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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.

SUBMITTER: Iacono R

PROVIDER: S-EPMC8477953 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.

Project description:BACKGROUND: The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs. RESULTS: We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods.We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence. CONCLUSIONS: Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.

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

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