KH176 Safeguards Mitochondrial Diseased Cells from Redox Stress-Induced Cell Death by Interacting with the Thioredoxin System/Peroxiredoxin Enzyme Machinery

Abstract

A deficient activity of one or more of the mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes leads to devastating diseases, with high unmet medical needs. Mitochondria, and more specifically the OXPHOS system, are the main cellular production sites of Reactive Oxygen Species (ROS). Increased ROS production, ultimately leading to irreversible oxidative damage of macromolecules or to more selective and reversible redox modulation of cell signalling, is a causative hallmark of mitochondrial diseases. Here we report on the development of a new clinical-stage drug KH176 acting as a ROS-Redox modulator. Patient-derived primary skin fibroblasts were used to assess the potency of a new library of chromanyl-based compounds to reduce ROS levels and protect cells against redox-stress. The lead compound KH176 was studied in cell-based and enzymatic assays and in silico. Additionally, the metabolism, pharmacokinetics and toxicokinetics of KH176 were assessed in vivo in different animal species. We demonstrate that KH176 can effectively reduce increased cellular ROS levels and protect OXPHOS deficient primary cells against redox perturbation by targeting the Thioredoxin/Peroxiredoxin system. Due to its dual activity as antioxidant and redox modulator, KH176 offers a novel approach to the treatment of mitochondrial (-related) diseases. KH176 efficacy and safety are currently being evaluated in a Phase 2 clinical trial.

Figure 1

Oxidative stress in Complex I deficient primary human skin fibroblasts. (a) Basal ROS levels in 3 healthy control cell lines (C1 to C3) and 7 patient cell lines (P1 to P7) bearing mutations in different nuclear encoded Complex I subunits. Bar graphs represent the average of at least 3 independent measurements ± SD, and are normalized on C1 response. AFI = average fluorescence intensity. (b) Cell viability of the same panel of cell lines upon 24 h treatment with 100 µM BSO. Bar graphs represent the average of at least 3 independent measurements ± SD, and are normalized on the untreated condition per cell line. For statistical analysis each patient cell line was tested against the average of the 3 control cell lines. **p < 0.01, ***p < 0.001.

Figure 2

Screening strategy for the selection of KH176. (a) Chemical structures of the hit compound Trolox and the lead compound KH176. The scaffold used for the synthesis of the 226 new chemical entities is also shown. (b) P4 cell line was used to evaluate the ability of the newly synthesized Trolox-derivative compounds to reduce ROS levels after 72 h treatment with semi-logarithmic dilutions of the test compound, and (c) protect cell viability from BSO-induced toxicity. Four illustrative curves are shown for selected compounds with each data point representing the average of a triplicate measurement, normalized on the untreated condition. (d) IC₅₀ (ROS assay) and EC₅₀ values (Redox Stress Survival assay) were used to generate a correlation plot indicating KH176 with the highest overall potency among the screened compounds.

Figure 3

Effects of KH176, KH176m and KH176i in cellular assays. (a) Cell viability of P4 cell line treated for 24 h with 200 µM BSO in co-incubation with different concentrations of KH176, KH176m, or the redox-silent KH176i. EC₅₀ KH176 = 2.7 × 10⁻⁷ M; EC₅₀ KH176m = 3.87 × 10⁻⁸ M. (b) ROS assay performed on P4 cell line after 24 h compound treatment showing that KH176m is a more potent ROS scavenger than KH176 and KH176i is inactive. IC₅₀ KH176m = 2.5 × 10⁻⁷ M. SD is indicated but is for most data points smaller than the symbol. (c) The P4 cell line was treated with 3 μM of KH176, KH176m or KH176i just before the addition of 100 µM H₂O₂ (white bars) or 1 mM KO₂ (black bars). Neutralization of both H₂O₂ and O₂^.− by KH176 and KH176m is shown by a decreased CM-H₂DCFDA oxidation. Normalization was performed on the basal condition (no exogenous oxidant). Compounds effects were compared to vehicle; ***p < 0.001; n.s. = non-significant. (d) Cellular superoxide levels were detected using HEt. Basal levels were decreased in the presence of KH176m (EC₅₀ KH176m = 1.7 × 10⁻⁶ M). KH176 had no effect. Results are the average of two independent experiments, SD is indicated. (e) Mitochondrial superoxide levels, detected with mitoSOX, could be decreased by KH176m (IC₅₀ = 1.4 × 10⁻⁶ M). KH176 was much less potent. (f) CumOOH induced lipid peroxidation measured with bodipy 581/591 C11 could be inhibited by KH176 (IC₅₀ = 6.4 × 10⁻⁵ M) and KH176m (IC₅₀ = 7.1 × 10⁻⁸ M), KH176i had no effect. 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.

Figure 4

KH176 and KH176m can rescue BSO-induced cell toxicity without increasing the GSH level. GSH level and corresponding cell viability of C1 and P4 cell lines treated for 24 h with increasing concentrations of (a) BSO alone or (b) in co-incubation with 3 µM KH176 or (c) 1 µM KH176m. Dashed lines represent the average GSH level of at least 2 independent measurements ± SD, while solid lines show the parallel cell viability average value ± SD. Data are all normalized on the untreated condition. Statistical significance between C1 and P4 viability is indicated.

Figure 5

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.

Figure 6

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.