High-Throughput Luminescence Assay for Sulfotransferase Activity

Living things develop and grow, adapt to constant changes in their environment and energy sources, reproduce and resist disease. These things they achieve through communication between organisms and between cells within an organism, often by changing the chemical make-up of molecules. A common route is to add or remove a small charged chemical such as phosphate or sulfate, which act as switches, altering the information that is communicated. This dictates the biological outcome, which may be as varied as cell movement, cell death, and successful (or not) infection. The addition of a negative sulfate group is performed by specific enzymes called sulfotransferases, but we lack the technology to easily measure the activity of these enzymes.

Sulfotransferases catalyse the transfer of sulfate to alcohols (-OH) on proteins, large sugars (glycans) and many small molecules, including hormones. There has been focussed research on small selective subsets of these enzymes and on some of their sulfated products. Glycan sulfation in animals plays key roles in development, wound repair and is associated with ageing, neurodegenerative diseases and infection by viruses. Sulfation of proteins on the amino acid tyrosine results in changes in protein-protein interactions related to immune function and virus infectivity. In plants, tyrosine sulfation produces hormones, and can be used by pathogens as part of their infection strategy. Bacterial sulfotransferases can sulfate a wider range of small molecule targets than animals, and some of these enzymes have potential as replacements of currently unsustainable chemical production of personal care and food products.

Efforts to understand the global significance of sulfation and to harness it have faced an unsurmountable obstacle: there is no simple and accessible means to measure the sulfation of a target by a sulfotransferase. The consequence is piecemeal research, which is largely unable to deliver the depth and breadth of fundamental knowledge and advances necessary to harness sulfation for our benefit in animal and plant health, and in product synthesis.

Our proposal aims to develop a simple, cheap and versatile assay technology that will allow all researchers to measure the addition of a sulfate to any biological or indeed man-made molecule by a sulfotransferase. Such assays have proven to be the key to understanding and exploiting related chemical modifications to biological molecules, such as phosphorylation.

Sulfotransferases use a universal sulfate donor, PAPS, which upon transfer of its sulfate group becomes PAP. Measuring PAP provides a direct measure of sulfation. We will develop molecular probes based on a rare earth element, Europium, which emit red light upon bind reversibly to PAP. Measurement of the emitted light is very sensitive and provides a real-time readout of the activity of the sulfotransferase. This will be independent of the molecule the sulfotransferase is attaching the sulfate to. In this way the assay will be fast, real-time and eliminate the need for chemically modified reagents, radioactivity, antibodies and expensive equipment not generally available.

Our proposed molecular probes have enormous potential to make the measurement of sulfotransferase activity routine and reduce the cost and time required to conduct high-throughput screening assays. These probes will provide a vital step towards the rapid, accurate determination of sulfotransferase kinetics and mechanism. This will enable better selection and validation of sulfotransferase inhibitors (drug leads), and of mutant enzymes for industrial use. The new technology will pave the way for understanding biological sulfation and exploiting it in the contexts of health and disease, and of product manufacture.

Sulfation of proteins, sugars and other substrates is an important regulatory modification, which is directly linked to homeostasis and disease in animals and plants. Moreover, many industrial products are sulfated using non-sustainable chemical routes, which for polymers are poorly controlled. Sulfation is catalysed by sulfotransferases (STs), which utilise the sulfonyl donor PAPS to sulfate a variety of acceptors (polysaccharides, proteins, metabolites and xenobiotics), generating PAP in the process.

STs have been demonstrated to be druggable, yet this aspect of drug discovery is underexplored whereas engineering of STs to optimize activity with industrially relevant substrates is not currently possible. Progress in ST research is limited by the lack of rapid, label-free tools for conducting high throughput screens of ST activity. Existing medium throughput assays rely upon expensive equipment and require fluorescently labelled acceptor substrates. This restricts the acceptors that can be used and hence the range of STs that can be analysed. Due to the lack of a generic assay, sulfation is understudied in living organisms, and requires technological innovation.

This project will develop a generic assay technology for real-time monitoring of ST reactions. Luminescent probes will be synthesised to reversibly bind and discriminate PAP and PAPS, providing a real-time readout of the PAP/PAPS ratio. This novel approach to ST monitoring will overcome significant limitations in existing assays, obviating the need for: (1) chemically labelled acceptors; (2) expensive equipment, radioactivity or antibodies; and (3) isolation or purification steps. We will transform HTS assay development to provide the first cost-effective method for quantifying ST activity, revolutionising the discovery of ST inhibitors and engineered STs, and pave the way for measuring PAP/PAPS intracellularly.

Success in this project will deliver a generic and accessible technology for the measurement of sulfotransferase (ST) activity. This is analogous to the popular 'Kinase Glo" assays used for the analysis of kinases. The main beneficiaries of the knowledge generated from the pump priming project are:

1. Academic biologists, clinicians and chemists. The major bottleneck in biological sulfation and its enzymology is the inability to quantify sulfotransferase activity without a specialised small synthetic substrate. This generally prevents the correlation of enzyme activity with phenotype. In structural biology, there may be a need to sulfate sugars, proteins or other molecules in a controlled manner to produce molecules with the appropriate modifications. This would be particularly important in the analysis of co-crystals of functional complexes and in enzyme engineering. High throughput assays of STs, where different acceptors can be used with the same enzyme, would facilitate the discovery of tool compounds that inhibit the enzyme.

The assay would be of interest to pharmacologists and clinicians looking to prolong the pharmacokinetics of drugs inactivated by sulfation and so reduce frequency of administration. Other clinicians might need to measure sulfation as biomarkers in a pathological or medical process, whereas medicinal chemists would integrate the assay into screening platforms with synthetic chemistry to produce and identify well-validated probe compounds.

2. Industrial biotechnology. A range of sulfated molecules, particularly alcohols and polysaccharides (e.g., xyloglucans, carrageenans) are widely used in the food and personal care industries. Current synthesis methods are resource-inefficient, not sustainable, particularly 'green' or precise, with post-sulfation tests necessary to determine the commercial viability of batches. Enzymatic sulfation, using designer enzymes with novel specificities produced via high throughput screening of mutants with the acceptor-independent PAP/PAPS assay would contribute towards a green and sustainable production of these consumer molecules.

3. Pharmaceutical industry. The development of selective ligands with the necessary specificity and efficacy is important in developing new resources in response to the challenges of ageing, including inflammation and neuronal stress. STs are druggable, but progress has been hampered by the lack of generic high throughput assays. One goal of this project is for our assay to be adopted by pharmaceutical companies to facilitate identification and validation of new potent ST ligands.

4. Agritech. The discovery of sulfated peptide hormones in plants and of a rice pathogen ST provides new strategies to ensure food security, dependent on ST assays. Further, the proposed probes can be tuned to detecting different anions and adapted readily for applications in environmental monitoring organisations, e.g., detecting harmful phosphates and sulfates (herbicides, pesticides) in watercourses, enabling better management of agricultural resources to reduce these pollutants.

5. Government agencies involved in drug safety and agriculture, working to understand how targeting drugs to subpopulations (personalised medicine) might be more efficacious clinically and ensuring food and water security.

6. The general public. The proposed research will contribute towards personalised medicine, sustainable sulfation of industrial products, improved crop growth and pathogen control, enhanced pharmacokinetics of existing drugs and new drugs, which will positively impact on health and environmental sustainability. More effective treatments of diseases and improved food security are areas that capture public interest and support, enabling us to engage the general public and young audiences, raising awareness of the power of synthetic chemical biology to advance drug discovery, agitech and biotechnology research.