Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia

Abstract

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreich's ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron-sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA.

Figure 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.

Figure 2.

Dyclonine induces FXN in cultured FA patient cells and FA mouse model cerebellum in vivo. (A) Dyclonine increases FXN protein expression in FA patient lymphoblast cell lines. To test if the mechanism of protection from diamide toxicity for dyclonine was through an increase in FXN protein levels, FA lymphoblasts were treated with 0.3–10 µM dyclonine or vehicle control (0.1% DMSO). Total protein was collected after 48 h, and lysates were probed by western blot analysis for FXN expression and normalized to β-actin. The plotted data represent the mean fold change in FXN protein in drug-treated cells, normalized to vehicle control. Error bars represent SEM. **P < 0.01, t-test (n = 2–15). A representative blot is shown for FA patient lymphoblast line 15850 treated with 10 µM dyclonine for 48 h. (B) Dyclonine increases FXN transcript levels in FA patient lymphoblasts. FA lymphoblast cell line 14518 was treated with 3–30 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. The plotted data represent the mean fold change in FXN transcript in drug-treated cells normalized to vehicle control. Error bars represent SEM. *P < 0.05, **P < 0.01, (n = 4), t-test. (C) Dyclonine increases FXN protein concentration in FA-YG8 mouse cerebellum. To determine the ability of dyclonine to reverse the in vivo FXN protein defect, FA-YG8 transgenic mice [hFXN^+/− with FXN (GAA)₁₉₀ expansion; mFxn^−/−] were treated with 1–10 mg/kg dyclonine for 1 week i.p. or p.o. Cerebellar lysates were probed by western blot analysis for FXN 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 = 3–10 and 11–12 months of age). A representative blot is shown for cerebellum from FA-YG8 mice treated with 10 mg/kg dyclonine for 1 week. FA-YG8 [hFXN^+/+ with FXN (GAA)₁₉₀ expansion; mFxn^−/−] mice with two copies of transgene used as a control. (D) Dyclonine increases FXN protein levels in FA-PandKIKO mouse cerebellum. To determine the ability of dyclonine to reverse the in vivo FXN protein defect in an additional FA model at a higher dose and duration, FA-PandKIKO mice [mFxn^+/− with FXN (GAA)₂₃₀ expansion; mFxn^−/−] were treated with 25 mg/kg dyclonine for 1 or 4 weeks p.o. Cerebellar lysates were probed by western blot analysis for FXN 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 < 0.01, t-test. (n = 3–17 and 8–10 months of age). A representative blot is shown for cerebellum from FA-YG8 mice treated with 25 mg/kg dyclonine for 1 week. Wild-type C57BL/6 mice used as a control.

Figure 3.

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).

Figure 4.

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).

Figure 5.

Dyclonine recovers downstream effects of FXN deficiency in vitro and in vivo. (A) Dyclonine increases aconitase activity in FA patient lymphoblast cell lines. FA lymphoblasts (GM16205, GM16197 and GM16243) were treated with 10 µM dyclonine or vehicle control (0.1% DMSO). Cell pellets were collected after 48 h, and aconitase activity was measured in lysates over 90 min to ensure linear range was captured. A healthy volunteer lymphoblast cell line was used as a control. The plotted data represent the mean rate change in fluorescence (dF/dt) from 25 to 30 min. Error bars represent SEM. *P < 0.05, t-test. (n = 3). (B) Dyclonine increases aconitase activity in FA mouse model cerebellum in vivo. FA-YG8 transgenic mice [hFXN^+/− with FXN (GAA)₁₉₀ expansion; mFxn^−/−] were treated with 5 mg/kg dyclonine for 1 week p.o. Aconitase activity was measured in cerebellar lysates over 90 min to ensure linear range was captured. Positive controls were vehicle-treated FA-YG8 transgenic mice [hFXN^+/+ with FXN (GAA)₁₉₀ expansion; mFxn^−/−]. The plotted data represent the mean rate change in fluorescence (dF/dt) from 15 to 20 min. Error bars represent SEM. *P < 0.05, t-test (n = 4 mice per group, 11–12 months of age). (C) Dyclonine increases succinate dehydrogenase activity in FA mouse model liver in vivo. FA-PandKIKO mice [mFxn^+/− with FXN (GAA)₂₃₀ expansion; mFxn^−/−] were treated with 25 mg/kg dyclonine for 1 week p.o. Succinate dehydrogenase activity was measured in liver lysates over 90 min. Positive controls were vehicle-treated WT C57/Bl6 mice. The plotted data represent the mean rate change in fluorescence (dF/dt) from 25 to 30 min. Error bars represent SEM. *P < 0.05, t-test (n = 4 mice per group, 8–9 months of age). (D) Dyclonine improves progressive behavioral defect of FA mice. FA-PandKIKO mice [mFxn^+/− with FXN (GAA)₂₃₀ expansion; mFxn^−/−] showed a pronounced increase in beam crossing time compared with WT controls at 13 months of age. FA-PandKIKO mice were treated with 25 mg/kg dyclonine or vehicle for 4 weeks p.o. Latency to cross a 16 mm beam was the recorded time from start to finish of the beam, with the experimenter blinded to treatment groups. The plotted data represent the mean latency to cross a beam post-dosing minus the time to cross pre-dosing for each individual mouse and serve as a change from baseline measurement. As a change from baseline, a negative number means that the animal was faster after dosing than it was before dosing. Positive controls were vehicle-treated WT C57/Bl6 mice. *P < 0.05, t-test (n = 7–9 mice per group, 12–14 months of age).

Figure 6.

Dyclonine induction of FXN in buccal cells of FA patients correlates with disease severity. FA patients were dosed with a 1% dyclonine oral rinse, twice daily for 30 s. Buccal cells were collected by cheek swabbing before and after 1 week of dosing. Cell lysates were probed by dipstick immunoassay for analysis of FXN expression and normalized to mitochondrial complex IV (n = 4 replicate experiments of FXN analysis per patient, summarized in Table 1). The plotted data represent the mean fold change in FXN protein in dyclonine-treated FA patient buccal cells normalized to expression for each individual patient before drug dosing (healthy volunteers not shown). This fold FXN induction is plotted against (A) FARS score, (B) FDS, (C) Z2 ataxia functional composite score and (D) FXN gene GAA-repeat length for each FA patient who participated in this clinical study (obtained through a natural history database). For A and B, a higher score indicates greater disease severity. For C, a lower score indicates greater disease severity. A P-value of <0.05 is considered significant, Pearson's correlation coefficient.