Attorney Docket No. AGS-013C2
`
`Technology), and IDH2 antibody (Abeam). Metabolites were extracted from cultured
`
`cells and from tissue samples according to close variants of a previously reported
`
`method (Lu, W., Kimball, E. & Rabinowitz, J. D. J Am Soc Mass Spectrom 17, 37-50
`
`(2006)), using 80% aqueous methanol (-80 °C) and either tissue scraping or
`
`homogenization to disrupt cells. Enzymatic activity in cell lysates was assessed by
`
`following a change in NADPH fluorescence over time in the presence of isocitrate
`
`and NADP, or aKG and NADPH. For enzyme assays using recombinant IDHl
`
`enzyme, proteins were produced in E. coli and purified using Ni affinity
`
`chromatography followed by Sephacryl S-200 size-exclusion chromatography.
`
`Enzymatic activity for recombinant IDHl protein was assessed by following a change
`
`in NADPH UV absorbance at 340 nm using a stop-flow spectrophotometer in the
`
`presence of isocitrate and NADP or aKG and NADPH. Chirality of 2HG was
`
`determined as described previously (Struys, E. A., Jansen, E. E., Verhoeven, N. M. &
`
`Jakobs, C. Clin Chem 50, 1391-5 (2004)). For crystallography studies, purified
`
`recombinant IDHl (R132H) at 10 mg/mL in 20 mM Tris pH 7.4, 100 mM NaCl was
`
`pre-incubated for 60 min with 10 mM NADPH, 10 mM calcium chloride, and 75 mM
`
`aKG. Crystals were obtained at 20°C by vapor diffusion equilibration using 3 µL
`
`drops mixed 2:1 (protein:precipitant) against a well-solution of 100 mM MES pH 6.5,
`
`20% PEG 6000. Patient tumor samples were obtained after informed consent as part
`
`of a UCLA IRE-approved research protocol. Brain tumor samples were obtained
`
`after surgical resection, snap frozen in isopentane cooled by liquid nitrogen and stored
`
`at -80 C. The IDHl mutation status of each sample was determined using standard
`
`molecular biology techniques as described previously (Yan, H. et al. N Engl J Med
`
`360, 765-73 (2009)). Metabolites were extracted and analyzed by LC-MS/MS as
`
`described above. Full methods are available in the supplementary material.
`
`Supplementary methods
`
`Cloning, Expression, and Purification of ICDHl wt and mutants in E. coli. The
`
`open reading frame (ORF) clone of human isocitrate dehydrogenase 1 (cDNA) (IDHl;
`
`ref. ID NM_005896) was purchased from Invitrogen in pENTR221 (Carlsbad, CA)
`
`and Origene Inc. in pCMV6 (Rockville, MD). To transfect cells with wild-type or
`
`mutant IDHl, standard molecular biology mutagenesis techniques were utilized to
`
`alter the DNA sequence at base pair 395 of the ORF in pCMV6 to introduce base pair
`
`change from guanine to adenine, which resulted in a change in the amino acid code at
`
`-142-
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`position 132 from arginine (wt) to histidine (mutant; or R132H), and confirmed by
`
`standard DNA sequencing methods. For 293T cell transfection, wild-type and R132H
`
`mutant IDHl were subcloned into pCMV-Sport6 with or without a carboxy-terminal
`
`Myc-DDK-tag. For stable cell line generation, constructs in pCMV6 were used. For
`
`expression in E. coli, the coding region was amplified from pENTR221 by PCR using
`
`primers designed to add NDEI and XHOl restrictions sites at the 5' and 3' ends
`
`respectively. The resultant fragment was cloned into vector pET41a (EMD
`
`Biosciences, Madison, WI) to enable the E. coli expression of C-terminus His8-tagged
`
`protein. Site directed mutagenesis was performed on the pET41a-ICHD1 plasmid
`
`using the QuikChange® MultiSite-Directed Mutagenesis Kit (Stratagene, La Jolla,
`
`CA) to change G395 to A, resulting in the Arg to His mutation. R132C, R132L and
`
`R132S mutants were introduced into pET41a-ICHD1 in an analogous way.
`
`Wild-type and mutant proteins were expressed in and purified from the E. coli
`
`Rosetta™ strain (Invitrogen, Carlsbad, CA) as follows. Cells were grown in LB (20
`
`µg/ml Kanamycin) at 37°C with shaking until OD600 reaches 0.6. The temperature
`
`was changed to 18°C and protein expression was induced by adding IPTG to final
`
`concentration of 1 mM. After 12-16 hours ofIPTG induction, cells were resuspended
`
