Mucopolysaccharidosis type IVA (MPS IVA) is a rare disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We report here two GALNS pharmacological chaperones, ezetimibe and pranlukast, identified by molecular docking-based virtual screening. These compounds bound to the active cavity of GALNS and increased its thermal stability as well as the production of recombinant GALNS in bacteria, yeast, and HEK293 cells. MPS IVA fibroblasts treated with these chaperones exhibited increases in GALNS protein and enzyme activity and reduced the size of enlarged lysosomes. Abnormalities in autophagy markers p62 and LC3B-II were alleviated by ezetimibe and pranlukast. Combined treatment of recombinant GALNS with ezetimibe or pranlukast produced an additive effect. Altogether, the results demonstrate that ezetimibe and pranlukast can increase the yield of recombinant GALNS and be used as a monotherapy or combination therapy to improve the therapeutic efficacy of MPS IVA enzyme replacement therapy.

Mucopolysaccharidosis IV A (MPS IV A) is a lysosomal storage disease produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Although genotype-phenotype correlations have been reported, these approaches have not enabled to establish a complete genotype-phenotype correlation, and they have not considered a ligand-enzyme interaction. In this study, we expanded the in silico evaluation of GALNS mutations by using several bioinformatics tools. Tertiary GALNS structure was modeled and used for molecular docking against galactose-6-sulfate, N-acetylgalactosamine-6-sulfate, keratan sulfate, chondroitin-6-sulfate, and the artificial substrate 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate. Furthermore, we considered the evolutionary residue conservation, change conservativeness, position within GALNS structure, and the impact of amino acid substitution on the structure and function of GALNS. Molecular docking showed that amino acids involved in ligand interaction correlated with those observed in other human sulfatases, and mutations within the active cavity reduced affinity of all evaluated ligands. Combination of several bioinformatics approaches allowed to explaine 90% of the missense mutations affecting GALNS, and the prediction of the phenotype for another 21 missense mutations. In summary, we have shown for the first time a docking evaluation of natural and artificial ligands for human GALNS, and proposed an update in genotype-phenotype correlation for Morquio A, based on the use of multiple parameters to predict the disease severity.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding for the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal accumulation of keratan sulfate (KS) and chondroitin-6-sulfate. In this study, we identified and characterized bromocriptine (BC) as a novel PC for MPS IVA. BC was identified through virtual screening and predicted to be docked within the active cavity of GALNS in a similar conformation to that observed for KS. BC interacted with similar residues to those predicted for natural GALNS substrates. In vitro inhibitory assay showed that BC at 50 μM reduced GALNS activity up to 30%. However, the activity of hrGALNS produced in HEK293 cells was increased up to 1.48-fold. BC increased GALNS activity and reduced lysosomal mass in MPS IVA fibroblasts in a mutation-dependent manner. Overall, these results show the potential of BC as a novel PC for MPS IVA and contribute to the consolidation of PCs as a potential therapy for this disease.