AID	Name	Type	Comment
602183	qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Summary	summary

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.

Hence, a HTS-compatible fluorescent, enzymatic assay to identify inhibitors of IDH1 to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR).

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

MLPCN Grant: DA032129
Assay Submitter (PI): Lenny Dang, Agios Pharmaceuticals

IDH1-R132H (0.5 ug/mL) was dispensed into black, solid-bottom 1536-well plates at 3 uL/well in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 4 mM beta-ME, and 0.05% protease-free BSA. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. A pre-read was run on the Viewlux to identify fluorescent compounds (540 nm excitation filter, 598 nm emission filter, bodipy mirror and 2 second exposure). After 30 minutes of incubation with compound, 3 uL of substrate mix were dispensed (0.016 mM NADPH, 2 mM alpha-KG, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, and 0.05% protease-free BSA). Plates were spun for 1 min at 1000xg and incubated at room temperature for 80 min. Then 1.5 uL of each detection reagent component for the coupling system were added (0.12 mg/mL diaphorase and 0.072 mM resazurin, each in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, and 0.05% protease-free BSA ) The protein, substrate and two detection components were maintained at 4 oC throughout the screen. Additionally, the substrate and resazurin reagents were protected from light. The plates were measured on an Envision plate reader for fluorescence signal using a 540 nm excitation filter and 590 nm emission filter with 10 flashes. 1x and 0x (no-enzyme) IDH1-R132H enzyme control were used to normalize %Activity of identified inhibitors; 0x enzyme values corresponded to 100% Activity (full inhibition), while 1x IDH1-R132H 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-effect 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 increases in fluorescence, concordant with their blocking the consumption of the NADPH. Inactive compounds showed no effect on the fluorescence signal.

Keywords: isocitrate dehydrogenase, IDH1, IDH1-R132H, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC

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.

Grant Number: DA032129