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 ..
BioActive Compounds: 66
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
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
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)