qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): WT assay for probe SAR
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 . By and large the standard of care of gliomas remains the use of the oral alkylating agent temozolomide and radiotherapy following surgical tumor resection . There is an urgent unmet medical need for novel therapeutics for gliomas. ..more
Depositor Specified Assays
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 . By and large the standard of care of gliomas remains the use of the oral alkylating agent temozolomide and radiotherapy following surgical tumor resection . 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 . Interestingly, IDH mutations are also present in ~10% of AML (acute myeloid leukemia) patients . Wild-type (WT) IDH1 catalyzes the conversion of isocitrate to -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+ . 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 . 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 selectivity assay is to identify small-molecule inhibitors of WT IDH1 using a biochemical assay that measures product NADPH production through the use of a diaphorase/resazurin-coupled system. Inhibition of the WT IDH1 enzymatic reaction 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 concentration-titration series that ranged from 100 microM to 5 nM.
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: R03 DA032129
PI Name: Dr. Lenny Dang
Compounds or DMSO were dispensed into black, solid 96-well plates at 1 microL/well. Then, 40 microL/well enzyme buffer was dispensed 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 and 0.65 mM WT IDH1. These were incubated at RT for 60 minutes. The substrate and detection buffer (10 microL; 20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.05% BSA, 0.05 mM NADP+, 0.00053 mM diaphorase, 0.04 mM resazurin and 0.07 mM isocitrate) was added to start the enzymatic reaction. The reaction progressed for 30 minutes at RT and which point 25 microL/well of 2% SDS was added as a stop solution. Following 1 minute of shaking on an oribital shaker, the fluorescence intensity was monitored on a Spectramax M5 plate reader (Ex 544, Em 590, bodipy filter). 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
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.
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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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)