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• W2050178904 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. 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. Recent advances in tumor genome analysis have suggested novel roles for previously unappreciated genes in establishment and maintenance of the oncogenic state (1Mardis E.R. Wilson R.K. Cancer genome sequencing: a review.Hum. Mol. Genet. 2009; 18: R163-R168Crossref PubMed Scopus (160) Google Scholar, 2Thompson C.B. Metabolic enzymes as oncogenes or tumor suppressors.N. Engl. J. Med. 2009; 360: 813-815Crossref PubMed Scopus (192) Google Scholar, 3Yates L.R. Campbell P.J. Evolution of the cancer genome.Nat. Rev. Genet. 2012; 13: 795-806Crossref PubMed Scopus (413) Google Scholar). Mutations in isocitrate dehydrogenase 1 (IDH1), 3The abbreviations used are:IDH1isocitrate dehydrogenase 1α-KGα-ketoglutaratePKpharmacokinetics2-HG2-hydroxyglutaraterac-ML309racemic ML309DMSOdimethyl sulfoxide. a metabolic enzyme responsible for the conversion of isocitrate to α-ketoglutarate (α-KG), were annotated at a high frequency in an analysis of 22 human glioblastoma multiforme tumor samples. Interestingly, all these mutations were G395A, resulting in the conversion of an arginine at position 132 to a histidine (4Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. Olivi A. McLendon R. Rasheed B.A. Keir S. Nikolskaya T. Nikolsky Y. Busam D.A. Tekleab H. Diaz Jr., L.A. Hartigan J. Smith D.R. Strausberg R.L. Marie S.K. Shinjo S.M. Yan H. Riggins G.J. Bigner D.D. Karchin R. Papadopoulos N. Parmigiani G. Vogelstein B. Velculescu V.E. Kinzler K.W. An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (4549) Google Scholar). Because proteins with the IDH1 R132H missense mutation display decreased efficiency in the conversion of isocitrate to α-KG in vitro because Arg-132 is one of the substrate-binding arginine triads in the enzyme active site, these were at first believed to be loss-of-function mutations (5Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. Friedman H. Friedman A. Reardon D. Herndon J. Kinzler K.W. Velculescu V.E. Vogelstein B. Bigner D.D. IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (4247) Google Scholar). However, the discovery of gain of function where IDH1 R132H results in a neomorphic enzymatic activity (Fig. 1), namely the conversion of α-KG to 2-hydroxyglutarate (2-HG), has profound implications for the role of IDH1 and its close homologue IDH2 in the metabolic activities of the cancer cell (6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar). As a dead-end metabolite, 2-HG accumulates to millimolar levels in cells with neoactive IDH1 (i.e. R132H or R132C) and IDH2 mutations (i.e. R172K) (7Gross S. Cairns R.A. Minden M.D. Driggers E.M. Bittinger M.A. Jang H.G. Sasaki M. Jin S. Schenkein D.P. Su S.M. Dang L. Fantin V.R. Mak T.W. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations.J. Exp. Med. 2010; 207: 339-344Crossref PubMed Scopus (602) Google Scholar), and acts as an inhibitor of the α-KG-dependent epigenetic machinery (8Chowdhury R. Yeoh K.K. Tian Y.M. Hillringhaus L. Bagg E.A. Rose N.R. Leung I.K. Li X.S. Woon E.C. Yang M. McDonough M.A. King O.N. Clifton I.J. Klose R.J. Claridge T.D. Ratcliffe P.J. Schofield C.J. Kawamura A. The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.EMBO Rep. 2011; 12: 463-469Crossref PubMed Scopus (745) Google Scholar, 9Koivunen P. Lee S. Duncan C.G. Lopez G. Lu G. Ramkissoon S. Losman J.A. Joensuu P. Bergmann U. Gross S. Travins J. Weiss S. Looper R. Ligon K.L. Verhaak R.G. Yan H. Kaelin Jr., W.G. Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation.Nature. 