A Potent PDK4 Inhibitor for Treatment of Heart Failure with Reduced Ejection Fraction

Abstract

Heart failure with reduced ejection fraction (HFrEF) is characterized not only by reduced left ventricular ejection fraction (EF) but is also combined with symptoms such as dyspnea, fatigue, and edema. Several pharmacological interventions have been established. However, a treatment targeting a novel pathophysiological mechanism is still needed. Evidence indicating that inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may be cardioprotective has been accumulating. Thus, we focused on vitamin K₃ and used its framework as a new PDK4 inhibitor skeleton to synthesize new PDK4 inhibitors that show higher activity than the existing PDK4 inhibitor, dichloroacetic acid, and tested their cardioprotective effects on a mouse heart failure model. Among these inhibitors, PDK4 inhibitor 8 improved EF the most, even though it did not reverse cardiac fibrosis or wall thickness. This novel, potent PDK4 inhibitor may improve EF of failing hearts by regulating bioenergetics via activation of the tricarboxylic acid cycle.

Keywords: 1,4-naphthoquinone; PDK4 inhibitor; TCA cycle; heart failure; quinol monooxygenase; vitamin K3.

Figure 1

(a) The structure of Vitamin K₃ and (b) the Vitamin K₃ (R¹ and R² = H) binding site in QuMo.

Scheme 1

Synthesis of C7-substituted derivatives. (a) Dimethyl succinate, ^tBuOK, ^tBuOH, reflux, 40 min, 74%; (b) Pd/C, H₂, MeOH, rt, 5 h, 98%; (c) PPA, 85 °C, 20 min, 86%; (d) trimethyl silane, TFA, rt, 5 min, 98%; (e) DDQ, benzene, 70 °C, 1 h, 81%; (f) CAN, MeCN/H₂O, rt, 5 min, 94%; (g) LDA, I₂, THF, −10 °C, 2 h, 85%; (h) Ac₂O, DMAP, pyridine, rt, 3 h, 96%; (i) CAN, MeCN/H₂O, rt, 5 min, 90%; (j) HCl/MeOH, rt, 2 days, 53%.

Scheme 2

Synthesis of C6-substituted derivatives. (a) KOH, O₂, DMF, 68 °C, 3 h, 72%; (b) AcCl, MeOH, rt, 4 h, 64%; (c) CAN, MeCN/H₂O, rt 5 min, 68%; (d) 9a: Gly-O^tBu·HCl, PyBOP, DIPEA, DCM, 10 min, 53%; 9b: Ser(^tBu)-O^tBu·HCl, PyBOP, DIPEA, DCM, 10 min, quant.; (e) 10a: CAN, MeCN/H₂O, rt, 5 min, 97%; 10b: CAN, MeCN/H₂O, rt, 5 min, 94%; (f) 10a: TFA, DCM, rt, 20 min, 97%; 10b: TFA, DCM, rt, 20 min, 97%.

Figure 2

Vitamin K₃ derivatives (5, 8, 11, 13a, and 13b) improve the ejection fraction (EF). (a) Study design. Echocardiographs were acquired before, at 4 weeks, and 5 weeks after TAC induction. Starting from week 4, each derivative was intraperitoneally (i.p.) injected into mice whose EF was lower than 55% once a day (q.d.) for a week (indicated by red arrows). Mice were euthanized 5 weeks after TAC induction. (b) Scatter dot plot showing means ± SD of the change in EF (after a week of daily PDK4 inhibitor injection). The change in magnitude was calculated by dividing the EF at week 5 (EF5) using the EF at week 4 (EF4). The Kruskal–Wallis test, followed by Dunn’s multiple comparison test. *** p < 0.001.

Figure 3

Recovery from heart failure with reduced ejection fraction (HFrEF) after 7 days of daily treatment with 8. (a) Scatter dot plot showing means ± SD of EF 4 weeks after a transverse aortic constriction operation (per-injection) and EF after 7 days of daily vehicle (n = 8) or 8 (n = 9) treatment following a 4-week incubation after the TAC operation (post-injection). Two-way ANOVA with Bonferroni’s multiple comparisons test. **** p < 0.0001. (b) EF of individual mice before and after injections of 8. (c) Example of M-Mode echocardiography of left ventricles of vehicle-injected and 8-injected mice that received the TAC operation (TAC mice). (d) Left ventricular internal diameter at end-diastole (LVIDd) and left ventricular internal diameter at end-systole (LVIDs) of vehicle-injected and 8-injected TAC mice. Mann–Whitney U test. NS = not significant. ** p < 0.01. (e) Heart sections of TAC mice stained with Sirius red. Fibrotic areas are stained pink. Scale bar: 200 µm. (f) Statistical results for fibrotic areas of heart sections of vehicle-injected and 8-injected TAC mice. NS = not significant.

Figure 4

8 inhibits pyruvate dehydrogenase activity. (a) Scatter dot plot showing means ± SD of PDH activity in heart extracts from sham (n = 4), vehicle-treated TAC mice (n = 19), and 8-treated TAC mice (n = 5). Mann–Whitney U test. ** p < 0.01 (b,c) Densitometric analysis of a mean phospho-PDH(Ser300)/total PDH ratios (b) and a mean phosphor-PDH(Ser293)/total PDH ratio (c). Each bar represents the mean ± SD. Mann–Whitney U test. ** p < 0.01. (d) Densitometric analysis of PDK4 expression normalized with GAPDH expression. Each bar represents the mean ± SD. Mann–Whitney U test. NS = not significant. (e) Representative Western blot images of phosphor-PDH, total PDH, PDK4, and GAPDH.