Funktion-Selective Protein Scavenger Nanolayers

Aim of the work is to provide reliable and cheap glycosidase detecting systems for high-throughput as well as micro-array/chip applications for a wide range of diagnostic purposes in biochemistry, medicine and "functional proteomics". Selected REVERSIBLE glycosidase inhibitors carrying a fluorescent tag will be chemically synthesised, biologically evaluted, screened for applicability in various simple anylytical test systems and ultimately connected to a chip surface. Exposure to pure enzymes, mixtures or "general" biological matrices result in binding events which can directly be measured by fluorescence spectrometry analysis. From this, a "glycosidase atlas" will later be available to identify glycosidases by their unique, finger-print-like patterns of interaction with the chip-bound inhibitors. After clear "proof of concept", the method can be applied to special diagnostic purposes and problems. For example, "viral enzyme chips" (influenza, herpes, HIV), "hereditary metabolic disease chips" (brain metabolism, post translational glycoprotein processing) or "cancer-associated enzyme chips" can be envisaged to be based on this approach. Relying on the application of reversible inhibitors for detection, snap-shots for time-resolved studies/diagnostics will also be available. Furthermore, on the same basis, the chip may be used several times (as opposed to "one-way" devices), allowing for additional diagnostic options as well as "cost- cutting" in "low-budget" situations (poor income systems such as NGOs or under-developed countries and societies).

The project was targeting the design, synthesis, biological evaluation and subsequent immobilisation on `chips` of new glycosidase inhibitors of the iminoalditol type to from analytical micro-arrays. These `spots on chips` were aimed to work as function-selective scavengers to collect, detect and categorize proteins with glycosidase activities on chips. Ultimately, quick and efficient screening for glycosidases employing novel chips should be achieved. Based on previous successful work which had addressed fluorescent soluble glycosidase inhibitors as analytical and diagnostic tools for glycosidase detection and characterisation, in the course of the project we extended the nitrogen substituent by an additional chemical `handle` providing the connection point for spacer arms which should attach the fluorescent molecule to the respective surface, mainly surface-modified glass slides by a co-valent bond. As the proof of concept, in a typical experiment, different inhibitors of varying selectivity could be compared by exposure to fluorescently labelled glycosidases which were detected at different channels (blue, green, yellow, red). Enzyme binding was highly selective for the respective configuration, i.e., glucosidases were selectively bound by the immobilised glucosidase inhibitors whereas the mannosidases and hexosaminidases probed were not affected but bound to the corresponding immobilised mannosidase inhibitor and the hexosaminidase inhibitor, respectively, applied in the study. In a very rewarding spin-off, some of our new compounds were recently discovered to be excellent inhibitors of lysosomal glycosidases. Due to the fact that some known inhibitors of these vital enzymes the lack of which is associated with severe hereditary diseases such as Gaucher`s, Fabry`s, Sandhoff`s and around forthy other less known ones, our compounds were also screened as molecular chaperones for enzyme mutants. These mutants are not able to fold into catalytically competent glycosidases and undergo degradation resulting in the accumulation of their respective substrates in the lysosomes thus causing swelling of organs, neurodegenerative symptoms, and many others including bone destruction. In the presence of sub-inhibitory concentrations of suitable inhibitors, the protein now can fold around the inhibitor which provides a template for the active site. Thus, in some cases manifold increases of enzyme activity can be gained likely resulting in the relief of symptoms. With our compounds tested thus far in patients` cell-lines, up to 18fold increase of enzyme activity was observed very recently. This now may provide a sound basis for further investigations in this area.