`in Lysis Buffer (20mM Tris, pH7.4, 0.1 % Triton X-100, 500 mM NaCl, 1 mM PMSF,
`
`5 mM P-mercaptoethanol, 10 % glycerol) and disrupted by microfluidation. The
`
`20,000g supernatant was loaded on metal chelate affinity resin (MCAC) equilibrated
`
`with Nickel Column Buffer A (20 mM Tris, pH7.4, 500mM NaCl, 5 mM P(cid:173)
`
`mercaptoethanol, 10% glycerol) and washed for 20 column volumes. Elution from the
`
`column was effected by a 20 column-volume linear gradient of 10% to 100% Nickel
`
`Column Buffer B (20 mM Tris, pH7.4, 500 mM NaCl, 5 mM P-mercaptoethanol, 500
`
`mM Imidazole, 10% glycerol) in Nickel Column Buffer A). Fractions containing the
`
`protein of interest were identified by SDS-P AGE, pooled, and dialyzed twice against
`
`a 200-volume excess of Gel Filtration Buffer (200 mM NaCl, 50 mM Tris 7.5, 5 mM
`
`P-mercaptoethanol, 2 mM MnSO4, 10% glycerol), then concentrated to 10 ml using
`
`Centricon (Millipore, Billerica, MA) centrifugal concentrators. Purification of active
`
`dimers was achieved by applying the concentrated eluent from the MCAC column to
`
`a Sephacryl S-200 (GE Life Sciences, Piscataway, NJ) column equilibrated with Gel
`
`Filtration Buffer and eluting the column with 20 column volumes of the same buffer.
`
`Fractions corresponding to the retention time of the dimeric protein were identified by
`
`SDS-PAGE and pooled for storage at -80°C.
`
`-143 -
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`Cell lines and Cell Culture. 293T cells were cultured in DMEM (Dulbecco's
`
`modified Eagles Medium) with 10% fetal bovine serum and were transfected using
`
`pCMV-6-based IDH-1 constructs in six-well plates with Fugene 6 (Roche) or
`
`Lipofectamine 2000 (In vitro gen) according to manufacturer's instructions. Parental
`
`vector pCMV6 (no insert), pCMV6-wt IDHl or pCMV6-R132H were transfected into
`
`human glioblastoma cell lines (U87MG; LN-18 (ATCC, HTB-14 and CRL-2610;
`
`respectively) cultured in DMEM with 10 % fetal bovine serum. Approximately 24
`
`hrs after transfection, the cell cultures were transitioned to medium containing G418
`
`sodium salt at concentrations of either 500 ug/ml (U87MG) or 750 ug/ml (LN-18) to
`
`select stable transfectants. Pooled populations of G418 resistant cells were generated
`
`and expression of either wild-type IDHl or R132 IDHl was confirmed by standard
`
`Western blot analysis.
`
`Western blot. For transient transfection experiments in 293 cells, cells were lysed 72
`
`hours after transfection with standard RIPA buffer. Lysates were separated by SDS(cid:173)
`
`PAGE, transferred to nitrocellulose and probed with goat-anti-IDHc antibody (Santa
`
`Cruz Biotechnology sc49996) or rabbit-anti-MYC tag antibody (Cell Signaling
`
`Technology #2278) and then detected with HRP-conjugated donkey anti-goat or
`
`HRP-conjugated goat-anti-rabbit antibody (Santa Cruz Biotechnology sc2004). IDHl
`
`antibody to confirm expression of both wild-type and R132H IDHl was obtained
`
`from Proteintech. The IDH2 mouse monoclonal antibody used was obtained from
`
`Abeam.
`
`Detection of isocitrate, a.KG, and 2HG in purified enzyme reactions by LC(cid:173)
`
`MS/MS. Enzyme reactions performed as described in the text were run to completion
`
`as judged by measurement of the oxidation state of NADPH at 340 nm. Reactions
`
`were extracted with eight volumes of methanol, and centrifuged to remove
`
`precipitated protein. The supernatant was dried under a stream of nitrogen and
`
`resuspended in H20. Analysis was conducted on an API2000 LC-MS/MS (Applied
`
`Biosystems, Foster City, CA). Sample separation and analysis was performed on a
`
`150 x 2 mm, 4 uM Synergi Hydro-RP 80 A column, using a gradient of Buffer A (10
`
`-144-
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`mM tributylamine, 15 mM acetic acid, 3% (v/v) methanol, in water) and Buffer B
`
`(methanol) using MRM transitions.
`
`Cell lysates based enzyme assays. 293T cell lysates for measuring enzymatic
`
`activity were obtained 48 hours after transfection with M-PER lysis buffer
`
`supplemented with protease and phosphatase inhibitors. After lysates were sonicated
`
