Davis Mindy I, Gross Stefan, Shen Min, Straley Kimberly S, Pragani Rajan, Lea Wendy A, Popovici-Muller Janeta, DeLaBarre Byron, Artin Erin, Thorne Natasha, Auld Douglas S, Li Zhuyin, Dang Lenny, Boxer Matthew B, Simeonov Anton
From the NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892.
Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and.
J Biol Chem. 2014 May 16;289(20):13717-25. doi: 10.1074/jbc.M113.511030. Epub 2014 Mar 25.
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.
异柠檬酸脱氢酶1（IDH1）的两种突变形式（R132H和R132C）与包括胶质母细胞瘤和急性髓系白血病在内的多种癌症相关。这些突变赋予了产生2-羟基戊二酸（2-HG）的新功能，并且2-HG此前被认为是一种致癌代谢物。突变型IDH1抑制剂有可能用于治疗这些疾病。在本研究中，我们使用生化、细胞和生物物理方法研究了新发现的抑制剂ML309的作用机制。底物结合和产物抑制研究有助于进一步阐明IDH1 R132H催化循环。这种快速平衡的抑制剂在生化和细胞试验中均具有活性。（+）异构体具有活性（IC50 = 68 nM），而（-）异构体对IDH1 R132H的抑制活性则低400多倍（IC50 = 29 μM）。IDH1 R132C同样受到（+）-ML309的抑制。野生型IDH1在很大程度上不受（+）-ML309的影响（IC50 >36 μM）。动力学分析结合微量热泳和表面等离子体共振表明，这种可逆抑制剂相对于α-酮戊二酸与IDH1 R132H竞争性结合，相对于NADPH则为非竞争性结合。提出了ML309抑制IDH1 R132H的反应方案，其中ML309在IDH1 R132H NADPH复合物形成后与IDH1 R132H结合。ML309还能够抑制胶质母细胞瘤细胞系中2-HG的产生（IC50 = 250 nM），并且细胞毒性极小。在外消旋ML309存在的情况下，2-HG水平迅速下降。这种下降一直持续到48小时，此时将化合物洗脱，2-HG水平恢复。
