Investigating new mechanisms for cysteine-to-lysine transfer

Cysteine residues represent convenient sites to carry out selective chemical reactions on peptides and proteins. Such 'bioconjugation' is desirable for many applications, from the construction of antibody-drug conjugates as targeted cancer therapies to the functionalisation of surfaces with peptides and proteins for assays and arrays. However, cysteine bioconjugates can suffer some key drawbacks. For example, if the cysteine is present in the protein in the form of a disulfide bond, then its permanent modification leads to loss of this key structural motif. This PhD project aims to overcome limitations with existing cysteine conjugation methods, and offer new opportunities in the field, by investigating a promising new approach to achieve site-selective protein modification referred to as 'cysteine-to-lysine transfer' (CLT). In CLT the reagents initially hook on to cysteine residues and then undergo transfer conjugation to a nearby lysine amino-acid. The work will explore unprecedented new reagent classes to achieve this reactivity, and thus will develop our fundamental understanding of transfer chemistries whilst providing powerful new platforms for the construction of protein conjugates; for example, as prospective therapeutics such as antibody-drug conjugates which will be a key target application of this work. As such this project is within the EPSRC Research Area of Chemical Biology and Biological Chemistry, and sits between the Research Themes of Physical Sciences and Healthcare Technologies.
The project will involve extensive research and training in Organic Synthesis and Chemical Biology techniques. The initial reaction discovery phase will involve reagent design and synthetic planning, then multi-step synthesis, and finally small molecule reaction investigations in biologically compatible conditions. Next, the most promising reagents will be explored in bioconjugation experiments with select proteins, with associated extensive bioconjugate analysis. After investigating the reagent scope and optimising the bioconjugation protocols, final antibody conjugates aim to be constructed to demonstrate the improvements possible over the state-of-the art. In addition to the retention of key disulfide linkages, which will improve stability and homogeneity, it is proposed that the methods will enable unprecedented multifunctionalisation of these proteins in a controlled manner and this will be demonstrated to show exciting new opportunities are enabled.