New tools and technology to evaluate biological sulphation

The survival of organisms depends upon the ability of different cell types to communicate with each other by assembling the correct complexes of proteins and carbohydrates ('glycans') at the correct time in the correct place. One way this is achieved is to use the tricks of chemistry to change the biological properties of polymers, such as proteins, by adding and removing small charged chemicals as a means of regulation. These events, more accurately called 'post-translational modifications', act as switches to change information flow and dictate the types of different biological outcomes elicited, such as cell movement, growth, survival or death. Our proposal aims to develop tools to evaluate the addition of a specific chemical group, called sulphate, to glycans or proteins. We already know that sulphation is a modification on glycan polymers (e.g. glycosaminoglycans) and tyrosine amino acids (components of proteins), but we are currently unable to control sulphation chemically with the desired precision.

Hydroxyl group (-OH) sulphation is catalysed by a family of enzymes called sulphotransferases (STs), and is a central, yet poorly understood, regulator of many aspects of cell biology. Indeed, we already know that glycan sulphation is important for cell-cell and host-microbe interactions, supporting rate-limiting events in extracellular and intracellular cell signalling pathways, including processes critical for cellular ageing, bacterial infection and neurodegeneration. Protein sulphation, exemplified by intracellular tyrosine sulphation, also leads to poorly-studied changes in protein-protein interactions such as those that accompany viral infection and immune function. Enzymatic sulphation is thought to occur in the lumen of the Golgi apparatus, where proteins destined for secretion (i.e. function outside cells) are decorated with different numbers of sulphate groups in different regions. Since both occur on tyrosine, the potential for competition between tyrosine phosphorylation and tyrosine sulphation represents an example of the potential impact of sulphation on cellular signalling at the level of protein-protein interactions.

However, the analysis of sulphation is unfocused, it attracts little strategic funding, and is neither specific for glycan nor protein modifications, making efforts to study its global significance challenging. We are of the opinion that since it underpins so much of basic biology, sulphation research urgently requires a concerted research strategy to develop new chemical probes that can be used to perturb and analyse sulphation. To accomplish this, new high-throughput assays to measure protein and glycan sulphation are required to support chemical biology screens that might have considerable impact on the sulphation field. Indeed, technology-based approaches for the analysis of a different chemical group, phosphate, has led to a revolution in our understanding of how cells communicate, and has been important for biologists working in the areas of structural biology, cell signalling and communication and drug design, with remarkable knock-on effects on biotechnology and pharmaceutical industries across the world.

We have recently shown that the binding of small molecules to STs can be detected by a 'thermal stability assay' using the principles of differential scanning fluorimetry, where the ST is heated up (leading to unfolding) in the presence and absence of different chemicals. Binding of chemicals changes the response of sulphotransferase to unfolding, forming the basis for a new assay to discover the first cell permeable chemical inhibitors of these enzymes. Our proposal will build upon these assays to permit sulphation to be studied in real time using an a higher-throughput format, forming the basis for new screens using a large panel of optimised chemicals. Together, these new technological platforms will lead to the discovery of new probes for studying biological sulphation.

In marked contrast to phosphorylation analysis, sulphation studies currently lacks validated chemical probe compounds for linking substrates to enzymes or to perturb systems in which sulphation is thought to regulate a rate-limiting biological phenotype. By designing and assaying synthetic substrates for human glycan STs and protein tyrosine (PT) STs, we believe that we are in a unique positition to discover/repurpose small molecule compounds for sulphation modulation in a range of experimental systems. Our proposal builds on a novel Thermal Stability Assay (TSA) for the glycan ST 2-OST, and involves the evaluation of a new 384-well screening procedure using a mobility shift assay (MSA) to evaluate PAPS-dependent sulphation of fluorescent glycan and peptide substrates in real time. Since both these enzymatic modifications cause a sulphation-based decrease in chemical charge on the reaction product, sulphation can be quantified in real time by integrating relative substrate and product ratios. The employment of robotic-based screening with cell permeable compounds assembled from unique panels of protein kinase inhibitor scaffolds will constitute a new high-throughput screen for ST inhibitors. A second complementary approach (ligand-stabilisation) employs TSA to evaluate compound binding through different fluorescent read-outs, providing a back-up for our new mobility shift assays. Recent advances in glycan synthesis and enhanced understanding of the context of amino acids adjacent to sulphated tyrosines, means we are in a timely position to develop technology and discover the tools needed to revolutionise sulphation analysis. The investigators are active in the field of synthetic and cellular sulphation glycobiology (Yates/Fernig) and chemical biology for analysis of protein phosphorylation (Eyers), together amounting to >60 years of experience that we will deploy for the discovery of new tool compounds with utility across the Biotechnology for Health remit.

