Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate

Abstract

Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity.

Conflict of interest statement

None.

Figures

Fig. 1

PDHC activity in cells treated with phenylacetate or phenylbutyrate. PDHC activity in wild-type human fibroblasts incubated with 1 mM or 10 mM of phenylbutyrate (red) or phenylacetate (blue) for 24 hours, or untreated (black). Averages ± SD are shown; * p Abbreviations: NT= not treated (no drug); PA= phenylacetate; PB= phenylbutyrate

Fig. 2

PDHC activity in tissues of mice treated with phenylacetate, phenylbutyrate, DCA or combination of phenylbutyrate and DCA. PDHC activity in brain, muscle, and liver mitochondrial fractions of mice treated with saline (n = 20), 250 mg/kg/day of phenylacetate (n = 5), 250 mg/kg/day of phenylbutyrate (n = 10), 25 mg/kg/day of DCA (n = 5), 250 mg/kg/day of DCA (n = 10), or with a combination of both drugs each at the dose of 250 mg/kg/day (n = 10). Averages ± SD are shown. a: p Abbreviations: PA= phenylacetate; DCA= dichloroacetate; PB= phenylbutyrate

Fig. 3

Differential inhibition of PDKs by phenylbutyrate. Lineweaver-Burk plots of PDK1 (a), PDK3 (b), PDK4 (c) in the absence (■) or in the presence of 0.25 mM (●), 0.5 mM (□), and 1 mM (○) of phenylbutyrate. Averages and standard errors of the mean are shown. (d) Secondary plots of the intercepts of lines from A and B against the relative inhibitor concentrations to compare the inhibitory effect of phenylbutyrate on different enzymes

Fig. 4

Putative binding sites of phenylbutyrate on PDK2 (a, b) and PDK3 (c, d). (a) Ribbon representation of human PDK2 structure (PDB 2BU8) and bound ligands represented in a space-filling model: ATP/Mg2+ in gray; phenylbutyrate in green; DCA in red. ATP and DCA are present in the X-ray structure while the phenylbutyrate position was suggested by docking analysis. (b) Specific interactions of phenylbutyrate (red, green, and white) with amino acid residues (stick representation) at the binding sites of PDK2. (c) Ribbon representation of human PDK3 structure (PDB 2Q8I) and bound ligands represented in a space-filling model: ATP/Mg2+ in gray; phenylbutyrate in green. ATP is present in the X-ray structure while phenylbutyrate binding site was suggested by the docking analysis. (d) Specific interactions of phenylbutyrate with amino acid residues (stick representation) of PDK3 at the binding sites. Van der Waals interaction spheres of the amino acid residues (stick representation) in contact with the inhibitor have been removed for clarity in b and d. Supplementary Fig. 1 shows Van der Waals interaction spheres of the amino acid residues in contact with the inhibitor

Fig. 5

PDHC activity in wild-type fibroblasts incubated with phenylbutyrate, DCA, or a combination of phenylbutyrate and DCA. PDHC activity expressed as nmol/min/mg protein of mitochondrial fractions of two wild-type cell lines (BA1020 and NA489) incubated with 1 mM or 10 mM DCA, 1 mM or 10 mM of phenylbutyrate, with the simultaneous presence of 1 mM DCA and 1 mM phenylbutyrate, or with the simultaneous presence of 10 mM DCA and 10 mM phenylbutyrate. Averages ± SD are shown. a: p Abbreviations: NT= not treated (no drug); DCA= dichloroacetate; PB= phenylbutyrate

Fig. 6

PDHC activity and phosphorylation of PDHC deficient cells incubated with phenylbutyrate, DCA, or a combination of phenylbutyrate and DCA. (a) PDHC activity expressed as nmol/min/mg protein in a PDHC deficient patient harboring the mutation p.N135S in the PDHA1 gene incubated with 1 mM or 10 mM DCA, 1 mM or 10 mM of phenylbutyrate, with the simultaneous presence of 1 mM DCA and 1 mM phenylbutyrate, or with the simultaneous presence of 10 mM DCA and 10 mM phenylbutyrate. Averages ± SD are shown. a: p < 0.05 compared to N.T.; b: p < 0.05 compared to DCA 1 mM; c: p < 0.05 compared to phenylbutyrate 1 mM; d: p < 0.05 compared to DCA 10 mM; e: p < 0.05 compared to phenylbutyrate 10 mM. (b) Western blotting for phosphorylated E1α and for total E1α protein in cells incubated with the conditions reported in panel a. The images are representative of two independent experiments. (c) Relative band intensities of phosphorylated E1α normalized for total E1α from two independent experiments. Averages ± SD are shown. Abbreviations: DCA= dichloroacetate; PB= phenylbutyrate; NT= not treated (no drug)