`and centrifuged at 12,000g, supernatants were collected and normalized for total
`
`protein concentration. To measure IDH oxidative activity, 3 µg of lysate protein was
`
`added to 200 µl of an assay solution containing 33 mM Tris-acetate buffer (pH 7.4),
`
`1.3 mM MgCb, 0.33 mM EDTA, 100 µM P-NADP, and varying concentrations of D(cid:173)
`( + )-threo-isocitrate. Absorbance at 340 nm, reflecting NADPH production, was
`
`measured every 20 seconds for 30 min on a SpectraMax 190 spectrophotometer
`
`(Molecular Devices). Data points represent the mean activity of 3 replicates per
`
`lysate, averaged among 5 time points centered at every 5 min. To measure IDH
`
`reductive activity, 3 µg of lysate protein was added to 200 µl of an assay solution
`
`which contained 33 mM Tris-acetate (pH 7.4), 1.3 mM MgCb, 25 µM P-NADPH, 40
`
`mM NaHCO 3, and 0.6 mM aKG. The decrease in 340 nm absorbance over time was
`
`measured to assess NADPH consumption, with 3 replicates per lysate.
`
`Recombinant IDHl Enzyme Assays. All reactions were performed in standard
`
`enzyme reaction buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 10% glycerol, 5 mM
`
`MgCb and 0.03% (w/v) bovine serum albumin). For determination of kinetic
`
`parameters, sufficient enzyme was added to give a linear reaction for 1 to 5 seconds.
`
`Reaction progress was monitored by observation of the reduction state of the cofactor
`
`at 340 nm in an SFM-400 stopped-flow spectrophotometer (BioLogic, Knoxville, TN).
`
`Enzymatic constants were determined using curve fitting algorithms to standard
`
`kinetic models with the Sigmaplot software package (Systat Software, San Jose, CA).
`
`Determination of chirality of reaction products from enzyme reactions and
`
`tumors. Enzyme reactions were run to completion and extracted with methanol as
`
`described above, then derivatized with enantiomerically pure tartaric acid before
`
`resolution and analysis by LC-MS/MS. After being thoroughly dried, samples were
`resuspended in freshly prepared 50 mg/ml (2R,3R)-( + )-Tartaric acid in
`
`dichloromethane:acetic acid (4:1) and incubated for 30 minutes at 75°C. After cooling
`
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`to room temperature, samples were briefly centrifuged at 14,000g, dried under a
`
`stream of nitrogen, and resuspended in H20. Analysis was conducted on an API200
`
`LC-MS/MS (Applied Biosystems, Foster City, CA), using an isocratic flow of 90: 10
`
`(2 mM ammonium formate, pH 3.6:MeOH) on a Luna C18(2) 150 x 2 mm, 5 uM
`
`column. Tartaric-acid derivatized 2HG was detected using the 362.9/146.6 MRM
`
`transition and the following instrument settings: DP-1, PP -310, EP -4, CE-12, CXP-
`
`26. Analysis of the (R)-2HG standard, 2HG racemic mixture, and methanol-extracted
`
`tumor biomass (q.v.) was similarly performed.
`
`Crystallography conditions. Crystals were obtained at 20°C by vapor diffusion
`
`equilibration using 3 µL drops mixed 2: 1 (protein:precipitant) against a well-solution
`
`of 100 mM MES pH 6.5, 20% PEG 6000.
`
`Protein characterization. Approximately 90 mg of human cytosolic isocitrate
`
`dehydrogenase (HcIDH) was supplied to Xtal BioStructures by Agios. This protein
`
`was an engineered mutant form, R132S, with an 11-residue C-terminal affinity(cid:173)
`
`purification tag (sequence SLEHHHHHHHH). The calculated monomeric molecular
`
`weight was 48.0 kDa and the theoretical pl was 6.50. The protein, at about 6 mg/mL
`
`concentration, was stored in 1-mL aliquots in 50 mM Tris-HCl (pH 7.4), 500 mM
`
`NaCl, 5 mM P-mercaptoethanol and 10% glycerol at -80°C. As shown in FIG. 32A,
`
`SDS-PAGE was performed to test protein purity and an anti-histidine Western blot
`
`was done to demonstrate the protein was indeed his-tagged. A sample of the protein
`
`was injected into an FPLC size-exclusion column to evaluate the sample purity and to
`
`determine the polymeric state in solution. FIG. 32B is a chromatogram of this run
`
`showing a single peak running at an estimated 87.6 kDa, suggesting IDH exists as a
`
`dimer at pH 7.4. Prior to crystallization, the protein was exchanged into 20 mM Tris(cid:173)
`
