Journal: Journal of Medicinal Chemistry

Article Title: Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

doi: 10.1021/acs.jmedchem.3c01820

Figure Lengend Snippet: Small molecule inhibitors tested against TET1–3. The focused panel contains broad-spectrum 2OG oxygenase inhibitors (A, purple), HIF PHD inhibitors (B, green), KDM targeted inhibitors (C, black), HDAC inhibitor (D, red), TCA cycle intermediates and related compounds (E, blue), and relevant nucleosides (F, yellow).

Article Snippet: Recombinant TET1 CD (L1418-V2136) with an N-terminal 3 × Flag-tag produced in Sf9 cells was purchased from Epigentek (Cat#: E12002-1).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

doi: 10.1021/acs.jmedchem.3c01820

Figure Lengend Snippet: Representative AlphaScreen IC 50 curves and correlation curves for inhibitors tested against TET1–3 CD and TET2 CDΔLCI . Plots for TET1 CD (A,B), TET2 CD (C,D), TET3 CD (E,F), and TET2 CDΔLCI (G,H) of selected inhibitors are shown. IOX1 (3, orange), NOG (5, pink), JIB-04 (14, gray), S -2HG (22, purple), R -2HG (23, black), ML324 (15, green), Vadadustat (13, red), IOX4 (8, yellow) and Panobinostat (21, blue). I-L AlphaScreen pIC 50 correlation plots for inhibitors for TET1–3 CD , TET2 CDΔLCI . Pearson correlation and Spearman coefficients were calculated for comparisons. Standard conditions: 5m C (1, 10 nM), ascorbate (100 μM), Fe(II) (10 μM), 2OG (10 μM), with TET1 CD (10 nM, 30 min incubation), TET2 CD (1 nM, 10 min incubation), TET3 CD (10 nM, 10 min incubation), or TET2 CDΔLCI (1 nM, 10 min incubation). n = 2–4, error given as ± StDev. IC 50 values are displayed in Table ; compound structures are given in Figure . Circled dots—other compounds from Table .

Article Snippet: Recombinant TET1 CD (L1418-V2136) with an N-terminal 3 × Flag-tag produced in Sf9 cells was purchased from Epigentek (Cat#: E12002-1).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Incubation

Journal: Journal of Medicinal Chemistry

Article Title: Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

doi: 10.1021/acs.jmedchem.3c01820

Figure Lengend Snippet: Evidence that the TET isozymes have different sensitivities to 2-hydroxyglurate enantiomers (2HGs) in cells. Representative dose–response curves for IF cell assays for U2OS cells stably expressing TET1 CD (A), TET2 CD (B), and TET3 CD (C) dosed with inhibitors for 24 h. Data are normalized to 5hm C levels of doxycyclin-induced TET-expressing cells (+Dox) and uninduced (-Dox) cells treated with 1% DMSO. Data are plotted as mean, and the error is given as ± s.e.m ( n > 3000 cells). (D) Tabulated cellular pEC 50 values of inhibitors in IF assays for TET1 CD , TET2 CD , and TET3 CD . Data are shown as mean with error given as ± StDev. of independent biological replicates with the number of replicates in brackets. N.I.: no inhibition at 3 mM. See Figure S13 for associated FLAG staining, cell counts, and TET1 CD MUT data.

Article Snippet: Recombinant TET1 CD (L1418-V2136) with an N-terminal 3 × Flag-tag produced in Sf9 cells was purchased from Epigentek (Cat#: E12002-1).

Techniques: Stable Transfection, Expressing, Inhibition, Staining

Journal: Journal of Medicinal Chemistry

Article Title: Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate

doi: 10.1021/acs.jmedchem.3c01820

Figure Lengend Snippet: Small molecule inhibitors of TETs can reduce global 5hm C levels in cells. (A) Selected images of IF staining of Dox-inducible U2OS cells stably transfected with FLAG-tagged TET1 CD . An increase in FLAG (red) and 5hm C (green) staining, corresponding to overexpression of catalytically active TET1 CD , is observed only after Dox (1 mg mL –1 )-mediated induction. DAPI nuclear staining is in blue. Reduction in the 5hm C level is observed while FLAG staining is maintained when cells are treated with TET inhibitors (e.g., IOX4 8 ). This trend corresponds to observations with cells overexpressing a catalytically inactive TET1 CD mutant ( Figures S7–S9 ). (B) Representative EC 50 curves for IOX1 3 , IOX4 8 , JIB-04 14 , and DMOG 30 for Dox-induced U2OS cells overexpressing TET1 CD . All tested compounds reduce 5hm C levels in a dose-dependent manner. The 5hm C levels of Dox-induced and -uninduced control cells (1% DMSO) are indicated. Data are plotted as mean and error given as ± s.e.m ( n > 3000 cells). See Figures S9 and S11 for dosing data on TET1 CD MUT.

Article Snippet: Recombinant TET1 CD (L1418-V2136) with an N-terminal 3 × Flag-tag produced in Sf9 cells was purchased from Epigentek (Cat#: E12002-1).

Techniques: Staining, Stable Transfection, Transfection, Over Expression, Mutagenesis