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BioAssay: AID 623995

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

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(753)
 
 
Active(66)
 
 
Inactive(604)
 
 
Inconclusive(83)
 
 
 Tested Substances
 Tested Substances
All(753)
 
 
Active(66)
 
 
Inactive(604)
 
 
Inconclusive(83)
 
 
AID: 623995
Data Source: NCGC (IDH003)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-03
Modify Date: 2014-04-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 66
Related Experiments
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AIDNameTypeComment
602179qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): qHTSConfirmatorydepositor-specified cross reference
602183qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): SummarySummarydepositor-specified cross reference: Summary AID
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.

QHTS-compatible fluorescent, enzymatic assay was developed to identify inhibitors of the IDH1 mutant (AID 602179). To identify selectivity of the identified inhibitors, a secondary enzymatic assay against the WT IDH1 was also developed. Inhibition of the WT IDH1 leads to a decrease in the amount of NADPH relative to a DMSO control and a concomitant decrease in the fluorescence of the diaphorase/resazurin enzyme product resorufin at 590 nm. Compounds were screened as a concentration-titration series that ranged from 115 uM to 0.20 uM.

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

MLSCN Grant: R03 DA032129
PI Name: Dr. Lenny Dang
Protocol
Enzyme buffer was dispensed into black, solid 1536-well plates at 3 microL/well in 20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.05% BSA, 2 mM b-ME and 0.65 nM WT IDH1. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. The substrate and detection buffer (1 uL; 20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.05% BSA, 0.06 mM NADP+, 0.53 uM diaphorase, 0.012 mM resazurin and 0.08 mM isocitrate) was added to start the enzymatic reaction. The fluorescence intensity was monitored in kinetic mode on a ViewLux plate reader at 598 nm for 10 minutes (Ex 525, EM 598, bodipy mirror, 0.5 sec exposure). The % Activity was determined from the corrected fluorescence values. As no specific WT IDH1 inhibitors have been identified in the literature, 1x (0.65 nM) and 0x WT IDH1 enzyme controls (untreated) were included to normalize % Activity of identified inhibitors; 0x enzyme values corresponded to 100% Activity (full inhibition), while 1x WT IDH1 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-response 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 decrease in fluorescence production over time, concordant with a decrease in WT IDH1 activity and less product NADPH production. Inactive compounds showed no effect on fluorescence signal increase relative to the DMSO control.
Keywords: Isocitrate dehydrogenase, IDH1, IDH1 R132H, 2-HG, 2-hydroxyglutarate, AML, glioma, 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
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 0.224 uM (0.224μM**)% Activity at given concentration.Float%
16Activity at 0.449 uM (0.449μM**)% Activity at given concentration.Float%
17Activity at 0.898 uM (0.898μM**)% Activity at given concentration.Float%
18Activity at 1.800 uM (1.8μM**)% Activity at given concentration.Float%
19Activity at 3.590 uM (3.59μM**)% Activity at given concentration.Float%
20Activity at 7.180 uM (7.18μM**)% Activity at given concentration.Float%
21Activity at 14.40 uM (14.4μM**)% Activity at given concentration.Float%
22Activity at 28.70 uM (28.7μM**)% Activity at given concentration.Float%
23Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
24Activity at 115.0 uM (115μM**)% Activity at given concentration.Float%
25Compound 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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