Summary
The glycosyltransferase (GT) activity assay provides a fluorescent-based, high-throughput compatible format for investigating the enzyme substrate binding of glycosyltransferases that use uridine diphosphate (UDP) sugars as donor substrates. The key component of the assay is a novel type of fluorescent UDP-sugar derivative which facilitates dual utility of the assay; it can be used in i) a binding assay, e.g. for the identification of GT inhibitors (ligand displacement assay) or ii) the investigation of the sugar-nucleotide specificity of putative GTs and other sugar-nucleotide-dependent enzymes. One major advantage of the assay is that the acceptor substrate for the sugar is not required.
Technology Overview
UDP-Sugars are substrates for a large number of biologically and therapeutically important enzymes, including in particular the glycosyltransferase family. Glycosyltransferases (GTs) are nature’s glycosylation reagents: enzymes that catalyse the transfer of a mono- or oligosaccharide from a glycosyl donor, usually a sugar-nucleotide, to a suitable acceptor, e.g. a glycan, peptide or lipid. GTs and their biosynthetic products play a key role in many fundamental biological processes underpinning human health and disease, including cellular adhesion, blood group biosynthesis, and the generation of peptidoglycan and lipopolysaccharide structures in pathogenic bacteria. Individual GTs have been identified as promising therapeutic targets in infection, inflammation and cancer. One example is the bacterial alpha-1,4-galactosyltransferase LgtC, which is required for the biosynthesis of Gal-Gal epitopes, a known virulence factor in Gram-negative pathogens such as Haemophilus and Neisseria (Fig. 1). GTs are also used routinely in biotechnology for the generation of defined glycoforms of glycotherapeutics.
Chemical tools and assay kits for GTs are therefore of considerable scientific and commercial interest, in order to identify inhibitors for these enzymes, to study their role in health and disease, to characterize enzymes with putative GT function, to identify new GT enzymes, and to aid the use of GTs in biotechnology.
Applications
Mammalian cells use a limited pool of only nine different GT donor sugar-nucleotides, six of which are UDP-sugars, i.e. UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-glucuronic acid (UDP-GlcUA) and UDP-Xylose (UDP-Xyl). These UDP-sugars are the donor substrates for the majority of mammalian GTs.
We have recently developed a novel type of fluorescent UDP-Gal derivative [1,2]. We have shown that this fluorophore binds selectively at the donor binding site of several galactosyltransferases (GalTs), and that upon binding, its fluorescence is quenched. This phenomenon can be exploited for several applications, including in particular the following two assay types:
(i) Ligand-displacement assays. Displacement of the fluorophore from the donor binding site, e.g. by the natural ligand UDP-Gal, results in the regeneration of fluorescence, and thus the fluorophore can be used to screen compound libraries for GT inhibitors (Fig. 2).
(ii) Functional characterization of putative GTs and other UDP-sugar-binding proteins. GTs are an unusually large family of enzymes and in the majority of cases of reported enzymes the annotated function is only based on sequence similarity, with no biochemical evidence for the putative GT activity. The fluorophores provide a simple tool to investigate the capacity of a putative GT to bind a given UDP-sugar, even in the absence of acceptor. Results from such an experiment would give an indication of the type of sugar transferred by a putative GT. The application of the fluorophores could therefore become a routine first step in the functional characterisation of new, putative GTs and other sugar-nucleotide-dependent enzymes.
Benefits
The fluorophores can be used in a binding assay, e.g. for the identification of GT inhibitors (ligand displacement assay) or the investigation of the sugar-nucleotide specificity of putative GTs and other sugar-nucleotide-dependent enzymes.
A major obstacle for GT drug discovery to date is the continuous lack of drug-like inhibitors. A major source for such inhibitors would be from high-throughput screening (HTS).
The assay is non-radioactive, operationally simple (only a fluorimeter is required) and adaptable for a microplate format. In contrast to existing GT inhibitor assays, the assay does not require access to the acceptor substrate of a given GT. This is important, as many GT acceptors are not commercially available and frequently synthetically difficult to access. Access to acceptor substrates can therefore be a bottleneck for the application of other GT assays.
Development
Proof-of-concept for the application of fluorescent UDP-Gal with different GalTs has been demonstrated. An attractive feature of the fluorophores is the fact that their fluorescence emission is based on the chemical modification of the uracil base, not of the sugar. The same fluorescent UDP fragment can therefore be conjugated to different sugars. This opens the door towards the broad application of the fluorophores with proteins that bind different UDP-sugars (e.g. UDP-Glc, UDP-GlcNAc, UDP-GalNAc etc).
The Product
A commercial assay kit based on the fluorophores would include a package of 4-5 different UDP-sugar fluorophores (e.g. UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc etc). Such a kit would be highly useful not only for the HTS for inhibitors against different GTs (i.e. GalTs, GlcTs etc), but also for the rapid screening of the donor specificity of putative GTs with unknown function. For such an application, the protein of interest would be incubated separately with each fluorophore. It would be expected that quenching will occur selectively and exclusively with the UDP-sugar that is the natural donor of the target protein.
Opportunity
Scientists at King’s are using the assay in house to screen for novel inhibitors of specific galactosyl tansferases of interest.
KCL is seeking partners to commercialize this opportunity. Patent applications are available for licensing.
IP Status: Nationalised in US and EP. Priority application filed November 2009.
Keywords:
Platform technology, galactosyl transferase