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. 2023 Jun 22;14:3737. doi: 10.1038/s41467-023-39444-y

Fig. 3. Functional characterization of the inhibitors.

Fig. 3

a Reversibility of TGR inhibitors. Activity (5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) reduction) of the enzyme (3.7 nM) incubated with 250 µM inhibitor for 15 min was determined (red bars). TGR (370 nM) was incubated with 250 µM compound and 100 µM NADPH for 15 min. The sample was diluted 100-fold and the activity was determined immediately (blue bars) and after 60 min (light grey bars). AF = auranofin. Average ± standard deviation (n = 3) shown. b NADPH dependence of inhibitors. Activity of TGR after exposure to 6 (green circles), 7 (blue squares), or 8 (black triangles) with or without initial incubation with NADPH compared to control TGR incubated with NADPH and without inhibitor. Average ± standard deviation (n = 3) shown. c Time dependence of inhibition. Inhibition of DTNB reduction by TGR in the presence of 50 µM fast inhibitors 6 (orange), 7 (blue), 8 (black) compared to TGR incubated without compound. Average ± standard deviation (n = 3) shown. d Time dependence of inhibition. Time-dependent activity (DTNB reduction) of TGR in the presence of 50 µM slow inhibitors 1 (purple), 3 (orange), 4 (black), 5 (blue) compared to TGR activity incubated in the absence of compounds. Average ± standard deviation (n = 3) shown. e Compound effect on thermal stability of TGR. Melting temperature without (black) or with addition of 500 µM NADPH (red) of TGR alone or with 100 µM inhibitor 1, 3, 5, 7, 8, 9 or control compounds 11 or 12. Average ± standard deviation (n = 3) shown. f Oxidase activity after incubation with inhibitors. Consumption of NADPH (ΔA340/min) by TGR after exposure to inhibitors for 15 min in presence of NADPH. Average ± standard deviation (n = 3) shown except control n = 12. g Production of superoxide by TGR after incubation with inhibitors. Superoxide production was determined by measuring consumption of pyrogallol red (ΔA540/min) without added superoxide dismutase (blue circles) and with added superoxide dismutase (black squares) Average ± standard deviation, n = 3 except Auranofin n = 2, TRi-1 with SOD, n = 2, compound 4 without SOD, n = 2, and compound 6 with SOD, n = 2. Source data are provided as a Source Data file.