Role of dichloroacetate in the treatment of genetic mitochondrial diseases

Abstract

Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition.

Fig. 1

Pathways of pyruvate metabolism and oxidative phosphorylation. Pyruvate may be reduced to lactate in the cytoplasm or may be transported into the mitochondria for anabolic reactions, such as gluconeogenesis and lipogenesis, or for oxidation to acetyl CoA by the pyruvate dehydrogenase (PDH) complex (PDC). Reducing equivalents (NADH, FADH) are generated by reactions catalyzed by the PDC and the tricarboxylic acid cycle and donate electrons (e⁻) that enter the respiratory chain at NADH ubiquinone oxidoreductase (complex I) or at succinate ubiquinone oxidoreductase (complex II). Cytochrome c oxidase (complex IV) catalyses the reduction of molecular oxygen to water and ATP synthase (complex V) generates ATP from ADP.

Fig. 2

The pyruvate dehydrogenase (PDH) multienzyme complex (PDC). Pyruvate is decarboxylated by the PDH subunit (E₁) in the presence of thiamin pyrophosphate (TPP). The resulting hydroxyethyl-TPP complex reacts with oxidized lipoamide (LipS₂), the prosthetic group of dihydrolipoamide transacetylase (E₂), to form acetyl lipoamide. In turn, this intermediate reacts with reduced coenzyme A (CoASH) to yield acetyl CoA and reduced lipoamide (Lip(SH)₂). The cycle of reaction is completed when reduced lipoamide is reoxidised by the flavoprotein, dihydrolipoamide dehydrogenase (E₃). Finally, the reduced flavoprotein is oxidized by NAD and transfers reducing equivalents to the respiratory chain via NADH. PDC is regulated, in part, by reversible phosphorylation, in which the phosphorylated enzyme is inactive. Increases in the intramitochondrial ratios of NADH/NAD and acetyl CoA/CoA also stimulate kinase mediated phosphorylation of PDC. The drug dichloroacetate (DCA) inhibits the kinase responsible for phosphorylating PDH, thus ‘locking’ the enzyme in its unphosphorylated, catalytically active state.

Fig. 3

Biotransformation of DCA. The major route of metabolism is dechlorination to glyoxylate, whereby DCA enters the general carbon pool of the host. The conversion is catalyzed in cytosol by the bifunctional enzyme zeta-1 isoform of glutathione transferase (GSTz1)/maleylacetoacetate isomerase (MAAI). Reductive dehalogenation of DCA to monochloroacetate normally represents a very minor pathway of biotransformation. End products of DCA metabolism include carbon dioxide, oxalate and various glycine and hippuriate derivatives excreted in the urine.

Fig. 4

Tyrosine catabolic pathway and site of action of DCA. DCA depletes maleylacetoacetate isomerase (MAAI), causing accumulation of maleylacetoacetate, maleylacetone, fumarylacetoacetate and fumarylacetone. MAAI is identical to the z-1 isoform of glutathione transferase (GSTz1), which biotransforms DCA to glyoxylate. Succinylacetone also accumulates as a result of DCA and is a known inhibitor of δ- aminolevulinate dehydratase, causing buildup of δ-aminolevulinate and inhibition of heme biosynthesis. Perturbation of heme metabolism is thought to cause the neuropathic complications in patients with tyrosinemia type I.

Fig. 5

Reduction of myelin proteins in myelinating Schwann cell-dorsal root ganglion (SC-DRG) neuron co-cultures after DCA exposure. (a) Myelinating SC-DRG neuron cultures were treated with DCA for 12 days at the indicated concentrations and whole protein lysates (10μg/lane for MAG and P0; 20μg/ lane for PMP22 and MBP) were analyzed by Western blotting with the indicated antibodies. Tubulin is shown as a loading control. Molecular mass in kDa. (b) Representative cultures were immunolabeled with antibodies against MAG, P0 and MBP. Sample incubated with non-specific rabbit IgG is shown in the upper right corner. Scale bar, 10μm.

Fig. 6

Concentration-time profile of ¹³C-DCA in plasma of samples obtained in 5 previously drug-naïve young children (ages 2.2-7.1 years) after receiving an initial dose of 12.5 mg/kg (◆) and after 6 months of 12.5 mg/kg administered twice daily (▲).

Fig. 7

Concentration-time profile of ¹²C-DCA in plasma of samples obtained in 5 previously drug-naïve older subjects (ages 14.0-33.9 years) after receiving an initial dose of 12.5 mg/kg (◆) and after 6 months of 12.5 mg/kg administered twice daily (▲).

Fig. 8

Effect of scAAV serotype 6, with or without DCA, on expression of PDH complex proteins. Top panel: Immunoblot shows relative changes in steady-state levels of PDH E1α, E1β, E2 and E2/E3bp after AAV vectors delivery and/or combination of DCA administration. Cell lysates (15 μg) from three healthy subjects (C1, C2 and C3) were separated on 10% SDS-PAGE gels and immunoblotted with monoclonal Abs against E1α, E1β, E2 and E2/E3bp. Bottom panel: Quantitative analysis of immunoblot data. Using ImageJ the pixel densities in each band (E2, E2/E3bp, E1α and E1β) from the gel were quantitated and the amount of protein was standardized to the amount of β-actin (E2, E2/E3bp, E1α and E1β/β-actin) in each lane, respectively. Data are means of 2 independent experiments.