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BioAssay: AID 623995

qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Cherrypicks in 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. ..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: 2012-04-04

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 66
Depositor Specified Assays
AIDNameTypeComment
602183qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): SummarysummarySummary AID
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 a-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
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
References:

1. Ohgaki H: Epidemiology of brain tumors. Methods Mol Biol 2009, 472:323-342.
2. Dubbink HJ, Taal W, van Marion R, Kros JM, van Heuvel I, Bromberg JE, Zonnenberg BA, Zonnenberg CB, Postma TJ, Gijtenbeek JM et al: IDH1 mutations in low-grade astrocytomas predict survival but not response to temozolomide. Neurology 2009, 73(21):1792-1795.
3. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009, 360(8):765-773.
4. Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, Koboldt DC, Fulton RS, Delehaunty KD, McGrath SD et al: Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009, 361(11):1058-1066.
5. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC et al: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009, 462(7274):739-744.

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.
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)
Classification
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