Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket

Abstract

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.

Keywords: Diabetes; Drug Development; Enzyme Inhibitors; Glucose Metabolism; Hepatic Steatosis; Mitochondrial Protein Kinase; Pyruvate Dehydrogenase Complex; Pyruvate Dehydrogenase Kinase; Structure-based Inhibitor Design.

FIGURE 1.

Structure of PDK2 and known and novel inhibitors. A, PDK dimer showing AZD7545 and dichloroacetate-binding sites in N-terminal domain (pink) and radicicol bound to the ATP-binding pocket in the C-terminal domain (green). B, chemical structures of known PDK inhibitors as follows: DCA, AZD7545, compound 3, radicicol, and M77976; and novel PDK inhibitors as follows: DC23, PA1, PA7, PS2, PS8, and PS10. The resorcinol ring is indicated in red, and isoindoline moiety is in blue.

FIGURE 2.

Comparison of inhibitor-binding pockets in PDK2 and Hsp90. A, superimposition of the C-terminal domain of PDK2 (green) harboring PA7 (pink) with the N-terminal domain of Hsp90 (orange) with bound PA7 (cyan). B, PA7 in Hsp90. C, PA7 in PDK2. D, superimposition of Hsp90-bound (cyan) and PDK2-bound PA7 (pink) (left), and the structure of PDK2-bound PS2 (right).

FIGURE 3.

Stereo views of inhibitor-binding pockets in PDK2 and Hsp90. A, Hsp90-PA7 structure (30). B, PDK2-PA1 structure with F_o − F_c density map (green mesh) contoured to 4 σ. C, PDK2-PA7 structure contoured to 4 σ. D, PDK2-PS2 structure contoured to 4 σ. E, PDK2-PS8 structure contoured to 3 σ. F, PDK2-PS10 structure contoured to 3 σ. w, ordered water molecule.

FIGURE 4.

Thermodynamic analysis of inhibitor binding to PDK2 and Hsp90. A, thermograms of PS10 binding to PDK2 and Hsp90 obtained by ITC. B, thermodynamic signatures of inhibitor bindings to PDK2 (left panel) and Hsp90 (right panel); ΔG, Gibbs binding energy; ΔH, binding enthalpy; ΔS, binding entropy; T, absolute temperature.

FIGURE 5.

Kinase profiling of compound PS8. Inhibition of the 21 representative kinases, including PDK2, in the Human Kinome by PS8 were measured in the concentration range of 15 nm to 300 μm. IC₅₀ values for each kinase were derived from individual inhibition curves. The IC₅₀ for PDK2 is at least 3 orders of magnitude lower than the next lowest value for CDK1/cyclin B.

FIGURE 6.

Enhanced PDC activity with reduced phosphorylation level in PS10-treated DIO mice. A, short term response. C57BL/6J male mice were fed a high fat diet for 3 weeks and treated with vehicle (V, n = 4) or PS10 at 70 mg/kg (T, n = 4) by a single i.p. injection while they had free access to food. Animals were sacrificed at 10 a.m., i.e.10 h after the injection. Tissues were harvested and analyzed for PDC activity and phosphorylation levels of the E1α subunit. Upper panel, PDC activity in heart, liver, kidney, and muscle. Lower panel, amounts of the phosphorylated (p-E1) and total (E1) E1α subunit in different tissues determined by Western blotting analysis. B, long term response. C57BL/6J male mice were fed a high fat diet for 10 weeks and then treated with vehicle (n = 3) or PS10 at 70 mg/kg/day (n = 3) for 3 days. The remaining procedures and result presentation are as in A. **, p < 0.01; *, p < 0.05.

FIGURE 7.

Glucose- and lipid-controlling properties of PS10. A, glucose tolerance test. C57BL/6J male mice were fed a high fat diet for 10 weeks and treated with vehicle (n = 4) or PS10 at 70 mg/kg/day (n = 6) for 4 weeks and were fasted for 6 h followed by injection of 1.5 g of glucose/kg by i.p. injection. Blood glucose levels were monitored at 0–2 h after the glucose injection. B, food intake of DIO mice fed the high fat diet for 10 weeks followed by treatments with vehicle (n = 5) or PS10 at 70 mg/kg/day (n = 5) for 1 week. C, body weight change in DIO mice from A after 6 weeks of treatment with vehicle or PS10. D, plasma lactate under the “Experimental Procedures.” E, plasma cholesterol concentration in DIO mice from C. F, plasma triglycerides concentrations in DIO mice also from C. G, change in the fat mass. DIO mice were treated as in B. Fat mass was determined as described under “Experimental Procedures.” H, Oil Red O stains of liver slices from vehicle- and PS10-treated DIO mice as in B. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant statistically.

FIGURE 8.

Calculated volumes of the DCA-binding and ATP-binding pockets in PDK2. The N-terminal domain of the PDK2 monomer with the allosteric site occupied by DCA is derived from coordinates of PDB code 2BU8. PDK inhibitor PS10 was modeled into the ATP-binding pocket in the C-terminal domain of the same monomer, according to the PS10 coordinates of PDB code 4MPN from this study. The volumes of the DCA-binding (211 ų) and ATP-binding (865 ų) pockets, as represented by blue mesh, were computed using program CASTp (50).