Fig. 3: Napabucasin targets 2-oxoglutarate:acceptor oxidoreductase.
a Representative image of DATRS assay to test the binding between napabucasin (NPB) and OorD. The OorD protein together with BSA was treated with NPB (10, 100, and 1000 μg/ml) and DMSO, proteolyzed by pronase, and then detected by silver staining. The gel is representative of three independent experiments. b MST measurements of the OOR/OorD proteins binding with NPB at several concentrations (0.488 nM–8 μM). Normalized fluorescence (ΔFnorm) from three independent experiments, with SEM shown, was plotted against the concentration of napabucasin or metronidazole. Data analysis was performed with the MO Affinity Analysis software (NanoTemper Technologies) using the Kd model. c NPB inhibits menaquinone 6 (MK-6) reduction by OOR. The IC50 value for inhibition of MK-6 reduction activity was determined using OOR assays in vitro. d High performance liquid chromatography (HPLC) chromatograms show production of succinyl-CoA (S-CoA) by the OOR reactions with NPB and MK-6 as the electron acceptors, as seen by the peak at 22.4 min in the samples. e Kinetic characterization of OOR catalysis with NPB (red) and MK-6 (blue). The curves were fitted to the Michaelis–Menten equation. f Cell viability of H. pylori strain G27 transformed with pTM117 (Vec), pBHKP500 [p(oorDABC)] or pBHKP501 [p(oorD)], performed in Columbia blood agar plates supplemented with or without NPB. Growth with/without metronidazole was used as a control experiment. The figure is representative of 3 independent experiments. g Detection of ROS levels in H. pylori cells treated with NPB after 4 h of incubation. Statistical significance was calculated using a one-way ANOVA with Tukey’s post-hoc test. h Proposed MoA of NPB against H. pylori. i The anti-H. pylori activities of 1,4-naphthoquinone derivatives correlate with their efficiency in OOR-catalyzed reduction. MK-6 was used as a control and showed no antibacterial activity against H. pylori without inducing ROS generation. N.D., not detected. Data are represented as mean ± SEM (n = 3 biological replicates) in (b, c, e, g, i). Source data are provided as a Source Data file.
