Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Abstract

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

Keywords: carbohydrate active enzymes; glycogen storage disease type 2; lysosomal disease; orphan drugs; α-Glucosidase.

Figure 1.

Pharmacological chaperones for lysosomal storage diseases. deoxynojirimycin (DNJ) (1), 1-deoxy-galactonojirimycin (DGJ) (2), N-acetylcysteine (NAC) (3), N-acetylserine (NAS) (4), N-acetylglycine (NAG) (5), L-carnitine (L-CAR) (6), D-carnitine (D-CAR) (7), and acetyl-carnitine (A-D-CAR) (8).

Figure 2.

Comparison of the effect of L-carnitine on the stability of rhGAA. (a) Effect of L-CAR on the rhGAA stability; (b) Effect of L-CAR on the structural stability of rhGAA; (c) Summary of the T_ms measured by DSF; (d) Determination of the K_D rhGAA-L-CAR by DSF.

Figure 3.

Effect of a racemic mixture of D/L-CAR on the structural stability of rhGAA. (a) DSF analysis. L-CAR and D-CAR were incubated with rhGAA either alone (5 and 10 mM) or in combination (at 5 or 10 mM each). (b) Summary of the T_m measured by DSF.

Figure 4.

Comparison of the effect of allosteric and non-allosteric chaperones on the stability of rhGAA. (a) Analysis of the synergistic effect of L-CAR and NAC. rhGAA was incubated with L-CAR either alone (10 or 20 mM) or in combination with NAC, at 10 mM each. (b) Analysis of the synergistic effect of L-CAR and DNJ. L-CAR was incubated with rhGAA either alone (10 or 20 mM) or in combination with DNJ (0.1 mM).

Figure 5.

Effect of L-CAR in PD fibroblasts. (a) Effect of L-CAR on the residual activity of mutated GAA in fibroblasts. Fibroblasts derived from three PD patients were incubated in the presence and in the absence of 0.1–10 mM L-CAR before being harvested and used for GAA assay. The untreated cells (UT) were used as a control. The chaperone has significant effects on endogenous residual activity in the cells from patient 3.

Figure 6.

Synergy between L-CAR and rhGAA in PD fibroblasts. (a) Setting the conditions for evaluation of synergy between L-CAR and rhGAA. Different treatment protocols were evaluated: (i) pre-incubation of cells with L-CAR for 24 h, followed by co-incubation of L-CAR and rhGAA for an additional 24 h; (ii) co-incubation of L-CAR and rhGAA for 24 h. (b) Setting the optimal L-CAR concentrations for evaluation of synergy between L-CAR and rhGAA. Fibroblasts were incubated with rhGAA and different L-CAR concentrations (1–20 mM). GAA activity enhancements were observed at 5, 10, and 20 mM L-CAR concentrations with the highest and statistically most significant enhancements at 10 and 20 mM. (c) Effect of L-CAR on rhGAA processing in PD fibroblasts. Cells were incubated for 24 h with rhGAA alone or with rhGAA in combination with 10 mM L-CAR. In the cells treated with the combination of rhGAA and L-CAR the amount of the 70–76 kDa mature GAA active peptides were dramatically improved, as indicated by quantitative analysis by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is the loading control. (d) GAA activities measured in PD fibroblasts. The increase of GAA activity confirms the enhancing effect of L-CAR.

Figure 7.

Kinetics of GAA enhancements at different time-points in PD fibroblasts treated with rhGAA alone or in combination with 10 mM L-CAR. (a) GAA activity increased progressively over time and an enhancing effect of co-incubation with L-CAR was already detectable at 2 h and became progressively more pronounced up to 24 h (a). The amounts and the processing of rhGAA, analysed by western blot, also improved over time (b).

Figure 8.

Effects of rhGAA and L-CAR co-dosing on lysosomal trafficking of the recombinant enzyme. The cells of three Pompe patients were incubated under the conditions selected in the previous experiments, and co-localization of rhGAA with Lamp2 was analysed by confocal immune-fluorescence microscopy. In all three cells lines the co-localization was improved (a). This result was confirmed by a quantitative analysis of total GAA signal (b) and of GAA signal co-localized with Lamp2 (c).