2012; 483: 484-488Crossref PubMed Scopus (559) Google Scholar), blocking differentiation and promoting the proliferation of undifferentiated tumorous cells. It has recently been shown that 2-HG alone can promote leukemogenesis (10Losman J.A. Looper R.E. Koivunen P. Lee S. Schneider R.K. McMahon C. Cowley G.S. Root D.E. Ebert B.L. Kaelin W.G. (R)-2-Hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible.Science. 2013; 339: 1621-1625Crossref PubMed Scopus (551) Google Scholar). Additionally, 2-HG suppresses the tricarboxylic acid (TCA) cycle and results in enhanced lipid metabolism (11Miyata S. Urabe M. Gomi A. Nagai M. Yamaguchi T. Tsukahara T. Mizukami H. Kume A. Ozawa K. Watanabe E. An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.Neurol. Med. Chir. (Tokyo). 2013; 53: 645-654Crossref PubMed Scopus (10) Google Scholar). Inhibitors of 2-HG production by mutant IDH1 and IDH2 could have important clinical applications in the treatment of IDH mutated glioblastoma and acute myeloid leukemia (4Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. Olivi A. McLendon R. Rasheed B.A. Keir S. Nikolskaya T. Nikolsky Y. Busam D.A. Tekleab H. Diaz Jr., L.A. Hartigan J. Smith D.R. Strausberg R.L. Marie S.K. Shinjo S.M. Yan H. Riggins G.J. Bigner D.D. Karchin R. Papadopoulos N. Parmigiani G. Vogelstein B. Velculescu V.E. Kinzler K.W. An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (4549) Google Scholar, 5Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. Friedman H. Friedman A. Reardon D. Herndon J. Kinzler K.W. Velculescu V.E. Vogelstein B. Bigner D.D. IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (4247) Google Scholar, 12Paschka P. Schlenk R.F. Gaidzik V.I. Habdank M. Krönke J. Bullinger L. Späth D. Kayser S. Zucknick M. Götze K. Horst H.A. Germing U. Döhner H. Döhner K. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication.J. Clin. Oncol. 2010; 28: 3636-3643Crossref PubMed Scopus (658) Google Scholar, 13Xu W. Yang H. Liu Y. Yang Y. Wang P. Kim S.H. Ito S. Yang C. Wang P. Xiao M.T. Liu L.X. Jiang W.Q. Liu J. Zhang J.Y. Wang B. Frye S. Zhang Y. Xu Y.H. Lei Q.Y. Guan K.L. Zhao S.M. Xiong Y. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases.Cancer Cell. 2011; 19: 17-30Abstract Full Text Full Text PDF PubMed Scopus (1992) Google Scholar). Moreover, such inhibitors could help elucidate mechanism by which these mutations function in the context of the cancer cell metabolome. Therefore, there is a need for the development of inhibitors for mutant IDH1 and to gain an understanding of their mechanisms of action. isocitrate dehydrogenase 1 α-ketoglutarate pharmacokinetics 2-hydroxyglutarate racemic ML309 dimethyl sulfoxide. A previously reported high-throughput screen identified the first potent series of inhibitors of IDH1 R132H that were further optimized (14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar). The series consists of a phenyl-glycine scaffold with one stereocenter. One enantiomer was shown to be predominantly responsible for the activity of the racemic mixture. The inhibitor series was selective for mutant IDH1 over wild-type (WT) IDH1 and had excellent cell activity (IC50 = 70 nm), including the ability to lower 2-HG levels by ∼90% in an in vivo U87MG IDH1 R132H mouse tumor xenograft model (14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar). Recently, a member of this series was shown to delay growth and promote differentiation of glioma cells (15Rohle D. Popovici-Muller J. Palaskas N. Turcan S. Grommes C. Campos C. Tsoi J. Clark O. Oldrini B. Komisopoulou E. Kunii K. Pedraza A. Schalm S. Silverman L. Miller A. Wang F. Yang H. Chen Y. Kernytsky A. Rosenblum M.K. Liu W. Biller S.A. Su S.M. Brennan C.W. Chan T.A. Graeber T.G. Yen K.E. Mellinghoff I.K. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells.Science. 