title: Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer.
abstract: Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (K(m)=110 µM) compared with the R132H homodimer (K(m)= 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Eric
authors: E
authors: Brooks
authors: Exelixis, Inc, South San Francisco, CA, USA Quintara Discovery, 170 Harbor Way, Suite 100, South San Francisco, CA 94080, USA.
authors: Xiang
authors: X
authors: Wu
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Art
authors: A
authors: Hanel
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Shaun
authors: S
authors: Nguyen
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Jing
authors: J
authors: Wang
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Jeffrey H
authors: JH
authors: Zhang
authors: Exelixis, Inc, South San Francisco, CA, USA.
authors: Amanda
authors: A
authors: Harrison
authors: Exelixis, Inc, South San Francisco, CA, USA Quintara Discovery, 170 Harbor Way, Suite 100, South San Francisco, CA 94080, USA wzhang704@gmail.com.
authors: Wentao
authors: W
authors: Zhang
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005977:Glutarates / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D055503:Protein Multimerization
chemical_list: D004791:Enzyme Inhibitors; D005977:Glutarates; D007656:Ketoglutaric Acids; D050505:Mutant Proteins; C019417:alpha-hydroxyglutarate; D009249:NADP; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human
keywords: HTS; IDH1; R132H mutation; RapidFire–mass spectrometry