associated with the tricarboxylic acid cycle (TCA) that catalyzes the oxidative decarboxylation of isocitrate, yielding alpha-ketoglutarate and CO2 via NADP+ to NADPH conversion. Subsequent studies confirmed the recurrent more ..
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 Tested Compounds
 Tested Compounds
All(387596)
 
 
Active(365)
 
 
Inactive(385475)
 
 
Inconclusive(1777)
 
 
 Tested Substances
 Tested Substances
All(390606)
 
 
Active(365)
 
 
Inactive(388463)
 
 
Inconclusive(1778)
 
 
AID: 602179
Data Source: NCGC (IDH001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-01-11
Modify Date: 2012-04-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 365
Related Experiments
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AIDNameTypeComment
602183qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): SummarySummarydepositor-specified cross reference
623995qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Cherrypicks in WT IDH1Confirmatorydepositor-specified cross reference
623980qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Cherrypicks Autofluorescence InterferenceConfirmatorysame project related to Summary assay
624002qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Confirmation of CherrypicksConfirmatorysame project related to Summary assay
624019qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): 2HG production assay for probe SARConfirmatorysame project related to Summary assay
624021qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): WT assay for probe SARConfirmatorysame project related to Summary assay
624023qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): confirmation assay for probe SARConfirmatorysame project related to Summary assay
624059qHTS for Inhibitors of Mutant Isocitrate Dehydrogenase 1 (IDH1): U87 Cytotoxicity Assay for Probe SARConfirmatorysame project related to Summary assay
624462qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Aqueous Kinetic SolubilityOthersame project related to Summary assay
Description:
Unbiased genomic sequencing for 22 glioma genomes found recurrent mutation of isocitrate dehydrogenase 1 (IDH1) on chromosome 2q33-a gene encoding the cytosolic isoform of IIDH1associated with the tricarboxylic acid cycle (TCA) that catalyzes the oxidative decarboxylation of isocitrate, yielding alpha-ketoglutarate and CO2 via NADP+ to NADPH conversion. Subsequent studies confirmed the recurrent IDH mutations in up to 70% of secondary gliomas and in 10% of AML cases. We have found that the somatic mutation of cancer-associated IDH1 is a point mutation resulting in various amino-acid substituents at Arginine132 (IDH1 R132)-a key residue found in the enzyme's active site that when mutated, results in the loss-of-function in metabolizing isocitrate but confers a gain-of-function to produce the oncometabolite 2-hydroxyglutarate (2HG). This in effect defines IDH1 as an oncogene, and provides an extraordinary opportunity to discover chemical probes against mutant IDH1 that may translate into much needed new therapies for glioma and AML patients.

Hence, a HTS-compatible fluorescent, enzymatic assay to identify inhibitors of IDH1 to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR).

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

MLPCN Grant: DA032129
Assay Submitter (PI): Lenny Dang, Agios Pharmaceuticals
Protocol
IDH1-R132H (0.5 ug/mL) was dispensed into black, solid-bottom 1536-well plates at 3 uL/well in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 4 mM beta-ME, and 0.05% protease-free BSA. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. A pre-read was run on the Viewlux to identify fluorescent compounds (540 nm excitation filter, 598 nm emission filter, bodipy mirror and 2 second exposure). After 30 minutes of incubation with compound, 3 uL of substrate mix were dispensed (0.016 mM NADPH, 2 mM alpha-KG, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, and 0.05% protease-free BSA). Plates were spun for 1 min at 1000xg and incubated at room temperature for 80 min. Then 1.5 uL of each detection reagent component for the coupling system were added (0.12 mg/mL diaphorase and 0.072 mM resazurin, each in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, and 0.05% protease-free BSA ) The protein, substrate and two detection components were maintained at 4 oC throughout the screen. Additionally, the substrate and resazurin reagents were protected from light. The plates were measured on an Envision plate reader for fluorescence signal using a 540 nm excitation filter and 590 nm emission filter with 10 flashes. 1x and 0x (no-enzyme) IDH1-R132H enzyme control were used to normalize %Activity of identified inhibitors; 0x enzyme values corresponded to 100% Activity (full inhibition), while 1x IDH1-R132H enzyme values were used to normalize 0%Activity (no inhibition).
Concentration-response curves were fitted to the signals arising from the resulting fluorescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent increases in fluorescence, concordant with their blocking the consumption of the NADPH. Inactive compounds showed no effect on the fluorescence signal.
Keywords: isocitrate dehydrogenase, IDH1, IDH1-R132H, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
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 inhibitors are ranked higher than compounds that showed apparent activation.
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 0.00245 uM (0.00245μM**)% Activity at given concentration.Float%
15Activity at 0.012 uM (0.0123μM**)% Activity at given concentration.Float%
16Activity at 0.307 uM (0.307μM**)% Activity at given concentration.Float%
17Activity at 1.530 uM (1.53μM**)% Activity at given concentration.Float%
18Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
19Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
20Activity at 76.60 uM (76.6μM**)% Activity at given concentration.Float%
21Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA032129

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