Periodic Reporting for period 1 - GlypStx (Glycoprotein-based inhibitors of shiga-like toxin)
Okres sprawozdawczy: 2018-02-01 do 2020-01-31
Gastrointestinal infections have substantial impact in both the developing world and in Europe with 1.5 billion cases each year leading to approximately two million deaths each year, 760,000 of which are in children under five years old. Many of these deaths are caused by bacteria for example E. coli O157, that produces protein toxins, including shiga-like toxin (SLT). In the human body, the surfaces of living cells are covered in complex carbohydrate molecules. This “sugar coating” allows the cells to interact with viruses, bacteria, and toxins that have complementary protein receptors. The pentagonal shiga-like toxin produced by E. coli O157 bacteria can attach to five copies of a specific carbohydrate on the gut wall and enter cells lining the intestine. The result is bloody diarrhoea and the toxin entering the circulatory system to cause kidney failure.
In this project, the aim was to make protein-based inhibitors that can prevent this toxin from attaching to, and enter kidney cells. The inhibitors were to be synthetic glycoproteins that have 5/15 copies of the target sugar attached at specific sites so that they can bind to all 5 of the toxin’s receptor sites simultaneously providing an extremely strong interaction to prevent the toxin from entering cells. During the project a series of glycopeptides were to be synthesised and attached to pentameric protein scaffolds using site specific chemical and enzymatic modification. The glycoproteins would then be evaluated as inhibitors of toxin adhesion using a variety of biophysical assays. The optimized inhibitors will have potential as a new class of future biopharmaceuticals.
In this project, the aim was to make protein-based inhibitors that can prevent this toxin from attaching to, and enter kidney cells. The inhibitors were to be synthetic glycoproteins that have 5/15 copies of the target sugar attached at specific sites so that they can bind to all 5 of the toxin’s receptor sites simultaneously providing an extremely strong interaction to prevent the toxin from entering cells. During the project a series of glycopeptides were to be synthesised and attached to pentameric protein scaffolds using site specific chemical and enzymatic modification. The glycoproteins would then be evaluated as inhibitors of toxin adhesion using a variety of biophysical assays. The optimized inhibitors will have potential as a new class of future biopharmaceuticals.
The fellowship was focussed on developing neoglycoproteins that can target the binding with shiga-like toxin. So, glycopeptides were designed and synthesised successfully in the host’s lab. Proteins including a non-binding mutant of cholera toxin B-pentamer, shiga-like toxin B-pentamer, serum amyloid protein were expressed and purified. The method for ligation of glycopeptides with protein was optimised and the improved procedure was published as part of a collaboration with the Fascione lab at University of York. Final biochemical and biophysical characterisation was interrupted by the coronavirus lockdown, so some experiments will be completed after the end of the fellowship. As part of the fellow’s training in expertise in managing interdisciplinary research projects, and transfer of his expertise in carbohydrate chemistry, he also established a new collaboration with colleagues in the School of Geography at University of Leeds in a new project, on testing novel isotope approaches to reconstruct past precipitation regimes in the Amazon. The fellow developed a method to measure isotopic oxygen ratio at each position of glucose units of cellulose molecule without isotopic fractionation. After development of the method, training in the protocols were given to other colleagues for testing on samples from Amazon forests. Results generated so far were presented in multiple international conferences in the form of poster presentations. Two scientific papers were published and additional publications will be submitted.
This fellowship provided the opportunity to raise the fellow’s research profile to represent an attractive and convincing prospect when applying for independent funding schemes. The fellowship helped the fellow to acquire diverse scientific skills and complementary training needed to address problems in the multidisciplinary field of chemical biology. This research topic is relevant to modern synthetic glycopeptides chemistry and drug design strategies targeting gasterointestinal disease with poor prognosis using current approaches which is often in the spotlight of the general public. The methods for making the glycoproteins has been optimised and is now a robust procedure that can easily be adopted by other researchers. The novel glycoproteins prepared are more complex that any previously reported using these synthetic strategies and are initial prototypes for a new class of therapeutic that could in future be developed to prevent kidney damage in cases of E. coli food poisoning. Public communication activities to a broad audience focussed on relating the research objectives to current health problems, improve general understanding of targeting difficult diseases and emphasise the potential of this research to provide ground-breaking techniques and knowledge with the ultimate goal to improve health-care.