Fig 1: Interaction between KH176m and Peroxiredoxin. (a) Kinetic curves of KH176 and KH176m binding to immobilized human Prdx2 were obtained by Surface Plasmon Resonance. KH176m (left graph), but not KH176 (right graph), displayed a dose-dependent binding to Prdx2. (b) In this in silico model, KH176m interacts at a junction of the Peroxiredoxin 4 (Prdx4) dimer (A and B) in a pocket formed by 13 amino acids at a distance =10 Å. A nitrogen atom on the pyridine ring of KH176m interacts with Cys124 and Thr121 of the b monomer of Prdx4. On the same monomer, Val123 forms also a third hydrogen bond with the oxygen atom on the open ring of KH176m. KH176m forms also electrostatic interactions with Val244 and Leu118. (c) and (d) show different angles of the interaction between KH176m and Prdx4.
Fig 2: Involvement of the Thioredoxin System in the mode of action of KH176(m). (a) KH176 and KH176m can rescue P4 cells from BSO (200 µM)-induced death, their efficiency depends on an active Thioredoxin System since the presence of 100 nM AFN inhibits the rescue. ***p < 0.001 as compared to the indicated control. Increasing amounts of KH176 (b) or KH176m (c) rescue BSO (200 uM) treated cells, different concentrations of AFN affect the efficacy, but not potency of KH176(m). (d) the antioxidant activity of KH176(m) is not affected by inhibition of the Thioredoxin System by AFN (100 nM). ***p < 0.001 and n.s. = non-significant both as compared to the indicated bars. (e) KH176m but not KH176 enhances the Thioredoxin System/Peroxiredoxin-dependent consumption of NADPH. TrxR1, Trx1 and Prdx2 were incubated with NADPH and H2O2 in the presence or absence of 100 µM of KH176 or KH176m. The graph reports the fluorescence signal of NADPH over time. − = no KH compounds. For all panels: unless otherwise indicated the data points represent the average of triplicate measurements and are normalized on the untreated condition, SD is indicated. ***p < 0.001, n.s. = non-significant.
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