GlycoPCs · How do Pharmacological Chaperones work? Molecular basis of the actions of glycomimetics on key glycosidases involved in lysosomal storage disorders

How do Pharmacological Chaperones work? Molecular basis of the actions of glycomimetics on keyglycosidases involved in lysosomal storage disorders

Numerous glycosidic enzymes present in the lysosome are required for metabolizing a wide variety of molecules, such as glycoproteins, glycolipids and oligosaccharides. These enzymes are synthesized in the endoplasmic reticulum and are transferred to the lysosome where they carry out their function. Lysosomal storage disorders are caused by mutations in these glycosidic enzymes. The mutants are synthesized at normal levels and may be functional, but they do not fold properly and are not trafficked correctly to the lysosome. Gaucher disease is caused by the absence of the functional enzyme β-glucocerebrosidase (GCase), leading to the storage of glucocerebroside in the macrophages. The presence and severity of neurological symptoms define three types of Gaucher disease as non-neuronopathic type I, neuronopathic type II and sub-acute neuronopathic type III. Fabry disease is another one of these rare diseases and is caused by a wide variety of mutations in the α-galactosidase A enzyme (α-Gal A). Due to the deficit of this enzyme in the lysosome, the glycosphingolipid accumulates in the vascular endothelium, skin, and heart. Pathological conditions associated with this accumulation include acroparesthesias, angiokeratomas, cardiomyopathy, stroke and renal failure. A novel and emerging therapeutic approach for the treatment of these diseases employs small molecules, called Pharmacological Chaperones (PCs). PCs selectively bind to the mutant enzyme in the endoplasmic reticulum and stabilize the correct 3D conformation allowing the mutant traffic to the lysosome (Figure 1). Some of these PCs has been approved or clinical trials are under way for their approval. The main objective of our proposal is the development and study of new PCs (iminosugars) in the treatment of Gaucher and Fabry disease. Understanding the mechanism of interaction of these PCs with mutants represents an important objective that would expand our knowledge on the protein target and help synthetic organic chemists in the rational design of new highly active and selective iminosugars. In this project, the molecular study of these protein-ligand (mutant enzyme-iminosugar) complexes involves different disciplines: (1) Study of ligand molecule interactions by Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) for identifying the main contacts of the ligand molecule with the protein. (2) Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) for mapping portions of the enzyme engaged in binding. (3) Molecular Modelling calculations: Docking to predict the mode of ligand binding and Molecular Dynamics simulation to represent the dynamic processes of the molecular systems.

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