2013; 340: 626-630Crossref PubMed Scopus (867) Google Scholar). ML309, described herein, is a newly identified and characterized member of the phenyl-glycine series. ML309 is active in both biochemical and cell assays. The time dependence of the effect on 2-HG levels in cells was explored. To gain a deeper understanding of how the substrates, and phenyl-glycine scaffold inhibitors, such as ML309, interact with IDH1 R132H enzyme, a detailed characterization using kinetic and biophysical approaches was undertaken. Based on these results, a compound binding model was proposed that provides a plausible explanation of the inhibitory mechanism and that can be used for future structure and activity relationship studies. All air- or moisture-sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents, such as dichloromethane, N,N-dimethylformamide, acetonitrile, methanol, and triethylamine, were purchased from Sigma-Aldrich. Analytical analysis was performed on an Agilent 1200 series LC/MS (Agilent Technologies, Santa Clara, CA). Sample dissolution in MeOH and analysis were performed at room temperature. The analytical column used was a Chiralcel OD column (4.6 × 150 mm, 5 μm). The mobile phase was 60:40 ethanol (absolute, 200 proof)/hexane (0.1% diethyl amine) at 1.0 ml/min. The sample was detected with a diode array detector at 220 and 254 nm. Optical rotation was determined with an in-line polarimeter (PDR-Chiral). Purity and enantiomeric excess were determined by this analytical method. All of the analogues tested in the biological assays have a purity greater than 95%. Preparative purification of racemic material was performed on an Agilent 1200 series Prep/LC (Agilent Technologies, Santa Clara, CA). The column used was a Chiralcel OD column (5 × 50 cm, 20 μm). The mobile phase was 60:40 ethanol (absolute, 200 proof)/hexane (0.1% diethyl amine) at 35 ml/min. Fraction collection was triggered by UV absorbance (220 nm). 1H NMR spectra were recorded on a Varian 400-MHz spectrometer. Chemical shifts are reported in ppm (CHD2OD at 3.31 ppm as internal standard) for MeOH-d4 solutions. High resolution mass spectrometry was recorded on Agilent 6210 time-of-flight LC/MS system. Confirmation of molecular formulae was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02). A solution of isocyanocyclopentane (196 mg, 2.064 mmol), 3-fluoroaniline (229 mg, 2.064 mmol), and 2-(1H-benzo[d]imidazol-1-yl)acetic acid (364 mg, 2.064 mmol) in MeOH (6.9 ml) was treated with 2-methylbenzaldehyde (248 mg, 2.064 mmol). The reaction was warmed to 40 °C and stirred for 4 h and then concentrated under reduced pressure. The crude product was purified by silica chromatography (2:98 to 5:95 MeOH/dichloromethane), which afforded racemic ML309 (rac-ML309) (216 mg, 0.446 mmol) in 22% yield. rac-ML309 (216 mg, 0.446 mmol) was separated by chiral chromatography to afford (−)-ML309 (73 mg, 0.151 mmol, >99% enantiomeric excess) and (+)-ML309 (66 mg, 0.136 mmol, >99% enantiomeric excess). (−)-ML309: LC-MS retention time: t1 (Chiralcel OD column; 60:40 EtOH/hexane isocratic) = 2.91 min; [α]D22 = −72.1° (c = 0.86, MeOH); 1H NMR (400 MHz, MeOH-d4) δ ppm 8.01 (s, 1 H), 7.89 (br s, 1 H), 7.65 (d, J = 7.8 Hz, 1 H), 7.41 (d, J = 7.8 Hz, 1 H), 7.32 (t, J = 6.4 Hz, 1 H), 7.27 (t, J = 6.8 Hz, 1 H), 7.14 (d, J = 6.8 Hz, 1 H), 7.07 (t, J = 7.2 Hz, 1 H), 6.99 (dt, J = 2.4, 8.0 Hz, 1 H), 6.88 (t, J = 7.2 Hz, 1 H), 6.81 (d, J = 7.2 Hz, 1 H), 6.56 (br s, 1H), 6.36 (s, 1H), 5.02 (d, J = 17.2 Hz, 1 H), 4.81 (d, J = 17.6 Hz, 1 H), 4.18 (tt, J = 6.8, 6.8 Hz, 1 H), 2.45 (s, 3 H), 1.94–1.83 (m, 2 H), 1.67–1.27 (m, 8 H); 19F NMR (282 MHz, MeOH-d4) δ, −113.03, −114.12 ppm; high resolution mass spectrometry (electrospray mass ionization) m/z (M+H)+ calculated for C29H29FN4O2, 485.2385; found 485.2335. (+)-ML309: LC-MS retention time: t1 (Chiralcel OD column; 60:40 EtOH/hexane isocratic) = 5.56 min; [α]D22 = + 75.3° (c = 0.76, MeOH); high resolution mass spectrometry (electrospray mass ionization) m/z (M+H)+ calculated for C29H29FN4O2, 485.2385; found 485.2367. rac-ML309 stability in aqueous solution, 5 mm glutathione, assay buffer, mouse and human microsomes, and human plasma and aqueous solubility were tested following methods described previously (16Avdeef A. High-throughput measurements of solubility profiles.Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical, and Computational Strategies, Verlag Helvetica Chimica Acta. 2007; (Zurich): 305-325Google Scholar, 17Di L. Kerns E.H. Li S.Q. Petusky S.L. High throughput microsomal stability assay for insoluble compounds.Int. J. Pharm. 2006; 317: 54-60Crossref PubMed Scopus (64) Google Scholar). A 20 mm DMSO stock of rac-ML309 was dispensed in assay buffer (final concentrations of 2% DMSO, 15% MeCN, 83% assay buffer), 5 mm glutathione (final concentrations of 2% DMSO, 49% MeCN, 49% PBS), or Dulbecco's PBS buffer (2% DMSO, 25% MeCN, 73% Dulbecco's PBS), and the remaining compound was monitored by the area under the curve of the peak at 220 nm by LC/MS at 0, 0.5, 1, 2, 6, 24, and 48 h. The half-life of rac-ML309 was tested by in vivo mouse pharmacokinetics (PK) using wild-type BALB/c mice (18Korfmacher W.A. Advances in the integration of drug metabolism into the lead optimization paradigm.Mini Rev. Med. Chem. 2009; 9: 703-716Crossref PubMed Scopus (25) Google Scholar). Full-length IDH1 (EC 1.1.1.42 and UniProt accession number O75874), IDH1 R132H, and IDH1 R132C were expressed and purified as described previously (6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar, 14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar). Both IDH1 R132H and IDH1 R132C protein were expressed with N-terminal His6 tags and purified by standard metal-chelate affinity chromatography techniques; IDH1 R132H was expressed in Escherichia coli, and IDH1 R132C was expressed in insect cells using the BaculoDirect (Invitrogen) baculovirus expression system. Proteins were buffer-exchanged by G25 gel-filtration chromatography into 500 mm NaCl, 50 mm Tris, pH 8.0, 10% glycerol and stored at −80 °C. NADPH, α-KG, and buffer components were purchased from Sigma. Experiments were run at room temperature unless otherwise specified. The activity of IDH1 R132H and IDH1 R132C was measured in 384-well plates by coupling NADPH consumption to a diaphorase/resazurin-based detection system and measuring resorufin production (Ex544/Em590) as described previously (6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar, 14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar). For testing the potency of small molecule inhibitors, an end point-coupled assay system was used, with 2–4 nm enzyme. In this assay, the reactions were run in the α-KG to 2-HG direction with the concomitant oxidation of NADPH to NADP. For IDH1 R132H, a final concentration of 1 mm α-KG and 4 μm NADPH was used; for IDH1 R132C, a final concentration of 200 μm α-KG and 4 μm NADPH was used. The NADPH remaining at the end of a 1-h incubation reaction was measured by the addition of 20 μm resazurin and 10 μg/ml diaphorase, which facilitated the stoichiometric conversion of resazurin to the highly fluorescent resorufin. To test the reversibility of the binding of compound to enzyme, 10× IC50 of the compound and 100× enzyme were incubated for 1 h, at which point the sample was diluted 100-fold and the enzyme reaction was run in kinetic mode monitoring the consumption of NADPH at 340 nm. Data were normalized to control columns representing maximum signal (no enzyme) and minimum signal (all components). WT