`HCl (pH 7.4) and 100 mM NaCl using Amicon centrifugal concentrators. At this time,
`
`the protein was also concentrated to approximately 15 mg/mL. At this protein
`
`concentration and ionic strength, the protein tended to form a detectable level of
`
`precipitate. After spinning out the precipitate, the solution was stable at -10 mg/mL
`
`at 4 °C.
`
`Initial attempts at crystallization. The strategy for obtaining diffraction-quality
`
`crystals was derived from literature conditions, specifically "Structures of Human
`
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`Cytosolic NADP-dependent Isocitrate Dehydrogenase Reveal a Novel Self-regulatory
`
`Mechanism of Activity," Xu, et al. (2005) ].Biol.Chem. 279: 33946-56. In this study,
`
`two crystal forms of HcIDH wildtype protein were produced. One contained their
`
`"binary complex", IDH-NADP, which crystallized from hanging drops in the
`
`tetragonal space group P432 12. The drops were formed from equal parts of protein
`
`solution (15 mg/mL IDH, 10 mM NADP) and precipitant consisting of 100 mM MES
`
`(pH 6.5) and 12% PEG 20000. The other crystal form contained their "quaternary
`complex", IDH-NADP/isocitrate/Ca2
`
`+, which crystallized in the monoclinic space
`
`group P2 1 using 100 mM MES (pH 5.9) and 20% PEG 6000 as the precipitant. Here
`
`they had added 10 mM DL-isocitrate and 10 mM calcium chloride to the protein
`
`solution. First attempts at crystallizing the R132S mutant in this study centered
`
`around these two reported conditions with little variation. The following lists the
`
`components of the crystallization that could be varied; several different combinations
`
`of these components were tried in the screening process.
`
`-147 -
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`In the protein solution:
`
`HcIDH(R132S)
`
`Tris-HCl (pH 7.4)
`
`NaCl
`
`always -10 mg/mL or -0.2 mM
`
`always 20 mM
`
`always 100 mM
`
`NADP+/NADPH
`
`absent or 5 mM NADP+ (did not try NADPH)
`
`DL-isocitic acid, trisodium salt
`
`absent or 5 mM
`
`calcium chloride
`
`In the precipitant:
`
`OR
`
`Drop size:
`
`Drop ratios:
`
`absent or 10 mM
`
`100 mM MES (pH 6.5) and 12% PEG 20000
`
`100 mM MES (pH 6.0) and 20% PEG 6000
`
`always 3 µL
`
`2:1, 1:1 or 1:2 (protein:precipitant)
`
`Upon forming the hanging drops, a milky precipitate was always observed. On
`
`inspection after 2-4 days at 20 °C most drops showed dense precipitation or phase
`
`separation. In some cases, the precipitate subsided and it was from these types of
`
`drops small crystals had grown, for example, as shown in FIG. 33.
`
`Crystal optimization. Once bonafide crystals were achieved, the next step was to
`
`optimize the conditions to obtain larger and more regularly-shaped crystals of IDH(cid:173)
`
`NADP/isocitrate/Ca2+ in a timely and consistent manner. The optimal screen focused
`
`on varying the pH from 5.7 to 6.2, the MES concentration from 50 to 200 mM and the
`
`PEG 6000 concentration from 20 to 25%. Also, bigger drops were set up (5-6 µl) and
`
`the drop ratios were again varied. These attempts failed to produce larger, diffraction(cid:173)
`
`quality crystals but did reproduce the results reported above. Either a dense
`
`precipitate, oily phase separation or small crystals were observed.
`
`Using a-Ketoglutarate. Concurrent to the optimization of the isocitrate crystals,
`
`other screens were performed to obtain crystals of IDH(R132S) complexed with a(cid:173)
`
`ketoglutarate instead. The protein solution was consistently 10 mg/mL IDH in 20
`
`mM Tris-HCl (pH 7.4) and 100 mM NaCL The following were added in this order: 5
`
`mM NADP, 5 mM a-ketoglutaric acid (free acid, pH balanced with NaOH) and 10
`
`mM calcium chloride. The protein was allowed to incubate with these compounds for
`
`at least an hour before the drops were set up. The precipitant was either 100 mM
`
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`MES (pH 6.5) and 12% PEG 20000 or 100 mM MES (pH 6.5) and 20% PEG 6000.
`
`Again, precipitation or phase separation was primarily seen, but in some drops small
`
`crystals did form. At the edge of one of the drops, a single large crystal formed,
