title: Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.
abstract: Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Mindy I
authors.initials: MI
authors.lastname: Davis
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Stefan
authors.initials: S
authors.lastname: Gross
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Min
authors.initials: M
authors.lastname: Shen
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Kimberly S
authors.initials: KS
authors.lastname: Straley
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Rajan
authors.initials: R
authors.lastname: Pragani
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Lea
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Janeta
authors.initials: J
authors.lastname: Popovici-Muller
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Byron
authors.initials: B
authors.lastname: DeLaBarre
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Erin
authors.initials: E
authors.lastname: Artin
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Natasha
authors.initials: N
authors.lastname: Thorne
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892, the Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Zhuyin
authors.initials: Z
authors.lastname: Li
authors.affiliation: Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
authors.forename: Lenny
authors.initials: L
authors.lastname: Dang
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Boxer
authors.affiliation: From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892, asimeono@mail.nih.gov.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D000081:Acetamides / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D055592:Biophysical Phenomena; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D050505:Mutant Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D054852:Small Molecule Libraries / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: C000612071:2-(2-(1H-benzo(d)imidazol-1-yl)-N-(3-fluorophenyl)acetamido)-N-cyclopentyl-2-o-tolylacetamide; D000081:Acetamides; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D054852:Small Molecule Libraries; D007521:Isocitrate Dehydrogenase
keywords: AML; Dehydrogenase; Drug Discovery; Enzyme; Enzyme Inhibitors; Glioblastoma; IDH1; Isocitrate Dehydrogenase; Small Molecules