Journal: Nature chemical biology

Article Title: Development of a chemical probe against NUDT15

doi: 10.1038/s41589-020-0592-z

Figure Lengend Snippet: a . Close-up view of the binding interactions between TH1760 with NUDT15. Hydrogen bonds are shown in black and relevant residues are shown in stick representation. NUDT15 is shown in green, TH1760 in magenta and 2Fo-Fc electron density map around TH1760 in blue. b . Comparison of the binding positions of TH1760 and 6-Thio-GMP. Structurally aligned 6-Thio-GMP (PDB ID: 5LPG) is shown in grey. c . Ligplot+ representation of interactions between NUDT15 and TH1760, with hydrophobic interactions shown as an arc with spokes and hydrogen bonds shown as dashed lines. d . TH7285, a close analogue of TH1760, could not inhibit NUDT15, shown using MG assay. Inhibition % of n=2 experiments performed in duplicate shown. e . TH7285 minimally stabilized NUDT15 at 10 μM, shown using DSF assay. Mean RFU ± SEM of n = 3 experiments shown.

Article Snippet: Since the Km of NUDT15 for 6-thio-dGTP was previously determined to 2 μM , a 6-thio-dGTP (Jena Bioscience, NU-1213S) concentration of 2.5 μM was used in the assay.

Techniques: Binding Assay, Inhibition

Journal: Nature chemical biology

Article Title: Development of a chemical probe against NUDT15

doi: 10.1038/s41589-020-0592-z

Figure Lengend Snippet: a . Schematic drawing of thiopurine activation and metabolism. In cells, thiopurines (Azathioprine, AZA-T; 6-mercaptopurine, 6-MP; and 6-thioguanine, 6-TG) are converted into 6-thio-(d)GTP before being incorporated into the genomic material, and eventually cause cell death. NUDT15 limits thiopurine efficacy by converting 6-thio-(d)GTP into the non-toxic 6-thio-(d)GMP. b . Depletion of NUDT15 sensitized AML-derived NB4 cells to thiopurine treatment. Expression of NUDT15-specific shRNA (shN15), but not the non-targeting control shRNA (shNT), sensitized the cells to 6-TG treatment. Cell viabilities were determined using resazurin viability assay and calculated by normalizing to no doxycycline (DOX), DMSO-treated controls. Mean % ± SEM of n=3 experiments shown. c . NUDT15 enzymatic activity is the key in modulating thiopurine cytotoxicity in NB4 cells. NB4 cells co-expressing DOX-inducible shN15 and shN15-resistant, HA-tagged NUDT15 overexpression constructs (wildtype, WT; catalytically dead, CD; or unstable, US) were assayed for viability under 6-TG treatment. Only the expression of WT, but not CD or US NUDT15 protected cells from 6-TG. Cell viability % was assayed using resazurin viability assay and calculated by normalizing to DMSO-treated controls. Mean ± SEM of n=3 experiments shown.

Article Snippet: Since the Km of NUDT15 for 6-thio-dGTP was previously determined to 2 μM , a 6-thio-dGTP (Jena Bioscience, NU-1213S) concentration of 2.5 μM was used in the assay.

Techniques: Activation Assay, Derivative Assay, Expressing, shRNA, Viability Assay, Activity Assay, Over Expression, Construct

