With this project the research groups of Prof. Hermen Overkleeft and Prof. Hans Aerts aim to develop chemical biology tools and approaches to understand, and interfere with, glucosylceramide metabolism in relation to the lysosomal storage disorder, Gaucher disease.

Gaucher patients are characterized by a genetic deficiency in the lysosomal hydrolase, GBA1, resulting in the accumulation of glucosylceramide. Levels of this glycolipid can be therapeutically corrected by either enzyme replacement therapy (administration of recombinant GBA1) or substrate reduction therapy (administration of GCS inhibitors). A third clinical approach currently under investigation is chemical chaperone therapy, aimed at stabilizing the proper fold of nascent, mutant GBA1 forms in the endoplasmic reticulum so that more copies of functional enzymes reach lysosomal compartments, thereby increasing residual lysosomal enzyme activity.

The underlying proposal builds on several discoveries the researchers recently made and that pertain glucosylceramide metabolism. The researchers developed a set of activity-based probes with which endogenous retaining β-glucosidases could for the first time and with high efficiency be monitored in living cells. The set of probes includes a cyclophellitol derivative with which GBA1 can be detected in healthy and Gaucher cells at pH 4-5, that is, in lysosomal environments where GBA1 activity is required. A corresponding aziridine derivative labels retaining β-glucosidase activity in general and reacts with GBA1 also at neutral pH, and thus also in the endoplasmic reticulum.

Prof. Aerts and Prof. Overkleeft propose three research lines, each of which can be executed independently but that are interconnected both with respect to the underlying biology and the nature of the approach taken, namely that of activity-based protein profiling as an enabling strategy in chemical biology research. In research line one they aim to deepen the insight in GBA1 enzymology in healthy and Gaucher cells by capitalizing on the efficacy of our probes. GBA1 stands apart from other human retaining β-glucosidases in that it accepts substrates/inhibitors bearing bulky functional groups at the glucose C6. The researchers will search for evolutionary related glycosidases in various organisms. They already have identified a microbial endoglucosidase lacking N-glycans that binds with high efficiency to their probes and they propose to develop fusion proteins by which a protein of interest is fused to this robust enzyme. Trafficking and localisation of the construct is then accomplished by addition of the probes in living cells, which with the aid of different fluorophores, can be accomplished as well in a pulse-chase setting. For the purpose of enzyme activity labelling in animal models the researchers will create activity-based probes containing near-infrared and/or pH-activatable dyes. These will also be of use to label the recombinant enzymes currently applied in enzyme replacement therapy, enabling visualisation and characterisation of their trafficking and half-life in vivo.

In research line two Hans Aerts and Hermen Overkleeft aim to develop activity-based probes by which ceramidase activities can be captured and studied. They have compelling evidence that in Gaucher cells glucosylceramide is processed to glucosylsphingosine and that this metabolite contributes to the pathology of Gaucher disease. They aim to identify the nature of this ceramidase activity and at the same time arrive at strategies by which ceramidases in general can be studied.

In research line three, the researchers aim for a conceptually new chemical chaperone therapy. With the aid of their probes they established that GBA1 active site inhibitors in Gaucher cells do increase the amount of active GBA1 molecules that reach the lysosome, but that this increase is less pronounced than what is described in the literature based on studies on cell extracts. The researchers will take the chemical chaperone strategy one step further by the development of a mechanism-based chemical chaperone. They envisage the development of GBA1 activity-based probes that bind in the endoplasmic reticulum, thereby stabilizing its appropriate fold. Once in the lysosome the enzyme-inhibitor construct is processed by a lysosomal ceramidase, thereby releasing a nucleophile that liberates active GBA1 enzymes.

• gaucher disease
• glucosylceramide

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