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).

GBM-associated mutations and altered protein expression are more common in young patients

“…As expected and consistent with the literature, IDH1 and ATRX are more frequently mutated in younger patients, which confers a more favorable prognosis [17, 18]. There is currently active searches for candidate drugs via high-output screening of compounds and robust activity on developing agents targeting the IDH mutant [19, 20]. Our data suggests that screening for this target in a younger patient population may be more likely to yield candidates for trials that select based on IDH mutational status.…”

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

“…The second is an orthosteric site, referred to as isocitrate site 1 35 . Additionally, several other IDH1 inhibitors have been disclosed 19,36,37 , however no ligand bound structures were reported and the MOI was found to be uncompetitive versus NADPH, contrasting with GSK849 and VVS.…”

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

“…Toward the ultimate goal of identifying new therapies for patients with IDH1 mutant cancers, several mutant-and isoform-selective inhibitors of mutant IDH1 have been described (Brooks et al, 2014;Deng et al, 2015;Levell et al, 2016;Okoye-Okafor et al, 2015;Popovici-Muller et al, 2012). However, a deep understanding of what drives this remarkable selectivity across compound classes is incomplete.…”

“…Pre-eminent among these 2-HG-modulated enzymes are several that regulate epigenetic state and cellular differentiation, providing a mechanism for how mutant IDH enzymes promote tumorigenesis . As such, blockade of 2-HG production via smallmolecule mutant IDH1 and IDH2 inhibitors is emerging as a promising therapeutic strategy (Brooks et al, 2014;Deng et al, 2015;Levell et al, 2016;Okoye-Okafor et al, 2015;Popovici-Muller et al, 2012). Remarkably, all published mutant IDH1 inhibitors are selective for R132 mutants versus wild-type (WT) IDH1, WT IDH2, and somatic cancer mutants of IDH2 (Ward et al, 2010;Yan et al, 2009).…”