Screening Carbohydrate-Active Enzymes - a Fluorescent Solution

Lead Research Organisation: King's College London
Department Name: Pharmaceutical Sciences

Abstract

In all domains of life, the formation of complex carbohydrates requires the concerted action of a multitude of glycosyltransferases (GTs), enzymes that catalyse the transfer of a sugar from a sugar-nucleotide donor onto an acceptor oligosaccharide chain. Such glycosylation reactions are central to many fundamental biological processes, such as cell signalling, cellular adhesion, carcinogenesis, the evasion of the immune system by human pathogens and, of particular interest in the context of this proposed study, the generation of the complex and dynamic 3D architecture that is the plant cell wall. In contrast to other protein families of comparable size and biological significance, our understanding of GT biology remains fragmented, due to a lack of molecular tools for the investigation of GT substrate specificity. Existing GT assays are generally labour- and/or cost-intensive and/or require specialist equipment. Our approach to this problem is through the design, synthesis and application of novel fluorescent sugar probes, which can be used to identify GTs and to determine their substrate specificity.

Technical Summary

Novel fluorescent sugar nucleotide probes developed in the Wagner group possess several advantages over conventional fluorophores previously used in protein bioassays: (i) the structural difference to the natural donor substrate is minimal; (ii) the binding characteristics are very similar to those of the natural ligand; (iii) the chemical synthesis of the fluorescent sugar-nucleotides is straightforward; and (iv) the same synthetic strategy can be used for the development of different fluorescent UDP-sugars, both with regard to the identity of the sugar and the properties of the fluorophore. The novel fluorophores proposed herein are therefore ideally suited for the efficient development of fluorescent probes for the investigation of sugar-nucleotide- dependent enzymes.

Publications

10 25 50
 
Description Small chemical probes can differentiate between different members of the same, large enzyme family, using a simple fluorescent read-out
Exploitation Route These findings can be used for the development of new assays, inhibitors and biotechnological tools
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The findings have led to a new academic/industry collaboration with Ludger Ltd. The goal of this collaboration is the development and application of new biotechnological tools for affordable, high-performance anti-cancer immunotherapeutics
First Year Of Impact 2015
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title GT fluorescent probes 
Description A series of ca 10 new fluorescent probes for glycosyltransferases (GTs) has been prepared. The biological evaluation of the new materials as potential substrate analogues or inhibitors is ongoing. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact The new fluorescent probes are currently under investigation in recombinant and cellular assays, in collaboration with other research groups (Palcic/Copenhagen, Page/London) 
 
Description Carlsberg Research Centre 
Organisation Carlsberg Group
Department Carlsberg Research Centre
Country Denmark 
Sector Private 
Start Year 2006
 
Description JIC 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution supply of synthetic reagents and intermediates for the enzymatic synthesis of novel fluorescent probes
Collaborator Contribution enzymatic synthesis of novel fluorescent probes and structure-activity studies of carbohydrate-active enzymes
Impact Exploration of carbohydrate-active enzymes for the synthetic preparation of a novel class of non-natural sugar-nucleotides as fluorescent biological probes
Start Year 2010
 
Description Ludger Ltd 
Organisation Ludger Ltd
Country United Kingdom 
Sector Private 
PI Contribution provision of glycosyltransferase inhibitors for the development of optimised glycoprotein therapeutics
Collaborator Contribution provision of unique glycoanalytics platform
Impact no outputs yet
Start Year 2015
 
Title DERIVATIVES OF URIDINE PHOSPHATE AND THEIR USES IN PROTEIN BINDING ASSAYS 
Description development of fluorescent nucleotides and nucleotide-conjugates for applications in protein binding assays, e.g. for small molecular inhibitor screening 
IP Reference WO2011051733 
Protection Patent granted
Year Protection Granted 2015
Licensed No
Impact this IP forms part of the ongoing collaboration with Ludger Ltd