| Glycosphingolipids, a subclass of lipids found at the cell surface, play crucial roles in health and disease. Glucosylceramide is the starting material in the biosynthesis of the large majority of glycosphingolipids. Turnover of these glycoconjugates takes place in lysosomes and involves the consecutive action of lysosomal exoglycosidases that recognize specific monosaccharides. More than ten inherited deficiencies in these hydrolytic activities exist, each characterized by impaired activity of a specific glycosidase and accompanying storage of the respective substrate(s). Gaucher disease, in which glucosylceramide accumulates due to deficiency of the lysosomal glucosylceramidase (GBA1), is the most common lysosomal storage disorder. Gaucher disease and related lysosomal storage disorders have been subject to extensive studies in our laboratories over the decades. We have actively participated in the development of the two existing therapies for the treatment of Gaucher patients. These are the enzyme replacement therapy, in which patients are treated with recombinant GBA1, and the substrate reduction therapy, in which the glucosylceramide biosynthesis is reduced by inhibition of the enzyme glucosylceramide synthase (GCS). Both therapies were firstly applied in Europe in our centre. In the course of our fundamental investigations on enzymes synthesizing and degrading glucosylceramide, we made several important discoveries. We first purified to homogeneity the lysosomal glucocerebrosidase (GBA1) and characterized its life cycle and inhibitor sensitivity. We unearthed the nature of a second, non-lysosomal glucosylceramidase (GBA2) that is situated near the plasma membrane and which compensatory enhanced activity contributes to the patholophysiology of Gaucher disease. We developed two hydrophobic iminosugars (MZ21 and MZ31) with superior GCS inhibitory properties compared to the substrate deprivation drug, Zavesca. Next, we identified GCS as a new pharmaceutical target for the treatment of diabetes II and amassed evidence that optimal therapeutic effect (elevated insulin sensitivity, lowered blood glucose levels, loss of weight and body fat) in obese diabetic animal models is achieved by the concomitant inhibition of GCS and intestinal glycosidases by iminosugars. We have very recently obtained evidence that some hydrophobic iminosugars affect FGF signaling by their interference with the FGF co-receptor beta-klotho, a glucosidase-like protein. FGF signaling is of importance in body lipid metabolism and particularly relevant in relation to obesity. The aim of the here proposed research is to dissect the biological pathways involved in glucosylceramide metabolism, in relation to both lysosomal storage disorders and type II diabetes/obesity. We will approach this by the synthesis of new iminosugar derivatives using both combinatorial chemistry and target oriented synthesis approaches. We will screen the activity and selectivity of these as inhibitors of GCS, GBA1, GBA2 and other glycoprocessing enzymes, and also as antagonists of beta-klotho mediated FGF signaling. To this end we will use advanced biochemical assays that are either in place or will be developed in the course of the proposed research. From a fundamental point of view we intend to shed light on the structure-activity-relationships that govern enzyme inhibition/beta-klotho binding of modified iminosugars. A reason to opt for a compound synthesis approach is that most factors (GBA2, GCS, beta-klotho) that are subject to our studies defy structural analysis, because they are membrane associated. The identification of selective inhibitors for each of these enzymes will help us to further pinpoint their exact contribution in normal physiology and in various pathologies (GBA2 in lysosomal storage disorders and beta-klotho/GCS in diabetes II). Finally it will help us in the definition of new and improved leads for the development of substrate reduction therapies, and possibly also enzyme stabilization therapies aimed at a variety of lysosomal storage disorders (Gaucher, Fabry, Niemann-Pick), as well as diabetes mellitus type II/obesity. |