IDH1 activity was also measured with this coupled diaphorase/resazurin system, but in this case product (NADPH) could be measured directly. Readout interference was tested with a counterscreen assay that contained all components except the proteins. The data were fit in GraphPad Prism 5 (La Jolla, CA) using nonlinear least-squares curve fitting. For determination of the Km(app) of substrates, IDH1 R132H was assayed in 150 mm NaCl, 20 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 0.03% BSA. Reactions where studied in an SFM-400 stop-flow spectrophotometer by monitoring the oxidation state of the co-factor at Ex340/Em450 and used 5–100 nm recombinant protein. Typical reactions were monitored for ∼5 s, and steady-state rates were extracted by linear regression with fluorescence converted to NADPH concentration by a standard curve of NADPH in assay buffer. The equations used to fit the substrate inhibition and product inhibition are described in detail in Ref. 19Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis. Wiley-VCH, New York2000Crossref Google Scholar. IDH1 R132H was labeled with a fluorescent dye NT-647 using the manufacturer's protocol (NanoTemper Technologies, München, Germany). A 16-point titration series of rac-ML309 in DMSO, NADPH in water, α-KG in water, or combinations thereof was transferred to the protein (20 nm) in a buffer containing 0.05% BSA, 0.01% Tween 20, 2 mm β-mercaptoethanol, 20 mm Tris, pH 7.5, and 150 mm NaCl and equilibrated for 15 min. The final top concentrations were 25 μm for NADPH, 25 mm α-KG, or 500 μm rac-ML309 for the single agent tests. For the competition tests, the substrate was held constant and rac-ML309 was titrated, i.e. α-KG was fixed at 150 mm and rac-ML309 was titrated down from 500 μm or NADPH was fixed at 50 μm and rac-ML309 was titrated down from 500 μm. The final DMSO concentration for each protein-compound sample was 0.5% DMSO. Samples were loaded into Monolith NT hydrophilic treated capillaries (NanoTemper Technologies). A capillary scan was performed followed by the successive measurement of thermophoresis in each capillary on a NanoTemper Monolith NT.115 instrument at room temperature. The IR laser power and the LED power conditions are described in the figure legends. A laser on-time of 30 s and a laser-off time of 5 s were used. The experiment was performed in duplicate, and the fits are reported as mean ± S.D. Data normalization and curve fitting were performed using GraphPad Prism 5. Binding experiments between IDH1 R132H and either NADPH or NADPH/rac-ML309 were performed using SPR measurements on a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.) at 25 °C. IDH1 R132H (“ligand”) was immobilized at high surface densities (6000–7000 resonance units) on an activated ProteOn HTE nickel-nitrilotriacetic acid sensor chip (Bio-Rad Laboratories, Inc.) through the His tag on the IDH1 R132H protein according to the manufacturer's instructions. The ligand was injected five times at a concentration of 20 μg/ml at a flow rate of 30 μl/min for 1000 s across two of the six available channels. To perform the binding assays, the various compounds (“analytes”) were injected at different concentrations in running buffer at a flow rate of 100 μl/min for 120 s (NADPH or rac-ML309 alone) or 180 s (rac-ML309 with NADPH), and sensorgrams were recorded. Blank surfaces and interspots (a feature of the ProteOn interaction array system) were used for background corrections using the ProteOn software. The sensorgrams were fit using the ProteOn analysis software. The protein rapidly lost activity on the chip, and therefore the chip was regenerated and the ligand was reloaded for each enzyme-compound test. The analytes tested included NADPH (a five-point 