`
`pictured below. This was the single crystal used in the following structure
`
`determination. FIG. 34 shows crystal obtained from a protein solution contained 5
`
`mM NADP, 5 mM a-ketoglutarate, 10 mM Ca2+. Precipitant contained 100 mM
`
`MES (pH 6.5) and 12% PEG 20000.
`
`Cryo conditions. In order to ship the crystal to the X-ray source and protect it during
`
`cryo-crystallography, a suitable cryo-protectant was needed. Glycerol is quite widely
`
`used and was the first choice. A cryo solution was made, basically as a mixture of the
`
`protein buffer and precipitant solution plus glycerol: 20 mM Tris-HCl (pH 7.5), 100
`
`mM NaCl, 5 mM NADP, 5 mM a-ketoglutaric acid, 10 mM calcium chloride, 100
`
`mM MES (pH 6.5), 12% PEG 20000 and either 12.5% glycerol or 25% glycerol. The
`
`crystal was transferred to the cryo solution in two steps. First, 5 µL of the 12.5%
`
`glycerol solution was added directly to the drop and incubated for 10 minutes,
`
`watching for possible cracking of the crystal. The liquid was removed from the drop
`
`and 10 µL of the 25% glycerol solution was added on top of the crystal. Again, this
`
`incubated for 10 minutes, harvested into a nylon loop and plunged into liquid nitrogen.
`
`The crystal was stored submerged in a liquid nitrogen dewar for transport.
`
`Data collection and processing. The frozen crystal was mounted on a Rigaku
`
`RAXIS IV X-ray instrument under a stream of nitrogen gas at temperatures near
`-170 °C. A 200° dataset was collected with the image plate detector using 1.54 A
`wavelength radiation from a rotating copper anode home source, 1 ° oscillations and
`
`10 minute exposures. The presence of 25% glycerol as a cryoprotectant was
`
`sufficient for proper freezing, as no signs of crystal cracking (split spots or
`superimposed lattices) were observed. A diffuse ring was observed at 3.6 A resolution,
`most likely caused by icing. The X-ray diffraction pattern showed clear lattice planes
`
`and reasonable spot separation, although the spacing along one reciprocal axis was
`rather small (b = 275.3). The data was indexed to 2.7 A resolution into space group
`P2 12 12 with HKL2000 (Otwinowski and Minor, 1997). Three structures for HcIDH
`
`are known, designated the closed form (1 T0L), the open form (1 T09 subunit A) and
`
`semi-open form (1 T09 subunit B). Molecular replacement was performed with the
`
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`CCP4 program PHASER (Bailey, 1994) using only the protein atoms from these three
`
`forms. Only the closed form yielded a successful molecular replacement result with 6
`
`protein subunits in the asymmetric unit. The unit cell contains approximately 53.8%
`
`solvent.
`
`Model refinement._Using the CCP4 program REFMAC5, rigid-body refinement was
`
`performed to fit each of the 6 IDH subunits in the asymmetric unit. This was followed
`
`by rigid-body refinement of the three domains in each protein subunit. Restrained
`
`refinement utilizing non-crystallographic symmetry averaging of related pairs of
`
`subunits yielded an initial structure with Royst of 33% and Rfree of 42%._Model
`
`building and real-space refinement were performed using the graphics program
`
`COOT (Emsley and Cowtan, 2004). A difference map was calculated and this showed
`
`strong electron density into which six individual copies of the NADP ligand and
`
`calcium ion were manually fit with COOT. Density for the a-ketoglutarate structure
`
`was less defined and was fit after the binding-site protein residues were fit using a
`
`2F0 -Fc composite omit map. Automated Ramachandran-plot optimization coupled
`
`with manual real-space density fitting was applied to improve the overall geometry
`
`and fit. A final round of restrained refinement with NCS yielded an Royst of 30.1 %
`
`and Rfree of 35.2%.
`
`a,A
`
`b,A
`
`c,A
`
`116.14
`
`275.30
`
`96.28
`
`('J,
`
`90°
`
`B
`
`90°
`
`y
`
`Unit cell
`volume, A3
`
`90°
`
`3.08 X 106
`
`z
`
`24
`
`Reflections in working set / test
`set
`Rcrvst
`Rfree
`
`68,755 / 3,608 (5.0%)
`
`30.1%
`35.2%
`
`- 150 -
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`

