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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

Fig. 4

CNDAC triphosphate (CNDAC-TP) is a SAMHD1 substrate. (A) Normalised results from a colorimetric SAMHD1 activity assay carried out in presence of different combinations of GTP, dGTP and CNDAC-TP. Horizontal lines and error bars represent means ± SD from three independent experiments. (B) Surface view of SAMHD1 tetramer with each subunit in a different colour. CNDAC-TP in a catalytic pocket is shown in magenta sticks. (B, inset) CNDAC-TP bound to the SAMHD1 catalytic pocket. Black asterisks indicate the site of nitrile modification. The SAMHD1 backbone is shown as ribbons with side chains shown as sticks. A magnesium ion is shown as a green sphere and coordinated waters are shown as red spheres. Portions of the structure are omitted for clarity. (C) Chemical structure of CNDAC with 2′S nitrile modification highlighted (left). 2Fo-Fc electron density (σ = 1.0) for CNDAC-TP co-crystallized in the catalytic pocket of SAMHD1 (right). Black asterisks indicate site of nitrile modification. (D) Concentrations of physiological dNTPs in THP-1 cells determined by LC-MS/MS after pre-treatment with VPX virus-like particles (VPX-VLPs, cause SAMHD1 depletion), VPR virus-like particles (VPR-VLPs, negative control), and with or without cytarabine (Ara-C) or CNDACs. Bars and error bars represent means ± SD from three independent measurements. The Lower Limit of Quantification (LLOQ) for dGTP was 0.2 ng/Pellet, so values below the LLOQ were set to 0.2 ng/Pellet (CNDAC and CNDAC + VPR). p-values were determined by two-tailed Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001). (E) AraC IC50s in THP-1 cells in the presence of different CNDAC concentrations (0, 0.375, 0.75, 1.5 μM). Horizontal lines and error bars represent means ± SD of three technical replicates of one representative experiment out of three. p-values were determined by two-tailed Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001)

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