Journal: Nature chemical biology

Article Title: Development of a chemical probe against NUDT15

doi: 10.1038/s41589-020-0592-z

Figure Lengend Snippet: a. b . NUDT15 depletion sensitized NB4 cells to 6-MP (a), and HL-60 cells to 6-TG (b). Cell viabilities assessed using resazurin viability assay after 96 (a) or 72 (b) h of treatment and calculated by normalizing to no DOX, DMSO-treated controls. Mean ± SEM of n=3 (a) or mean of n=2 (b)experiments performed in triplicates shown. Left panels: resazurin viability curve; right panels: Western blot demonstrating DOX-induced NUDT15 knockdown. c . d . Depletion of NUDT15 in HL-60 (c) or NB4 (d) did not affect DNA replication, evidenced by EdU incorporation. Cells expressing DOX-inducible NUDT15-specific (N15) or non-targeting (NT) shRNA were treated with DOX for 48 h, before EdU labelling. Left panels: Mean EdU+ve population% of n=2 experiments shown. Right panels: representative FACS histogram showing EdU signal intensity. e . E67A variant of NUDT15, compared to the wildtype (WT) construct, is catalytically inactive against 6-thio-dGTP (tested at 50μM), shown using enzyme-coupled MG assay. Mean activity of a representative experiment shown with individual repeat values. f . RT-qPCR analysis of NUDT15 mRNA levels in NB4 cells co-expressing DOX-inducible shRNA, and shRNA-resistant, HA-tagged NUDT15 constructs (wildtype, WT; unstable, US; or catalytically dead, CD), with GAPHD as the house keeping gene. NUDT15 mRNA levels were normalized to cells expressing WT NUDT15 construct. Mean of n=2 experiments performed in triplicate shown. g . Doxycycline treatment induced the co-expression of shRNA (shNT and shN15) and shN15-resistant, HA-tagged NUDT15 overexpression constructs (WT, CD, or US) in NB4 cells. h . NB4 cells co-expressing DOX-inducible shNT shRNA and shRNA-resistant, HA-tagged NUDT15 overexpression constructs (WT, CD, or US) were assayed for viabilities under 6-TG treatment. Overexpression of WT NUDT15 conferred marginal resistance to 6-TG Mean ± SEM of n=3 independent experiments performed in duplicates shown.

Article Snippet: Since the Km of NUDT15 for 6-thio-dGTP was previously determined to 2 μM , a 6-thio-dGTP (Jena Bioscience, NU-1213S) concentration of 2.5 μM was used in the assay.

Techniques: Viability Assay, Western Blot, Expressing, shRNA, Variant Assay, Construct, Activity Assay, Quantitative RT-PCR, Over Expression

Journal: Nature chemical biology

Article Title: Development of a chemical probe against NUDT15

doi: 10.1038/s41589-020-0592-z

Figure Lengend Snippet: a. b . TH1760 significantly enhanced the intracellular accumulation and incorporation of thiopurines and their metabolites. HL-60 cells were treated with thiopurines alone or combined with 10 μM TH1760 for 16h, before levels of 14 C-labbeled 6-MP metabolite in DNA were determined via radioactive counts (a), or 6-thio-dGTP lesions in DNA were measured via mass spectrometry (b). Mean ± SEM of n=3 experiments shown. In a, DMSO Vs. TH1760 group: at 1 μM 6-MP, **p = 0.00027, t ratio=4.638, df=16; at 2μM 6-MP, **p = 0.00047, t ratio=4.381, df=16. In b, DMSO Vs. TH1760 group: at 0.5μM 6-TG, **p = 0.00117, t ratio=8.267, df=4; at 1μM 6-TG, *p = 0.02, t ratio=4.532, df=3. All performed with multiple t-test (two-tailed, Holm-Sidak correction). c . TH1760 potentiated 6-TG-induced DNA damage in NB4 cells. NB4 cells treated with 6-TG alone or combined with 10 μM TH1760 for 48 h were assayed for DNA damage by alkaline comet assay. Top panel: Quantification of the tail moment of a representative experiment performed in duplicate (200 cells per condition). Lines represent geometric mean tail moments. DMSO Vs. TH1760 group: at 0 nM 6-TG, n.s., p=0.7054; 50 nM 6-TG, ****p < 0.0001, Z=9.652; 200 nM 6-TG, ****p < 0.0001, Z=11.78 (Kruskal–Wallis test, Dunn’s correction, GraphPad Prism). Bottom panel: Representative, pseudo-colored images of treated NB4 cells following the alkaline comet assay. d . Western blot of DNA damage and apoptotic markers in HL-60 cells treated as described in c, confirming that TH1760 potentiated 6-TG-induced cellular responses. Two experiments performed.

Article Snippet: Since the Km of NUDT15 for 6-thio-dGTP was previously determined to 2 μM , a 6-thio-dGTP (Jena Bioscience, NU-1213S) concentration of 2.5 μM was used in the assay.

Techniques: Mass Spectrometry, Two Tailed Test, Alkaline Single Cell Gel Electrophoresis, Western Blot