`

`X-ray data and refinement statistics for IDH(R132S)-NADP/a-ketoglurate/Ca2
`
`+
`
`Attorney Docket No. AGS-013C2
`
`Crystal parameters
`
`Space group
`
`Unit cell dimensions
`
`a, b, c,A
`
`a,~, y, o
`
`I
`
`I
`
`I
`
`I
`
`I
`
`P21212
`
`I
`116.139,275.297, 96.283
`
`90.0, 90.0, 90.0
`
`Volume,A3
`
`3,078,440
`
`6
`
`24
`
`No. protein molecules in
`asymmetric unit
`
`No. protein molecules in
`unit cell, Z
`
`Data collection
`
`Beam line
`
`Date of collection
`
`Apr 25, 2009
`
`A,A
`
`1.5418
`
`Detector
`
`Rigaku Raxis IV
`
`Data set (phi), 0
`
`200
`
`Resolution, A
`
`25-2.7 (2.8-2.7)
`
`Unique reflections (N, F
`> 0)
`
`73,587
`
`Completeness, %
`
`85.4 (48.4)
`
`<I> I <JI
`
`9.88 (1.83)
`
`- 151 -
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`Attorney Docket No. AGS-013C2
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`R-merge
`
`0.109 (0.33)
`
`Redundancy
`
`4.3 (1.8)
`
`Mosaicity
`
`Wilson B factor
`
`Anisotropy B factor, A2
`
`Refinement Statistics
`
`0.666
`
`57.9
`
`-1.96
`
`Resolution limit, A
`
`20.02-2.70
`
`No. of reflections used
`for R-worka / R-freeb
`
`68,755 I 3608
`
`Protein atoms
`
`19788
`
`Ligand atoms
`
`No. of waters
`
`Ions etc.
`
`Matthews coeff. A3/
`Dalton
`
`Solvent,%
`
`348
`
`357
`
`6
`
`2.68
`
`53.8
`
`R-worka / R-freeb, (%)
`
`30.1 I 35.2
`
`Figure-of-merit=
`
`0.80 (0.74)
`
`Average B factors
`
`Coordinates error
`(Luzzati plot), A
`
`RM.S. deviations
`
`31.0
`
`0.484
`
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`Attorney Docket No. AGS-013C2
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`Bond lengths, A
`
`Bond angles, 0
`
`0.026
`
`2.86
`
`I
`
`I
`
`Completeness and R-merge are given for all data and for data in the highest resolution
`
`shell. Highest shell values are in parentheses.
`
`aR factor= LhkJ IF0 -Fcl / Lhk!Fo, where F0 and Fe are the observed and calculated
`
`structure factor amplitudes, respectively for all reflections hkl used in refinement.
`
`b R-free is calculated for 5 % of the data that were not used in refinement.
`
`cFigure of merit= v'i.~~ +]J2, where x = ( LE'TP(o:)cos o:)/( LE'TP(o:)), y = ( I:i""P(o:.
`)sin o:.)/ ( Lif P(o:)), and the phase probability P(x) = exp(A cos o:+ B sin o:+ C cos(2o:)
`+ D sin(2o:.)), where A, B, C, and Dare the Hendrickson-Lattman coefficients and o: is
`
`the phase.
`
`Stereochemistry of IDH(R132S)-NADP/a-ketoglurate/Ca2
`+
`
`Ramachandran plot statistics
`
`No.of
`amino
`acids
`
`%of
`Residues
`
`Residues in most favored regions [A, B, L]
`
`1824
`
`82.2
`
`Residues in additional allowed regions [a, b, 1, p]
`
`341
`
`15.4
`
`Residues in generously allowed regions [-a, -b, -1, -p]
`
`Residues in disallowed regions
`
`38
`
`17
`
`1.7
`
`0.8
`
`Number of non-glycine and non-praline residues
`
`2220
`
`100
`
`Number of end-residues (excl. Gly and Pro)
`
`Number of glycine residues
`
`Number of praline residues
`
`Total number of residues
`
`Overall <G> -factord score ( > -1.0)
`
`387
`
`198
`
`72
`
`2877
`
`-0.65
`
`- 153 -
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`
`

`

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`
`Generated by PROCHECK (Laskowski RA, MacArthur MW, Moss DS, Thornton JM
`
`(1993) J Appl Crystallogr 26:283-291.)
`
`ct G-factors for main-chain and side-chain dihedral angles, and main-chain covalent
`
`forces (bond lengths and bond angles). Values should be ideally -0.5 or above -1.0.
`
`Radiation wavelength, A
`
`1.54
`
`Resolution, A ( outer shell)
`
`20-2.70 (2.80-2.70)
`
`Unique reflections
`
`73,587
`
`Completeness ( outer shell)
`
`85.4% (48.4%)
`
`Redundancy (outer shell)
`
`4.3 (1.8)
`
`Rmerge (outer shell)
`
`10.9% (33%)
`
`<I> I <a(I)> ( outer shell)
`
`9.88 (1.83)
`
`Clinical Specimens, metabolite extraction and analysis. Human brain tumors
`
`were obtained during surgical resection, snap frozen in isopentane cooled by liquid
`
`nitrogen and stored at -80 C. Clinical classification of the tissue was performed using
`
`standard clinical pathology categorization and grading as established by the WHO.
`
`Genomic sequence analysis was deployed to identify brain tumor samples containing
`
`either wild-type isocitrate dehydrogenase (IDHl) or mutations altering amino acid
`
`132. Genomic DNA was isolated from 50-100 mgs of brain tumor tissue using
`
`standard methods. A polymerase chain reaction on the isolated genomic DNA was
`
`used to amplify a 295 base pair fragment of the genomic DNA that contains both the
`intron and 2nd exon sequences of human IDHl and mutation status assessed by
`
`standard molecular biology techniques.
`
`Metabolite extraction was accomplished by adding a lOx volume (m/v ratio) of -80 °C
`
`methanol:water mix (80%:20%) to the brain tissue (approximately l00mgs) followed
`
`by 30 s homogenization at 4 C. These chilled, methanol extracted homogenized
`
`tissues were then centrifuged at 14,000 rpm for 30 minutes to sediment the cellular
`
`and tissue debris and the cleared tissue supernatants were transferred to a screw-cap
`
`freezer vial and stored at -80 °C. For analysis, a 2X volume of tributylamine (10 mM)
`
`acetic acid (10 mM) pH 5.5 was added to the samples and analyzed by LCMS as
`
`follows. Sample extracts were filtered using a Millex-FG 0.20 micron disk and 10 µL
`
`were injected onto a reverse-phase HPLC column (Synergi 150mm x 2 mm,
`
`- 154 -
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`
`

