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BioAssay: AID 623980

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

Malignant glioblastomas (WHO grade IV), including primary and secondary glioblastomas, are among the most lethal with a median survival of one year, and unfortunately they are also the most prevalent type of brain tumors [1]. By and large the standard of care of gliomas remains the use of the oral alkylating agent temozolomide and radiotherapy following surgical tumor resection [2]. There is an urgent unmet medical need for novel therapeutics for gliomas. ..more
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 Tested Compounds
 Tested Compounds
All(753)
 
 
Inactive(753)
 
 
 Tested Substances
 Tested Substances
All(753)
 
 
Inactive(753)
 
 
AID: 623980
Data Source: NCGC (IDH004)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-03
Modify Date: 2012-04-04

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeComment
602183qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): SummarySummarydepositor-specified cross reference: Summary AID
602179qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): qHTSConfirmatorysame project related to Summary assay
623995qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Cherrypicks in WT IDH1Confirmatorysame 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:
Readout Interference Assay for Inhibitors of IDH1 R132H and WT IDH1

Malignant glioblastomas (WHO grade IV), including primary and secondary glioblastomas, are among the most lethal with a median survival of one year, and unfortunately they are also the most prevalent type of brain tumors [1]. By and large the standard of care of gliomas remains the use of the oral alkylating agent temozolomide and radiotherapy following surgical tumor resection [2]. There is an urgent unmet medical need for novel therapeutics for gliomas.

IDH mutations occur in up to 70% of grade II-IV secondary glioblastomas, and IDH1 R132H is the most prevalent mutation [3]. Interestingly, IDH mutations are also present in ~10% of AML (acute myeloid leukemia) patients [4]. Wild-type (WT) IDH1 catalyzes the conversion of isocitrate to a-ketoglutarate with the concomitant reduction of NADP+ to NADPH. In contrast, IDH1 R132H catalyzes the conversion of -ketoglutarate to 2-hydroxyglutarate (2-HG) with the concomitant oxidation of NADPH to NADP+ [5]. Recently, a second significant advancement for the field was the finding that IDH is an oncogene, and that its metabolic product 2-HG may contribute to the pathogenesis of IDH-mutated cancers. Indeed, unbiased metabolite profiling of a U87MG stable cell line engineered to express IDH1 R132H mutant protein demonstrated that mutated IDH1 confers a gain-of-function to produce the onco-metabolite 2-HG, and in effect classifying IDH1 as an oncogene [5]. These studies demonstrated the power of utilizing 2-HG as a biomarker for IDH-mutated cancers, as well as adding to the wealth of data linking the metabolite 2-HG to cancer. Although this link to cancer is tantalizing, the direct evidence of how 2-HG drives pathogenesis of gliomas and AML remains a subject of further research.

The goal of this detection interference assay is to identify whether the potential small-molecule inhibitors of IDH1 R132H or WT IDH1 confound the fluorescence readout by interfering with the detection reagent. Inhibition of the detection reagent would lead to a decrease in observed fluorescence of the product resorufin relative to the DMSO control. Compounds were screened as concentration-titration series that ranged from 76 uM to 0.15 uM.

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

MLSCN Grant: R03 DA032129
PI Name: Dr. Lenny Dang
Protocol
Detection reagent buffer was dispensed into black, solid 1536-well plates at 9 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 beta-ME, 0.008 mM NADPH, 1 mM alpha-ketoglutarate, and 0.008 mM resorufin. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. The fluorescence intensity was monitored on an Envision plate reader at 590 nm (Ex 540, Em 590, bodipy mirror, 10 flashes). The %Activity was determined from the corrected fluorescence values. Resorufin at 0x was included to normalize %Activity of identified inhibitors; 0x resorufin values corresponded to 100%Activity (full inhibition), while 1x resorufin 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. 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 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
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 0.400 uM (0.4μM**)% Activity at given concentration.Float%
16Activity at 0.799 uM (0.799μM**)% Activity at given concentration.Float%
17Activity at 1.600 uM (1.6μM**)% Activity at given concentration.Float%
18Activity at 3.200 uM (3.2μM**)% Activity at given concentration.Float%
19Activity at 6.390 uM (6.39μM**)% Activity at given concentration.Float%
20Activity at 12.80 uM (12.8μM**)% Activity at given concentration.Float%
21Activity at 25.60 uM (25.6μM**)% Activity at given concentration.Float%
22Activity at 51.20 uM (51.2μM**)% Activity at given concentration.Float%
23Compound 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 All Data
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