Fig. 1: ART558 inhibits Polθ polymerase activity.
From: Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance
a Structure and biochemical IC50 activity of the DNA polymerase Polθ inhibitor ART558 and its inactive isomer, ART615. b Biochemical dose–response curves for ART558 and ART615. c dNTP competition assay. ART558 exhibits non-competitive inhibition with respect to dNTPs. d DNA competition assay. ART558 exhibits uncompetitive inhibition with respect to DNA. e Differential scanning fluorimetry assay. ART558 shows DNA-dependent thermal stabilisation of Polθ, ART615 does not. Each data point represents the mean ± SD from two technical replicates. f DNA damage localization assay. YFP-Polθ was recruited to sites of laser-microirradiation after pre-incubation with the indicated compounds or DMSO. Each data point represents the mean ± SEM from n cells (DMSO n = 28; 12 µM ART615 n = 21; 3 µM ART558 n = 17, 12 µM ART588 n = 27). ART558 (orange, red), but not ART615 (grey), enhanced YFP-Polθ recruitment indicating direct target engagement. g Left: YFP-Polθ expression in U2OS Flp-In Trex cells. Right: images of YFP-Polθ localization to DNA damage. Frames at indicated time points show representative recruitment of YFP-Polθ to damage after compound treatment, scale bar = 20 μm. White arrow indicates micro-irradiation site. h Schematic describing a luminescent, extrachromosomal, TMEJ reporter assay. Structure of the extrachromosomal TMEJ repair reporter substrate is shown. i ART558 suppresses TMEJ-mediated repair of an extrachromosomal DNA substrate. ART558 suppressed the formation of the MMEJ product, but not the NHEJ product. j ART558, but not ART615, inhibits TMEJ-mediated repair of a NanoLuciferease-encoding TMEJ reporter substrate. ART558 inhibited TMEJ in a dose-dependent manner, with a cellular EC50 of 150 nM. Data represent mean ± SEM of n = 4 (technical replicates) and are representative of biological replicates using indicated compounds. k, l Clonogenic survival of Polq wildtype and Polq null mouse embryonic stem cells after exposure to different doses of ionizing radiation (IR). k ART558 elicits radio-sensitivity in Polq wildtype but not Polq null mouse embryonic stem cells. l ART615 does not elicit radio-sensitivity. Cell survival seven days after irradiation is shown. Data are mean ± SD, n = 3 Surviving Fractions. Two-way ANOVA with Tukey post hoc test was used to calculate p values, p = ns for p values >0.1.