`

`Attorney Docket No. AGS-013C2
`
`Phenomenex Inc.) and eluted using a linear gradient LCMS-grade methanol (50%)
`
`with 10 mM tributylamine and 10 mM acetic acid) ramping to 80 % methanol: 10 mM
`
`tributylamine: 10 mM acetic acid over 6 minutes at 200 µUmin. Eluted metabolite
`
`ions were detected using a triple-quadrupole mass spectrometer, tuned to detect in
`
`negative mode with multiple-reaction-monitoring mode transition set (MRM's)
`
`according to the molecular weights and fragmentation patterns for 8 known central
`
`metabolites, including 2-hydroxyglutarate as described above. Data was processed
`
`using Analyst Software (Applied Biosystems, Inc.) and metabolite signal intensities
`
`were obtained by standard peak integration methods.
`
`EXAMPLE 9 COMPOUNDS THAT INHIBIT IDHl R132H
`
`Assays were conducted in a volume of 76 ul assay buffer (150 mM NaCl, 10
`
`mM MgC12, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in a
`
`standard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mM aKG), 1 ul
`
`of test compound was added in DMSO. The plate was centrifuged briefly, and then 25
`
`ul of enzyme mix was added (0.2 ug/ml ICDHl R132H) followed by a brief
`
`centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at
`
`room temperature, then 25 ul of detection mix (30 uM resazurin, 36 ug/ml ) was
`
`added and the mixture further incubated for 5 minutes at room temperature. The
`
`conversion of resazurin to resorufin was detected by fluorescent spectroscopy at
`
`Ex544 Em590 c/o 590.
`
`Table 24a shows the wild type vs mutant selectivity profile of 5 examples of
`
`IDH1R132H inhibitors. The IDHl wt assay was performed at lx Km of NADPH as
`
`opposed to IDHR132H at lOx or lO0x Km of NADPH. The second example showed
`
`no inhibition, even at 100 uM. Also, the first example has IC50=5.74 uM but is
`
`shifted significantly when assayed at lO0x Km, indicating direct NADPH-competitive
`
`inhibitor. The selectivity between wild type vs mutant could be >20-fold.
`
`Table 24a
`
`STRUCTURE
`
`LDHa
`IC50
`
`LDHb
`IC50
`
`ICDH
`IC50
`(uM)@
`4uM
`(10x
`Km)
`NADPH
`
`ICDH
`IC50
`(uM)@
`40 uM
`NADPH
`
`IC50
`Ratio
`(40/4)
`
`IDH1wt
`IC50@ 1x
`Km (uM)
`
`- 155 -
`
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`

`Attorney Docket No. AGS-013C2
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`25.43
`
`64.07
`
`5.74
`
`>100
`
`17.42
`
`16.22
`
`5.92
`
`17.40
`
`12.26
`
`41.40
`
`3.38
`
`NO
`inhibition
`
`H
`s~
`s
`
`0
`
`8.61
`
`>100
`
`12.79
`
`14.70
`
`1.15
`
`19.23
`
`- 156 -
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`

`

`Attorney Docket No. AGS-013C2
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`33.75
`
`>100
`
`14.98
`
`19.17
`
`1.28
`
`46.83
`
`12.76
`
`>100
`
`23.80
`
`33.16
`
`1.39
`
`69.33
`
`Additional exemplary compounds that inhibit IDH1R132H are provided below in
`
`Table 24b.
`
`OMe rN» 'lj "--
`oN~
`,0 y 0/
`(N)
`~'/,~ t~
`
`Compound
`No.
`0 ~ 0¥0
`I
`1
`
`/2
`
`2
`
`- 157 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`I
`
`No.
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Compound
`
`y O'/
`»'/,~
`(N)
`0 r~
`N y 0,,,,
`(N)
`0 r~ ~
`~'/,~
`y 0/
`(N)
`~ ' / ,~ s-
`0 t~
`
`y 0/
`(N)
`~}~
`0 r~
`y 0/
`»'/,~
`(N)
`0 r~
`
`F
`
`- 158 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`8
`
`9
`
`10
`
`11
`
`12
`
`Compound
`
`0
`
`~ I
`
`~
`
`0~TI
`0
`
`.0
`
`y 0/
`(N)
`~'?, TI
`0 t~
`F3C y 0/
`(N)
`N H
`o')Cr~s~
`y 0/
`(N)
`N
`~ I
`O~lj ~
`y 0/
`(N)
`y 0/
`(N)
`N H
`o')CrNnO~
`
`.0
`
`0
`
`.0
`
`- 159 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`13
`
`14
`
`15
`
`16
`
`17
`
`Compound
`
`-160-
`
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`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`18
`
`19
`
`20
`
`21
`
`22
`
`Compound
`
`-161 -
`
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`

