Fig 1: High-throughput screening reveals that dyclonine protects FA patient fibroblasts from diamide stress. (A) Effect of antioxidant inhibitors on 50B11 cell viability with FXN knockdown. Eleven inhibitors of thiol-related antioxidants were tested in an siRNA-mediated, FXN-deficient 50B11 DRG cell line [10 µM antimycin A, 1 µM auranofin, 100 µM BSO, 100 µM carmustine, 10 µM diamide, 0.1% diethyl maleate (DEM), 0.1% ethanol, 0.03% H2O2, 1 mm l-glutathione (l-GSH), 0.1% phenethyl isothiocyanate (PEITC), 100 µM dichloronitrobenzene (DCNB) and 1 µM N-methyl protoporphyrin (NMP)]. Cell viability was measured with Calcein-AM after 24 h and normalized to untreated control (n = 3). Increased sensitivity to cell death was induced by inhibitors of thiol-related antioxidants diamide and auranofin in FXN knockdown cells compared with AllStars non-targeting siRNA negative control. (B) FXN-dependent sensitivity to diamide is dose-dependent in 50B11 cells. Cell viability was measured with Calcein-AM after 24 h of treatment with 3–300 µM diamide and normalized to untreated control (n = 3). (C) Friedreich's patient cells are sensitive to diamide. To confirm these effects in patient cells with low FXN, we tested 100 µM diamide in fibroblasts and lymphoblasts and found that patient cells were more sensitive to diamide compared with healthy control cells. (D) Results of high-throughput screen for drugs that protect from diamide toxicity. Significance is shown comparing healthy volunteer and FA patient lines grouped together. This cell-based assay in FA patient fibroblast cell line 1134 was further optimized for high-throughput screening in 96-well plates, with a mean Z′-value of 0.75 (n = 25). This platform was used to screen a library of 1600 drugs that have been approved for clinical use. FA fibroblasts were pretreated with 10 µM test compound, DMSO (negative control) or 300 µM dithiothreitol (DTT) (positive control) for 24 h and followed by 100 µM diamide for 24 h. Cell viability was measured with Calcein-AM. Screening data (diamide + all drugs) are the mean of two replicates and presented as fold above DMSO + diamide control. Arrow indicates dyclonine response. Mean + SD for the 1600 drugs was 1 ± 0.3; mean + SEM was 1 ± 0.01. Compounds that rescued from diamide toxicity greater than mean + 2× SD advanced to secondary screening, which included replication of protective effect in a concentration-dependent manner, 0.01–10 µM. (E) An example of dose-dependent protection by dyclonine. Dyclonine was added to FA fibroblast line 1134 for 24 h before 100 µM diamide treatment, and Calcein-AM viability is shown as fold above DMSO + diamide control. Intrinsic effect: 2.1 ± 0.49-fold above DMSO + diamide control; EC50: 0.36 ± 0.25 μM (n = 3). (F) Chemical structure of dyclonine. The plotted data in A, B, C and E display mean responses and error bars represent SD (n = 3–4). *P < 0.05, **P < 0.001 relative to control, t-test.
Fig 2: Dyclonine drives an induction of Nrf2 through the AREs in FXN gene. (A) Dyclonine increases the expression of ARE-luciferase reporter gene. To explore the mechanism of FXN induction by dyclonine, effects on the nrf2-target ARE were evaluated in a reporter HeLa cell line transduced with ARE-luciferase. Cells were treated with 1.25–10 µM dyclonine or vehicle control (0.1% DMSO). After 24 h, cells were lysed and luciferase activity was measured on a plate reader. The plotted data represent the mean fold change in luminescence in drug-treated cells normalized to vehicle control. (+) control = 5 µM sulforaphane. Error bars represent SEM. **P < 0.01, t-test (n = 3). (B) Dyclonine increases nrf2-target protein expression in the FA mouse cerebellum. FA-YG8 transgenic mice [hFXN+/− with FXN (GAA)190 expansion; mFxn−/−] were treated with 1–10 mg/kg dyclonine for 1 week p.o. Cerebellar lysates were probed by western blot analysis for nrf2-target proteins HO1, NQO1, and GPX4 expression and normalized to β-actin. The plotted data represent the mean fold change in FXN protein in drug-treated mice normalized to vehicle control. Error bars represent SEM. *P < 0.05, **P < 0.01, t-test. (n = 3). A representative blot is shown for cerebellum from FA-YG8 mice treated with 10 mg/kg dyclonine for 1 week. (C) Dyclonine induces nrf2 binding to ARE sites in Fxn and Hmox1. Multiple ARE sites were found upstream of FXN gene (Supplementary Material, Fig. S2). The top two candidates 5597 and 16722 bp upstream were selected from position weight. ChIP assays were performed to determine the binding of Nrf2 to these sites and the promoter of Hmox1 as a positive control. FA lymphoblasts (GM14518, GM15850 and GM16220) were treated with vehicle (0.1% DMSO), 5 µM dyclonine or 5 µM of the nrf2-inducer dimethyl-fumarate for 24 h. Chromatin was immunoprecipitated with anti-nrf2 antibody or anti-IgG negative control and by PCR for target loci. The plotted data represent the mean fold enrichment of PCR product with Nrf2 pulldown compared with IgG control. This shows increased amplification of regions of DNA for both the ARE sites, 16 722 bp upstream of fxn and Hmox1 after dyclonine treatment. Error bars represent SEM. *P < 0.05, **P < 0.01, t-test (n = 5 individual pulldowns per condition, n = 3 per experiments).
