Journal: eLife

Article Title: A 2-Hydroxybutyrate- mediated feedback loop regulates muscular fatigue

doi: 10.7554/elife.92707

Figure Lengend Snippet: Figure 4. 2-Hydroxybutyrate feedback mecanism. (A) Relative reaction rate for recombinant human BCAT2 activity assays. Leu was kept at a constant concentration of 5 mM, 2HB concentrations are indicated in the legend. Data are normalized to the maximum reaction rate for a given experiment day and data represent three independent experiment days, mean ± SD. (B) Indicated metabolites were added to culture media for indicated time prior to cell harvest for NAD +and NADH assays, NAD+/NADH ratio displayed. N=8 independent assays, Dunnett’s multiple comparisons test, **** p<0.0001, * p<0.05. (C) C2C12 myoblasts were transfected with vector control or DDK-tagged hBCAT2 before repeating the 60-min time point NAD +and NADH

Article Snippet: Activity of BCAT was assessed using recombinant human BCAT2 (rhBCAT2; R&D Systems; Cooper et al., 2002).

Techniques: Recombinant, Activity Assay, Concentration Assay, Transfection, Plasmid Preparation, Control

Journal: Cell

Article Title: Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

doi: 10.1016/j.cell.2018.08.038

Figure Lengend Snippet: (A) BCAT reactions. (B and C) Immunoblots of HOG cells expressing HA-tagged wild-type (WT) or mutant IDH1 (R132H) (n = 3) (B) or the indicated shRNAs (n = 2) (C). EV = empty vector. (D) Heatmap of metabolites in HOG cells as in (B) (n = 3) and (C) (n = 4) or HOG cells treated with the BCAT inhibitors Compound 2 (Cmpd 2) or gabapentin (gaba) (n = 3) relative to DMSO, shControl, and EV controls. Numbers after metabolites indicate m/z. (E) In vitro BCAT1 assays. Data were normalized to 0 mM (R)-2HG sample at each concentration of 2OG. For 150 μM 2OG samples, n = 4. For all other samples, n = 3. (F) Lineweaver-Burk plot of data in (E). (G) In vitro BCAT1 assays with 50 μM 2OG and (R)-2HG (n = 3) or (S)-2HG (n = 2). (H to K) In vitro transaminase assays with 50 μM 2OG. For panels (H), (I), (J), and (K), n = 3, 4, 8, and 4, respectively. (L to N) In silico modeling of 2OG, (R)-2HG, and (S)-2HG binding to human BCAT2 in the PMPbound conformation. Dashed lines = predicted hydrogen bonds. Asterisks = 2 additional hydrogen bonds predicted to be formed by (R)-2HG relative to (S)-2HG. For all panels, data presented are means ± SD; *p < .05, **p < .01, ***p < .001. Two-tailed p values were determined by unpaired t-test. See also Figure S1.

Article Snippet: Recombinant transaminase activity assays Recombinant human BCAT1, BCAT2, GOT1, GOT2, and OAT enzymes were purchased from MyBioSource.

Techniques: Western Blot, Expressing, Mutagenesis, Plasmid Preparation, In Vitro, Concentration Assay, In Silico, Binding Assay, Two Tailed Test

Journal: Cell

Article Title: Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

doi: 10.1016/j.cell.2018.08.038

Figure Lengend Snippet: (A) Schema of GLS-dependent nitrogen transfer in α-15N-glutamine tracing assays. (B and C) α-15Nglutamine tracing assays (n = 3). (D) Immunoblot analysis (n = 2). (E) 15N-(amide)-glutamine tracing in HOG stable lines (n = 4). Fractional labeling of ammonia 8 hr after tracer addition is shown. (F) Glutamate levels in NHA and HOG stable cell lines treated with 100 nM CB-839 or DMSO for 2 hr (n = 4). (G) Glutamate levels in GSC lines treated with CB-839 or DMSO for 2 hr (n = 3). (H and I) Immunoblots (H) and 15N-leucine and α-15N-glutamine tracing assays (I) in HOG stable cells expressing the indicated sgRNAs (together with Cas9) and, where indicated, sgRNA-insensitive BCAT1 and BCAT2 cDNAs. n = 3. (J) Schema depicting the major routes of glutamate synthesis in glial cells. (K and L) 15N-BCAA and α-15N-glutamine tracing assays of NHA (K) and HOG (L) cells (n = 3). Fractional labeling of the glutamate pool at 6 hr is shown. (M) α-15N-glutamine (n = 6) and 15N-leucine (n = 3) tracing assays in HOG cells treated with 100 μM Compound 2 or 100 nM CB-839, respectively. Cells were given 15N-labeled amino acids for 20 minutes. (N) Immunoblot analysis of HOG stable lines infected to produce the GLS Y394L mutant or EV. KGA and GAC splice isoforms are indicated. n = 3. (O) Glutamate levels in HOG stable cell lines as in (N) (n = 3). For panels (N) and (O), see also Figures S3P to S3R. For all panels, data presented are means ± SD; *p < .05, **p < .01, ***p < .001. Two-tailed p values were determined by unpaired t-test. See also Figure S3.