If successful, the project will provide a new framework for measuring ST activity, and defining, and then working up, new classes of ST (inhibitory) ligands with the potential to influence all aspects of cell biology. As such, the main beneficiaries of the knowledge generated from the pump-prime project are:
1) Academic biologists, clinicians and chemists. These include structural, matrix, neuro, signalling and glycobiologists, all of whom are interested in how sulphation status affects model systems. Indeed, the inability to quantify neither glycan ST activity nor protein tyrosine ST activity are major drawbacks in biological sulphate enzymology, since changes in these enzymatic outputs cannot currently be correlated with biological phentoypes. At the biophysical level, structural biologists may need to sulphate sugars or enzymes in a controlled manor, or inhibit sulphation completely, to produce biomolecules compatible with specific approaches such as X-Ray analysis, NMR study or cryo-EM studies. Very few cell treatments to modulate sulphation are available, with chlorate, a non-specific cytotoxic inhibitor of protein sulphation from 1986, still dominating the literature. Cell biologists may wish to assay STs using rapid real-time assays such as those described here, rather than having to use 35S-based outputs. Matrix biologists and neurobiologists often grow cells with unknown mixtures of sulphated matrices, and our work might be important for improving controlled inputs into these experiments. An understanding of how sulphation pathways can be manipulated enzymatically would be very useful. To use an extreme example, the discovery of non-specific staurosporine (kinase) inhibitors affords few applications as a specific chemical biology probe, but generates a useful reagent to drive cells into apoptosis. In particular, glyobiologists will appreciate the availability of tool compounds with which to modify sulphation patterns using rapidly acting small molecules and correlate them with biological effects. The addition of STs to the canon of targetable enzymes will also have a major effect on clinicians (who might be driven to analyse sulphation, or sulphation changes, as biomarkers during a biological or medical process), and by medicinal chemists, who will rapidly integrate new screening platforms with synthetic chemistry to generate biologically 'fit' (i.e. well-validated) probe compounds.
2) Industrial biotechnology associated with sulphated products. These include major blue-chip companies who synthesise, or extract, sulphated products with tunable physiochemical properties linked to their function. The controlled analysis of sulphation in glycans or proteins provides a new useful chemical tool for development of innovative products with industry, including those with higher purity and known sulphation status
3) Pharmaceutical sector, who will see the potential for the development of ligands with the requisite specificity and cellular efficacy to be developed into new resources to respond to challenges in ageing populations, including neuronal stress and inflammation. A lack of progress in pharma is linked to a lack of technological innovation (i.e. ST assays are slow and low-throughput) rather than a lack of druggability of ST targets (e.g. purine based inhibitors (Ki = <100 nM) of beta-aryl sulphotransferase IV and a 500 nM inhibitor of oestrogen ST have been reported).
4) Government authorities, who are aware of drug-safety issues with small molecules that are approved as drugs, and are currently embedded in trying to understand how specifically targetting medicines might more usefully done in different populations ('personalised medicine')
5) The general public. Although currently unclear, sulphation status of products might well contribute/correlate with healthier lifestyles and/or environmental sustainability. An ability to analyse samples for sulphation activity is needed for these statements to be tested.