New tools and technology to evaluate biological sulphation

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Clubbs Coldron AKM (2020) Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria. in Methods in molecular biology (Clifton, N.J.)

 
Description We have screened libraries of small molecules to evaluate sulphation on both glycans (sugars) and polypeptides (proteins) and discovered many new classes of sulfotransferase inhibitors. We have now obtained follow-on funding from BBSRC responsive mode (panel D) to evalute the human sulfotyrosine proteome. Our work was recognised by an independent editorial at Biochemical Journal. We have also published new work in collaboration with University of Liverpool chemists, which looks at how inhibitors of sulfotransferases can be obtained from nucleoside starting points.
Exploitation Route Assays for screening glycan and tyrosyl sulphotransferases could be widely employed to screen libraries of ligands, and we believe that through both private and open-access approaches that this is happening. We have also begun to develop sulfatase assays for sulfated glycans, which assess real-time desulfation and can be used for enzymology and inhitibor screening.
Sectors Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://www.ncbi.nlm.nih.gov/pubmed/30291171
 
Description New tools to study biological sulphation are being taken up by industry to drive discovery and analysis of glycan and protein sulphation. In particular, screening of sulfatases has begun, and the work has led to a new TRDF grant, to Dr D Fernig
First Year Of Impact 2018
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description ArrestAD: Sulphation analysis in the Alzheimers' brain
Amount £740,000 (GBP)
Funding ID 737390 
Organisation European Union 
Sector Public
Country European Union (EU)
Start 03/2017 
End 02/2020
 
Title Kinome-wide databases for human cancer cells 
Description All mRNA kinome signature for 12 human cancer cell lines has been generated 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? Yes  
Impact The analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for multiple cancer patients. 
 
Title Small molecule Inhibitors of glycan sulphotransferase (specifically HS2ST) 
Description Screening of libraries using recombinant 2OST enzymes and small molecules previously designated as protein kinase inhibitors. We identified the susceptibility of HS2ST to a variety of cell permeable compounds, including the promiscuous protein kinase inhibitor rottlerin and a family of oxindole RAF kinase inhibitors identified in a PKIS screen. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact New impetus for drug discovery in the glycan sulphotransferase field 
 
Title Small molecule Inhibitors of tyrosyl sulphotransferase (specifically human TPST1 and TPST2) 
Description Screening of libraries using recombinant TPST1 and TPST2 enzymes in a new ratiometric sulphation assay led to the discovery of the susceptibility of HS2ST to a variety of cell permeable compounds, including the promiscuous protein kinase inhibitor rottlerin and a family of oxindole RAF kinase inhibitors identified in a PKIS screen. This might extend to clinical RAF inhibitors, although further work is required. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact New impetus for discovery of sulphotransferase inhibitors 
 
Title Sulphation-based detection technology for glycans 
Description We currently lack essential enzyme assays and chemical tool compounds for approaches to manipulate glycan sulphation experimentally. Using BBSRC funding from a TRDF award, we have new procedures for thermal shift and non-radioactive enzyme-based 2OST sulphation assays, permitting us to rapidly assess glycan sulphation catalysed by HS2ST on an artificial fluorescent glycan substrate. These rapid, quantifiable assays permitted the development of rapid screening approaches for the discovery of biochemical and chemical inhibitors of HS2ST enzyme activity. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Impact on field of glycan sulphation via open access publication. 
 
Title Sulphation-based detection technology for protein tyrosine modification 
Description The biological post-translational sulphation of tyrosine residues by Tyrosyl Protein Sulpho Tranferases (TPST1 and 2) regulates secreted human proteins. TPSTs catalyse the transfer of sulphate from the essential co-factor PAPS (3'-phosphoadenosine'5'-phosphosulphate) to a context-dependent tyrosine residue in protein substrates. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches to discover inhibitors of tyrosine sulphation, We have developed new assays that permit the development of thermal and non-radioactive mobility-based enzymatic assays to quantify protein tyrosine sulphation within proteins and synthetic fluorescent peptides. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact New impetus in the field of protein sulphation 
 
Description Indian Institute of Science, Bangalore 
Organisation Indian Institute of Science, Bangalore
Country India 
Sector Academic/University 
PI Contribution New partnership driven through analysis of biological sulphation
Collaborator Contribution Provide bioinformatics software
Impact Publications are currently being written
Start Year 2017
 
Description University of Vicosa 
Organisation Federal University of Viçosa
Country Brazil 
Sector Academic/University 
PI Contribution A grant entitled "Capacity building for kinome technology in Brazil and India" was awarded by BBSRC GRCF ODA. Our partnership will strengthen international links and knowledge exchange with scientists in Brazil (specifically working on Leishmanias and kinase inhibitors) who are generating 'big data' approaches to help explain how the process of protein phosphorylation controls cell signalling in a variety of neglected organisms.
Collaborator Contribution Brazillian scientists, led by Professor Gustavo Bressan, are revealing the mechanisms of splicing by SRPK1 and SRPK2 kinases, and working on Leishmanias and kinase inhibitors of SRPK2. We are working with them to understand how protein phosphorylation controls cell signalling in these and other neglected organisms.
Impact Currently, these are multi-disciplinary research collaborations, no impact yet, beyond the societal impact of meeting dozens of scientists and lay members of the public who attended my seminars, and the PhD viva that I was external examiner for.
Start Year 2017
 
Title KinView Kinome Viewer 
Description Kinview places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. KinView can identify differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. Kinome Viewer can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. 
Type Of Technology Webtool/Application 
Year Produced 2016 
Impact High impact publication and thousands of web hits at the online server ( 
URL https://omictools.com/kinome-viewer-tool