Fig 3: Induction of the FXN Gene in 4 Different Types of FRDA Cells with 3 Best plTALEVP64Fibroblasts of 4 different FRDA patients (i.e., FRDA66, with 240 and 640 GAA repeats; FRDA162, 355/805; FRDA4675, 255/1,140; and FRDA4743, 470/970) were treated with plTALEVP64-6-15, plTALEVP64-8-15, or plTALEVP64-F4-15 to increase the expression of the endogenous FXN gene. The FXN expression was compared to untreated cells and cells treated with empty pCR3.1. (A) The expression of the FXN gene in control cells and in treated FRDA cells is presented as the number of FXN mRNA copies per microgram RNA. (B and C) The FXN mRNAs are normalized with either GAPDH (B) or HPRT (C) mRNAs. (D–G) Frataxin protein in cells from these 2 FRDA (D and E in FRDA4675 and F and G in FRDA4743) patients were treated with the best plTALEVP64s and compared with negative controls (untreated cells or cells treated with empty plasmid). *p < 0.05, **p < 0.003, ***p < 0.0003, and ****p < 0.0001. (A–C) Results are the average ± SEM.
Fig 4: Induction of the FXN Gene by the 2 Best plTALEST10X and Their Synergistic Effect in Fibroblasts of 4 Different FRDA Patients Compared with Those of 4 Normal Subjects(A) Number of copies of FXN mRNA in 4 different normal fibroblasts (NED) and in 4 FRDA cells (FRDA66 cells, with 240/640 GAA repeats; FRDA162 cells, with 355/805 GAA repeats; FRDA4675 cells, with 255/1,140 GAA repeats; and FRDA4743 cells, with 470/970 GAA repeats) in negative control fibroblasts and after induction of the endogenous FXN gene with the 2 best plTALESTs (i.e., plTALEST10X-6-15 and plTALEST10X-8-15). (B and C) FXN mRNA normalized with GAPDH (B) or HPRT (C) mRNAs in various FRDA cells not treated or treated with plTALEST10X-6-15, plTALEST10X-8-15 alone or in pair. (D and E) Induction of the expression of the frataxin protein in cells from these 4 FRDA patients treated with 1 or 2 plTALEST10X with respect to the negative controls. Western blots for frataxin protein (D) were quantified by densitometry and normalized with the GAPDH band (E). *p < 0.05, **p < 0.003, ***p < 0.0003, and ****p < 0.0001. (A–C and E) Results are the average ± SEM.
Fig 5: Dyclonine exerts epigenetic effects. (A) Dyclonine induction of FXN transcript levels in healthy and FA patient lymphoblast cells with varying GAA-repeat length. Healthy and FA patient lymphoblasts (GM16216, GM16197 and GM14518) were treated with 30 µM dyclonine or vehicle control (0.1% DMSO). RNA was analyzed after 24 h by RT-PCR with expression with primers for FXN normalized to β-actin. GAA, number of GAA repeats on FXN allele with less insertions. The plotted data represent the mean fold change in FXN transcript in drug-treated cells normalized to vehicle control for that cell line. Error bars represent SEM. *P < 0.05, **P < 0.01, t-test (n = 4). The induction of Fxn mRNA increases with GAA repeat length. (B) Dyclonine inhibits histone methyltransferase activity. Histone methyltransferase G9a (G9aHMTase) activity, which specifically methylates histone H3K9, was measured in nuclear extracts of healthy lymphoblast cells treated with drug for 60 min. Negative controls are 0.1% DMSO vehicle-treated cells; positive controls are cells treated with G9aHMTase-specific inhibitor 1 µM Bix01294. The plotted data represent the mean absorbance at 450 nm which reflects G9aHMTase activity. Error bars represent SEM. *P < 0.05, **P < 0.01, t-test. (n = 4).
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