Use of activity-based probes to study the action of glycosidases

Lead Research Organisation: University of York
Department Name: Chemistry

Abstract

The development of new approaches to dissect the diverse and wonderful roles for carbohydrates in living cells is a major challenge for modern cell biology, biochemistry and medicine. The colossal potential of carbohydrates is reflected in the multiplicity of functions for glycans; in addition to acting as energy sources, carbohydrates play central roles in cell and organism structure, communication signalling and epigenetic modification. This project will build upon recent developments in the activity-based profiling of carbohydrate-degrading enzymes termed gycosidases; notably in the context of lysosomal storage diseases. The student will initially analyse binding of "next-generation" probes to the human Gaucher and Fabry disease enzymes. 3-D structure will inform the synthesis of improved probes. Probes will be used to quantity enzyme activity in healthy and disease cells, working in collaboration with Overkleeft and Aerts, Leiden possibly leading to the development of new diagnostic technologies. As the project evolves, we expect to see the probe concept rolled-out to other enzyme classes and applications and ultimately, we would like to combine such techniques with cryo-electron tomography approaches to probe the activity of enzymes within living cells.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1941670 Studentship BB/M011151/1 30/09/2017 31/12/2021 Rhianna Rowland
 
Description We've applied activity-based probes to the detection, identification and quantification of enzymes in a range of processes from biomass degradation to human health and disease, specifically to study lysosomal storage disorders. This research brings together chemical, biochemical and structural analysis to drive the development of more selective and potent inhibitors.

We have used activity based probes, inspired by the ß-glucosidase inhibitor cyclophellitol, to detect and identify ß-xylosidases and endo-ß-1,4-xylanases in the secretomes of Aspergillus niger. Furthermore, we demonstrated the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.

We have been able to expand the concept of activity based probes to the generation of potent inhibitors for enzymes such as beta-glucocerebrosidase (GBA) which is associated with the most common lysosomal storage disorder Gaucher Disease. Supported and driven by structural studies, we have shown that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these compounds. Generation of animal models is vital to improving our understanding of this disease and the development of new therapeutic strategies.

In additions to the developments of activity based probes and inhibitors, we also branched into the field of molecular chaperones. We have designed, synthesized and structurally evaluated a new class of neutral, conformationally constrained competitive glycosidase inhibitors that act by mimicry of the Michaelis complex conformation. Specifically, we found that D-galactose-configured a-cyclosulfamidate effectively stabilizes recombinant human a-D-galactosidase, the enzyme associated with Fabry's Disease, both in vitro and in cellulo and have provided protein crystal structures to support this work.
Exploitation Route The structural aspects of this work should aid future design of inhibitors, probes and chaperones for enzymes such as GBA1 and GLA.

This research should also provide a reliable route for the production of GBA and reduce reliance upon costly enzyme replacement therapy formulations for structural and mechanistic chemistry.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://pubs.rsc.org/en/content/articlelanding/2019/sc/c9sc03342d#!divAbstract