Thomas Pesnot

Structural and mechanistic basis for a new mode of glycosyltransferase inhibition.

Glycosyltransferases are carbohydrate-active enzymes with essential roles in numerous important biological processes. We have developed a novel donor analogue for galactosyltransferases which locks a representative target enzyme in a catalytically inactive conformation, thus almost completely abolishing sugar transfer. Results with other galactosyltransferases suggest that this novel and unique mode of glycosyltransferase inhibition is, very likely, generally applicable to other members of this very important enzyme family also.

Glycosyltransferases and their Assays

Glycosyltransferases (GTs) are a large family of enzymes that are essential in all domains of life for the biosynthesis of complex carbohydrates and glycoconjugates. GTs catalyse the transfer of a sugar from a glycosyl donor to a variety of acceptor molecules, for example, oligosaccharides, peptides, lipids or small molecules. Such glycosylation reactions are central to many fundamental biological processes, including cellular adhesion, cell signalling and bacterial‐ and plant‐cell‐wall biosynthesis. GTs are therefore of significant interest as molecular targets in chemical biology and drug discovery. In addition, GTs have found wide application as synthetic tools for the preparation of complex carbohydrates and glycoconjugates. In order to exploit the potential of GTs both as molecular targets and synthetic tools, robust and operationally simple bioassays are essential, especially as more and more protein sequences with putative GT activity but unknown biochemical function are being identified. In this minireview, we give a brief introduction to GT biochemistry and biology. We outline the relevance of GTs for medicinal chemistry and chemical biology, and describe selected examples for recently developed GT bioassays, with a particular emphasis on fluorescence‐based formats.

Novel derivatives of UDP-glucose: concise synthesis and fluorescent properties.

A series of novel 5-substituted UDP-glucose derivatives with interesting fluorescent properties and potential applications as sensors for carbohydrate-active enzymes is reported. An efficient synthesis of the target molecules was developed, centred around the Suzuki-Miyaura reaction of (hetero)arylboronic acids with 5-iodo UDP-glucose. Interestingly, the optimised cross-coupling conditions could also be applied successfully to 5-bromo UMP, but not to 5-bromo UDP-glucose.

Inhibition of galactosyltransferases by a novel class of donor analogues.

Galactosyltransferases (GalT) are important molecular targets in a range of therapeutic areas, including infection, inflammation, and cancer. GalT inhibitors are therefore sought after as potential lead compounds for drug discovery. We have recently discovered a new class of GalT inhibitors with a novel mode of action. In this publication, we describe a series of analogues which provide insights, for the first time, into SAR for this new mode of GalT inhibition. We also report that a new C-glycoside, designed as a chemically stable analogue of the most potent inhibitor in this series, retains inhibitory activity against a panel of GalTs. Initial results from cellular studies suggest that despite their polarity, these sugar-nucleotides are taken up by HL-60 cells. Results from molecular modeling studies with a representative bacterial GalT provide a rationale for the differences in bioactivity observed in this series. These findings may provide a blueprint for the rational development of new GalT inhibitors with improved potency.

Two-Step Synthesis of Novel, Bioactive Derivatives of the Ubiquitous Cofactor Nicotinamide Adenine Dinucleotide (NAD)

We report the design and concise synthesis, in two steps from commercially available material, of novel, bioactive derivatives of the enzyme cofactor nicotinamide adenine dinucleotide (NAD). The new synthetic dinucleotides act as sirtuin (SIRT) inhibitors and show isoform selectivity for SIRT2 over SIRT1. An NMR-based conformational analysis suggests that the conformational preferences of individual analogues may contribute to their isoform selectivity.

A Novel Fluorescent Probe for Retaining Galactosyltransferases

Glycosyltransferases (GTs) are a large class of carbohydrate‐active enzymes that are involved, in both pro‐ and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high‐throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP‐galactose (UDP‐Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read‐out in ligand‐displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP‐Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor.

Base-modified Donor Analogues Reveal Novel Dynamic Features of a Glycosyltransferase.

Background: Modified UDP-Gal donor substrates with 5-formylthienyl and 5-phenyl substituents on the uracil base exhibit differential inhibition patterns for glycosyltransferases. Results: Structural studies reveal a new enzyme loop folding mode for the 5-formylthienyl analogue. Conclusion: Differential inhibition is attributed to alternate enzyme conformational changes and interactions with the respective inhibitors. Significance: The conformational plasticity of glycosyltransferases can be exploited in designing novel inhibitors. Glycosyltransferases (GTs) are enzymes that are involved, as Natures “glycosylation reagents,” in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.

Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and limitations

Graphical abstract

Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the “tucked under” conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.