Article Snippet: Recombinant transaminase activity assays Recombinant human BCAT1, BCAT2, GOT1, GOT2, and OAT enzymes were purchased from MyBioSource.

Techniques: Western Blot, Labeling, Stable Transfection, Expressing, Infection, Mutagenesis, Two Tailed Test

Journal: Cell

Article Title: Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

doi: 10.1016/j.cell.2018.08.038

Figure Lengend Snippet: (A) GSH levels and reduced/oxidized glutathione ratios in HOG orthotopic gliomas. n = 8 and 6 IDH1 wild-type and mutant tumors, respectively. (B) Total glutathione levels in IDH1 mutant HOG (n = 3) and MGG152 (n = 5) cells treated with 3 μM AGI-5198 or DMSO for 3 days. (C) Schema for regulation of glutathione synthesis by mutant IDH. (D and E) Volcano plots of metabolites in NHA cells treated with 500 μM (R)-2HG-TFMB for 24 hr (D) or expressing a BCAT1 shRNA (E) relative to DMSO and a control shRNA, respectively. p values were determined via t-test using MetaboAnalyst. n = 3. (F) Venn diagram of metabolites co-regulated by (R)-2HG and a BCAT1 shRNA. (G) 15N-BCAA tracing assays of HOG stable lines (n = 3). Fractional labeling of the glutamate and GSH pools is shown at 6 and 12 hr, with values normalized to HOG EV cells at 6 hr. (H and I) Total glutathione levels in HOG and NHA cells treated for 8 hr with Compound 2 (n = 3) (H) or CB-839 (I). In (I), n = 3 for cells treated with 100 nM CB-839 and n = 4 for all others. (J) Total glutathione levels in HOG (n = 3) and NHA stable lines treated for 8 hr with 100 nM CB-839, 250 μM BSO, or DMSO. For NHA lines, n = 3 for BSO-treated IDH1 mutant cells and n = 5 for all others. (K) Total glutathione levels in HOG stable cell lines as in Figure 3N (n = 3). See also Figure S5H. (L to N) Total glutathione levels in GSC lines that were untreated (L) or treated for 8 hr with 1 μM CB-839 (M) or 250 μM BSO (N). n = 3. (O) LC-MS/MS-based GSH quantification in IDH1 mutant and wild-type HOG orthotopic gliomas treated with 7 doses of CB-839 (200 mg/kg BID) or vehicle control. n = 8, 9, 8, and 6 mice in EV-Vehicle, EV-CB-839, R132H-Vehicle, and R132H-CB-839 cohorts, respectively. For all panels, data presented are means ± SD; *p < .05, **p < .01, ***p < .001. Two-tailed p values were determined by unpaired t-test. n.s. = nonsignificant. See also Figure S5.

Article Snippet: Recombinant transaminase activity assays Recombinant human BCAT1, BCAT2, GOT1, GOT2, and OAT enzymes were purchased from MyBioSource.

Techniques: Mutagenesis, Expressing, shRNA, Labeling, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Journal of Medicinal Chemistry

Article Title: BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe

doi: 10.1021/acs.jmedchem.2c00441

Figure Lengend Snippet: Hybrid design of compound 38 . (A) Structure of compound 38 , combining substructural elements of compound B 13 with the trifluoromethyl-pyrimidinedione core, exemplified by compound 12 . (B) Superposition of two X-ray co-crystal structures that inspired the design: Co-crystal structure of BCAT2 with the compound B 13 (PDB accession code 5HNE , carbon atoms in wheat) and BCAT1 structure in complex with 12 (chain A, ligand carbon atoms in cyan, PDB 7NWM ). (C) Co-crystal structure of BCAT1 with hybrid compound 38 (PDB accession code 7NY9 ), protein carbon atoms in gray and inhibitor carbon atoms in yellow).

Article Snippet: Recombinant human BCAT1 enzyme (catalogue no. ATGP1479) and recombinant human BCAT2 enzyme (catalogue no. ATGP1523) were purchased from ATGen (South Korea).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe

doi: 10.1021/acs.jmedchem.2c00441

Figure Lengend Snippet: BCAT1 and BCAT2 Activities and BCAA Measurement in U-87 MG and MDA-MB-231 Cell Lines for Inhibitors 35 and 36 and Their Respective Atropisomers a and b a

Article Snippet: Recombinant human BCAT1 enzyme (catalogue no. ATGP1479) and recombinant human BCAT2 enzyme (catalogue no. ATGP1523) were purchased from ATGen (South Korea).

Techniques: