Pantothenate kinase activation relieves coenzyme A sequestration and improves mitochondrial function in mice with propionic acidemia

Abstract

Propionic acidemia (PA) is a rare autosomal-recessive metabolic disease that arises from mutations in propionyl-CoA (C3-CoA) carboxylase. Reduced enzyme activity slows C3-CoA metabolism, leading to an elevated plasma C3:C2-carnitine ratio, the hallmark biomarker of PA. The metabolic imbalances experienced in PA are however poorly defined. Here, we used a hypomorphic PA mouse model to demonstrate that C3-CoA accumulation in liver reduced non-esterified CoA (CoASH) and acetyl-CoA (C2-CoA). Tricarboxylic acid (TCA) cycle intermediates that are normally metabolized instead accumulated in urine, providing direct evidence for compromised mitochondrial function in PA. Pantothenate kinase (PanK) is known to catalyze the rate-controlling step in CoA biosynthesis, and its inhibition by C3-CoA prevents an increase in CoA biosynthesis to alleviate CoASH sequestration. PZ-3022 is an allosteric PanK activator that counteracts C3-CoA inhibition. PZ-3022 therapy increased hepatic CoASH and C2-CoA and decreased C3-CoA in the PA mouse model, leading to improved intracellular C3:C2-CoA and plasma C3:C2-carnitine ratios. Elevated urinary malate is a major component of the metabolic signature for TCA cycle dysfunction in the PA mouse, and the 80% reduction in urine malate by PZ-3022 therapy indicates the restoration of mitochondrial function. Thus, CoASH sequestration in PA leads to reduced TCA cycle activity that is relieved by PZ-3022, providing preclinical proof of concept for PanK activators as a therapy to attenuate the underlying mitochondrial defect in PA.

Fig. 1.. CoASH sequestration in PA mice.

Wild-type (WT) and Pcca^−/−PCCA(A138T)^tg/0 (PA) mice were maintained on a standard rodent chow and samples were harvested at day 68-71. (A) Plasma carnitine. (B) Plasma C2-carnitine. (C) Plasma C3-carnitine. (D) Plasma C3:C2-carnitine ratio. (E) Liver carnitine. (F) Liver C2-carnitine. (G) Liver C3-carnitine. (H) Liver C3:C2-carnitine ratio. (I) Liver CoASH (non-esterified CoA). (J) Liver C2-CoA. (K) Liver C3-CoA. (L) Liver C3:C2-CoA ratio. Male mice are in blue and female mice are in red. [¹³C]C2-CoA was used as the internal standard (fig. S2). The p values are in red. ns indicates p > 05. Sample sizes of animals are in parentheses.

Fig. 2.. Transgenic PCCA expression and TCA cycle metabolites in PA mice.

(A) Western blot analysis of PCCA(A138T) transgene expression in WT and PA mice. This blot illustrates the tissue-specific distribution of murine PCCA in WT mice (10 μg/lane) compared to the expressed human transgene PCCA(A138T) protein in PA mice (60 μg/lane). Western blots for each tissue were obtained from triplicate mice (fig. S3), and a fourth blot was performed for this figure. The red triangle indicates a non-specific band. (B) A metabolomics screen of TCA cycle metabolites in plasma (upper panel) and urine (lower panel) of three male and three female PA mice. The p values are in red.

Fig. 3.. Properties of PZ-3022 and its impact on CoA in PA mouse liver.

(A) Chemical structures and relevant properties of PZ-2891 and PZ-3022. Purity and NMR spectra of PZ-3022 are shown in figs. S8 & S9. (B) Inhibition of PANK3 by PZ-3022. Data were fit to the Morrison equation (line) and EC₅₀ was calculated. (C) Crystal structure of the PanK3•AMPPNP•Mg²⁺•PZ-3022 complex (PDB ID: 6PE6) overlaid on the PZ-2891 structure (PDB ID: 6B3V). The two PANK3 protomers are colored gold and cyan. The Fo-Fc simulated annealing omit map is contoured at 3 σ (yellow mesh). (D) Elevation of total cellular CoA in C3A cells treated with 10 μM of either PZ-2891 or PZ-3022. (E) Half-life of PZ-2891 compared to PZ-3022 in mice. Complete pharmacokinetic profiles are in table S4. (F) Male C57BL/6 mice were orally gavaged every 24 h with Captisol containing 10 mg/kg of either PZ-2891 or PZ-3022 for three days, and liver total CoA was determined 4 h after the last dose using a fluorescent derivatization assay. The p values are in red. Number of biological replicates are in parentheses.

Fig. 4.. Acute treatment with PZ-3022 relieves CoASH sequestration in PA mouse liver.

Mice were orally gavaged every 24 h with either 10 or 30 mg/kg PZ-3022 plus 50 mg/kg pantothenate. Control animals received 50 mg/kg pantothenate. Four hours after the third dose, the impact of short-term PZ-3022 treatment on liver CoA pools was determined using mass spectrometry. (A) CoASH. (B) C2-CoA. (C) C3-CoA. (D) C3:C2-CoA ratio. Male mice are in blue and female mice are in red. (E) Elevation of total hepatic CoA in PA mice dosed with PZ-3022. (F) C57BL/6J male mice were administered PZ-3022 by gavage and at the indicated times samples were taken (3 mice per point) to determine plasma PZ-3022 and liver total CoA. (G) Envigo chow containing 1000 ppm pantothenate was formulated with 7.5, 37.5 and 75 ppm PZ-3022. C57BL/6J male mice were maintained on the diets for 1 week and total liver CoA was determined. (H) PZ-3022 in plasma and liver as a function of PZ-3022 in the diet. The p values are in red. ns indicates p > 0.05. Mouse sample sizes are in parentheses.

Fig. 5.. Metabolic parameters in mice treated with PZ-3022 for 70 days.

Animals were maintained on a defined diet supplemented with 1000 ppm pantothenate either with or without 75 ppm PZ-3022 beginning at weaning on day 21. On day 68-70, liver CoAs and carnitines were determined by mass spectrometry in male (blue) and female (red) mice. (A-H) Liver: CoASH (A), C2-CoA (B), C3-CoA (C), C3:C2-CoA ratio (D), carnitine (E), C2-carnitine (F), C3-carnitine (G), and C3:C2-carnitine ratio (H). (I-L) Plasma: carnitine (I), C2-carnitine (J), C3-carnitine (K), and C3:C2-carnitine ratio (L). The p values are in red. ns indicates p > 0.05. Mouse sample sizes are in parentheses.

FIG. 6.

TCA cycle intermediates in plasma and urine of treated PA mice.(A) The impact of PZ-3022 therapy on the relative abundance of plasma TCA cycle intermediates (Fig. 2). (B) Effect of PZ-3022 therapy on TCA cycle intermediates in urine. Urinary TCA cycle metabolites eliminated over 24 hours were quantified by mass spectrometry and normalized to mouse body weight. The two-tailed Student’s t test was used to compare two indicated groups, and P values <0.05 are reported in red. ns means P > 0.05. Three male (blue) and three female (red) mice were used.