1:3 dilution series starting at 1 μm) and NADPH/rac-ML309 (at 1 μm NADPH constant and a five-point 1:3 dilution of rac-ML309 starting at 3 μm). The running buffer used for ligand loading and testing of NADPH alone or rac-ML309 alone was TBS. For the tests with rac-ML309 and NADPH co-injections, the running buffer was TBS with 1 μm NADPH and 0.25% DMSO. U87MG (human glioblastoma) cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA). U87MG is cultured in RPMI 1640, 2 mm l-Glutamine, 10 units/ml penicillin/streptomycin, and 10% FBS (Life Technologies). Cells were maintained in 5% CO2 at 37 °C. U87MG cells were infected with lentivirus containing full-length IDH1 R132H (U87MG pLVX-IDH1 R132H-neo or U87MG IDH1 R132H for short). For infection, cells were plated with the viral supernatant supplemented with 8 μg/ml Polybrene (5.0 × 104 cells/ml viral supernatant) and incubated in 5% CO2 at 32 °C for 24 h. After infection, transduced cells were selected using G418 (1 mg/ml) for 2 weeks to generate stable expression cells. U87MG pLVX-IDH1 R132H-neo cells were maintained in DMEM containing 10% FBS, 1× penicillin/streptomycin, and 500 μg/ml G418. Cells were seeded at a density of 5000 cells/well into 96-well white solid-bottom microtiter plates and incubated overnight at 37 °C and 5% CO2. The next day compounds were prepared in 100% DMSO and then diluted in medium for a final concentration of 0.2% DMSO. Medium was removed from the cell plates, and 200 μl of the compound dilutions were added to each well. After 48 h of incubation with compound at 37 °C, 100 μl of medium was removed from each well and analyzed by LC-MS for 2-HG concentrations as described in Ref. 6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar. The cell plates were then allowed to incubate another 24 h. At 72 h after compound addition, Promega CellTiter-Glo reagent (Madison, WI) was added to each well, and the plates were read for luminescence to determine any compound effects on growth inhibition (GI50) as described in Refs. 6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar and 14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar). For compound time course and washout studies, HT1080 cells (cultured as described in Refs. 6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar and 14Popovici-Muller J. Saunders J.O. Salituro F.G. Travins J.M. Yan S. Zhao F. Gross S. Dang L. Yen K.E. Yang H. Straley K.S. Jin S. Kunii K. Fantin V.R. Zhang S. Pan Q. Shi D. Biller S.A. Su S.M. Discovery of the first potent inhibitors of mutant IDH1 that lower tumor 2-HG in vivo.ACS Med. Chem. Lett. 2012; 3: 850-855Crossref PubMed Scopus (162) Google Scholar) that have endogenous expression of IDH1 R132C were incubated with compound similar to the cell-based assays with the following exceptions. Cells were plated into 6-well dishes. Upon each time point, cells were washed three times with PBS and harvested, and 2-HG was extracted as described previously (6Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. Marks K.M. Prins R.M. Ward P.S. Yen K.E. Liau L.M. Rabinowitz J.D. Cantley L.C. Thompson C.B. Vander Heiden M.G. Su S.M. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2634) Google Scholar). Containing a chiral center, rac-ML309 was separated into its (+) and (−) enantiomers (Fig. 2A). A coupled biochemical assay was used to determine the IC50 values for IDH1 R132H for the (+)-ML309 and (−)-ML309 enantiomers, which were 68 ± 1.2 and 29,000 ± 1.3 nm, respectively (F" @default.
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• W2050178904 title "Biochemical, Cellular, and Biophysical Characterization of a Potent Inhibitor of Mutant Isocitrate Dehydrogenase IDH1" @default.
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