`

`Attorney
`
`D cket o.
`N AGS-013C2
`0
`
`No.
`
`23
`
`24
`
`25
`
`26
`
`27
`
`Compound
`
`-162-
`
`Rigel Exhibit 1002
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`28
`
`29
`
`30
`
`31
`
`32
`
`Compound
`
`- 163 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Compound
`
`-164-
`
`Rigel Exhibit 1002
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`38
`
`39
`
`40
`
`41
`
`42
`
`Compound
`
`- 165 -
`
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`
`

`

`Attorney
`
`D cket o.
`N AGS-013C2
`0
`
`No.
`
`43
`
`44
`
`45
`
`46
`
`47
`
`Compound
`
`-166-
`
`Rigel Exhibit 1002
`Page 433 of 1523
`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`48
`
`49
`
`50
`
`51
`
`52
`
`Compound
`
`- 167 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`53
`
`54
`
`55
`
`56
`
`57
`
`Compound
`
`- 168 -
`
`Rigel Exhibit 1002
`Page 435 of 1523
`
`

`

`N AGS-013C2
`Attorney Docket o.
`
`No.
`
`58
`
`59
`
`60
`
`61
`
`62
`
`Compound
`
`- 169 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`63
`
`64
`
`65
`
`66
`
`67
`
`Compound
`
`- 170 -
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`68
`
`69
`
`70
`
`71
`
`72
`
`Compound
`
`~ ' ? ,~
`
`y O''.,..
`(N)
`0 r~
`~'I,~
`y Cl
`(N)
`~}~
`0 r~
`Q
`(N)
`0 r~
`y 0/
`(N)
`~}~
`0
`~ 00_1__
`y 0/
`(N)
`»·~
`ro oov
`
`O
`
`- 171 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`73
`
`74
`
`75
`
`76
`
`77
`
`Compound
`
`-172-
`
`Rigel Exhibit 1002
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`78
`
`79
`
`80
`
`81
`
`82
`
`Compound
`
`oy
`
`O
`Cl
`
`❖0
`❖S......._
`0
`
`(N) :'.x)} ~
`ro o
`/?
`
`(N)
`~}~
`O t~
`
`" ) ) / N
`O
`
`/? N
`(N) 0 /
`ro o 0~
`oy
`(N)
`:'.x)} ~
`O t~
`Cl y:
`(N)
`~}~
`O t~
`
`- 173 -
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`
`

`

`Attorney Docket No. AGS-013C2
`
`No.
`
`83
`
`84
`
`85
`
`86
`
`87
`
`Compound
`
`- 174 -
`
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`Page 441 of 1523
`
`

`

`Compound
`
`~ ' / ,~
`
`Attorney Docket No. AGS-013C2
`
`No.
`
`88
`
`89
`
`90
`
`91
`
`92
`
`~'?.~
`
`y F
`(N)
`0 r~
`~ (N)
`0 r~
`9-0/
`(N)
`0 r~
`y 0/
`(N)
`N 0 H
`;/1: ~
`y 0/
`(N)
`~~'~
`0 r~
`0
`
`~ ' / ,~
`
`0 , ~N
`
`0
`
`- 175 -
`
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`
`

`

`Attorney Docket No. AGS-013C2
`
`EXAMPLE 10. The mutant enzyme IDH2-Rl 72K has elevated NADPH
`
`reductive catalysis activity as compared to wildtype IDH2 enzyme.
`
`NADPH reduction activity was measured for the enzymes IDH2-Rl 72K,
`
`IDH2-wildtype, IDH1-R132H and IDHl-wildtype. The final reactant concentrations
`
`for each reaction were as follows: 20 mM Tris 7.5, 150 mM NaCl, 2 mM MnCb,
`
`10% glycerol, 0.03% BSA, enzyme (1-120 µg/mL), 1 mM NADPH, and 5 mM aKG
`
`(alpha ketoglutarate). The resulting specific activities (µmol/min/mg) are presented in
`
`the graph in FIG. 35. The results indicate that the mutant IDH2 has elevated
`
`reductive activity as compared to wildtype IDH2, even though both the mutant and
`
`wildtype IDH2 enzymes were able to make 2HG (2-hydroxyglutarate) at saturating
`
`levels of reactants aKG and NADPH.
`
`EXAMPLE 11: 2-HG accumulates in AML with IDHl/2 mutations
`
`Patients and clinical data
`
`Peripheral blood and bone marrow were collected from AML patients at the time of
`
`diagnosis and at relapse, following REB approved informed consent. The cells were
`
`separated by ficol hypaque centrifugation, and stored at -150° C in 10% DMSO, 40%
`
`PCS and 50% alpha-MEM medium. Patient sera were stored at -80° C. Cyt