CORDIS - EU research results

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

Periodic Reporting for period 1 - GlycoPCs (How do Pharmacological Chaperones work? Molecular basis of the actions of glycomimetics on keyglycosidases involved in lysosomal storage disorders)

Reporting period: 2018-10-01 to 2020-09-30

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.
This multidisciplinary project has required the joint efforts of different research groups with specific and complementary expertise. One of the involved groups (Prof. Inmaculada Robina, University of Seville) has just synthesized iminosugars with PC activity. Robina´s group has characterized a pyrrolidine-aryltriazole iminosugars with PC activity in Gaucher´s cell line with β-glucocerebrosidase (GCase) mutation of N370S. In addition, a nonavalent pyrrolidine-based iminosugar has increased the activity of α-Gal A in a fibroblast cell line containing the mutation R301G; being the first example of a multivalent enzyme activity enhancer for Fabry disease. As an extension of both works, at the UEA we started studying these enzyme-PC complexes by STD NMR, Molecular Modelling and HDX-MS.
Explanation of the work carried per WP is shown bellow.
WP1: Cloning, expression and purification of glycosidic enzymes involved in Gaucher and Fabry disease.
Enzyme purity is fundamental for the structural characterization of the enzyme-PC complexes when NMR and MS technologies are being used. We focused on WT enzymes (GCase and α-Gal A) and relevant mutants which have been reported to strongly react to PCs. Mutations initially planned to test were: R301G α-Gal A, N370S GCase and L444P GCase. In collaboration with Dr. José Corchero (Autonomous University of Barcelona), we are expressing these enzymes in HEK 293 cells. So far, we have produced enough quantity of α-Gal A and GCase and we are developing new strategies for increasing the expression level of the mutants.
WP2: Study of molecular interactions between iminosugars and enzyme mutants by STD NMR.
We have just carried out STD NMR experiments on the complex α-Gal A with the PC provided by Prof. Robina (nonavalent pyrrolidine-based iminosugar) and the results showed that a specific interaction of the ligand with α-Gal A takes place. Although, we have carried out the initial studies with the α-Gal A (WT), the mutants that are been expressed in Corbero's laboratory will be key to study real molecular interactions.
WP3: Molecular Modelling calculations. So far, we have investigated, using advanced molecular dynamic simulations, different complexes between GCase or α-Gal A and a variety of substrates, inhibitors and the proposed chaperones in order to study the following properties: Protein stability, Protein-ligand complex strength, Intermolecular interactions between the ligand and the protein, Conformational changes in the protein structure and effect of conformational changes in the catalytic activity. These properties helped us to evaluate, and predict, the chaperone properties of the studied iminosugars with the most representative mutants for GCase (N370S and L444P) and the cellular-line available mutant for R301G α-Gal A. We could find a relationship between the presence of iminosugars and lower loop flexibility in N370S mutant for GCase with could cause the enhancement of the protein stability in the endoplasmic reticulum. Similar findings were observed for R301G mutant in α-Gal A.
WP4: HDX-MS. To expand our knowledge on the conformation of the complexes and to deepen our understanding of the mechanism of action of this PC, we will complete our project with HDX-MS experiments.
The COVID-19 outbreak negatively affected ongoing or planned activities. For this reason, we are still performing some experiments, and we hope to be able to prepare several manuscripts on these studies for publication in the near future.
IMPACT: Regarding society needs, the contribution to the full deployment of targeted therapies against lysosomal storage diseases will have a significant impact. First, it will give new, innovative treatment opportunities for an orphan pathology, with the subsequent increase on wellness of population, but also having a positive economic effect due to less loss of working hours, more independent living for the patients and less premature deaths.