Sugar-bisphosphates to cure PMM2-CDG?

INTRODUCTION PMM2-CDG is the most common congenital disorder of glycosylation (CDGs). It is caused by mutations in the gene PMM2, encoding the enzyme phosphomannomutase-2 (PMM2). A defective PMM2 leads to GDP- mannose deficiency and hypoglycosylation of numerous glycoproteins. At the present, PMM2-CDG has no cure. The use of pharmacological chaperones (PCs) was proposed as a therapeutic approach for many different diseases. PCs are small molecules able to bind unstable target proteins and stabilize them. Since a total lack of phosphomannomutase activity is considered incompatible with life, patients carry at least one misfolding mutation. Thus, PMM2-CDG is a potential candidate for the PC therapy. Phosphomannomutase-2 natural activator in cells is a-glucose-1,6-bisphosphate (aG16P) which stabilizes PMM2 in vitro but it is hydrolysed in vivo by phosphomannomutase-1 (PMM1), particularly in the presence of inosine monophosphate (IMP). OBJECTIVES We are pursuing three different paths in order to stabilize mutant enzymes and to prevent their premature degradation ?Looking for G16P analogs: b-glucose-1,6-bisphosphate (bG16P), the anomer of aG16P and its potential as therapeutic PC ?Improving the cellular uptake of G16P by using lipophilic ?G16P derivative ?Enhancing endogenous ?G16P by knocking out PMM1 METHODS ?-glucose-1-phosphate (?G1P) was synthesized from maltose with bacterial phosphorylase and ?G16P was produced by phosphorylation. ?G16P was purified on an ionic exchange column and was characterized by 31P nuclear magnetic resonance (31P-NMR). In silico docking of ?G16P on PMM2 was carried out by using PELE simulation. Limited proteolysis was performed by incubating PMM2 in the presence of trypsin with or without the ligand and analyzed by SDS-PAGE. Thermal shift assay was used to assess the stabilization in vitro of the recombinant enzyme. The phosphomannomutase activity was monitored by 31P-NMR and by fluorescence spectroscopy. The lipophilic ligand was synthesized by adding acetoxymethyl groups to G16P via standard organic chemistry procedures (collaboration with dott Sodano Univ. of Tourin and prof Rimoli Univ. of Naples). The acetoxymethyl groups would be hydrolyzed once the prodrug is inside the cells. PMM1-KO was edited by CRISPR/Cas9 system in PMM2-CDG fibroblasts.