New Reagents for Protein Modification

Cysteine oxidation has attracted increasing interest recently, due to cumulative evidence of its key role in several critical and complex cellular processes, notably oxidative stress response mechanisms and redox cellular signalling pathways. It is estimated that up to 12% of all cysteines in the human proteome is in an oxidised form in cells and tissues. Astonishingly, cysteine sulfinic acid (Cys-SO2H, "CSA") alone is thought to account for ~5% of accessible (i.e. not buried in the protein core) cysteines in the human proteome. While increasing evidence in recent years have suggested that CSA acts as a regulatory modification, the full scope of its biological formation and activity remains poorly understood.

A major obstacle is the lack of methods to incorporate CSA chemically within purified proteins to enable accurate biochemical studies. Indeed, current methods for the preparation of CSA have proven extremely laborious and have relied on treating a cysteine thiol (Cys-SH) containing peptide/protein of interest (POI) with oxidising agents such as hydrogen peroxide or peroxyacids. Such methods are particularly inefficient and produce complex mixtures of oxidised POIs, further hampering purification and accurate characterisation in biochemical and biophysical assays.

To address this critical caveat, we aim to develop new methods allowing the incorporation of CSA within recombinant proteins for functional studies, by modulating the nucleophilicity hence reactivity of the cysteine thiol towards oxidation. This programme of work will involve two main aspects: First, it will establish the design and synthetic routes towards novel cysteine chemoselective reagents in a CAPPING-OXIDATION-UNCAPPING one-pot sequence, along with biocompatible protocols for the efficient conversion of cysteine to CSA in short peptides. Second, we will then take advantage of these new reagents/protocols for the controlled introduction of CSA in an array of recombinant, therapeutically relevant proteins in proof-of-concept experiments.

An important focus of our investigation will be directed at shedding initial light on the influence of CSA on the structure, stability and molecular recognition properties of proteins. Overall, the successful development of these methodologies will provide the community with a valuable new tools to investigate the function and relevance of CSA in human physiology and disease. Finally, it will also open exciting avenues for the development of new generations of synthetic proteins/peptides encoding new activities and functions, for both fundamental science and therapeutic applications.

Synthetically modified proteins and the reagents to prepare them are critical tools to aid elucidation of molecular mechanisms governing living systems, but also for subsequent therapeutic intervention in disease. The development of novel biochemicals/biologicals is of high-value to the UK economy. Overall, the growing life science sector employs over 230,000 people and generates over £60 billion turnover annually in the UK. This project aims to pioneer the development of new tools to study the role and molecular basis of cysteine oxidation in human cellular processes. The primary impact of this research will be in the biochemical and biological sciences, and will be especially beneficial to researchers focusing on cysteine redox cellular signalling in both human physiology and disease. New tools for CSA generation will significantly expand the biochemical toolbox for protein cysteine modification, and allow the precise dissection of the impact of CSA on proteins' properties hence deepening our fundamental understanding of living systems. Accumulating evidence are pointing at unbalanced cysteine redox processes as drivers of diverse disease states, including cancer and neurodegeneration, two leading health related problems costing millions of lives and billions of pounds each year worldwide. Therefore, this research also has the potential to impact the health sector and wellbeing of patients through informing the development of new drugs on the long term. It has also been shown that cysteine oxidation can alter the binding efficiency of drugs to their protein targets. This study has the potential to impact drug discovery and medicine by allowing systematic and improved predictions of drug efficacy in cells, hence influencing decision making in lead optimisation and prioritisation in clinical trials. This will be of interest to researchers in medicinal chemistry in academia and the pharmaceutical industry sector. Reagents and methods to covalently target protein cysteine residues have attracted significant attention in the industrial sector, which recently culminated in the approval of the covalent anticancer drug molecule TAGRISSO. Our research will deliver new classes of covalent reagents targeting nucleophilic residues in proteins, opening new exciting avenues for the development of covalent chemical probes and drugs. Overall, while tangible intellectual and commercial impact is foreseeable, this will likely be achieved beyond the timescale of this grant (1.5 year PDRA). However, the landscape into which this work fits and impact it is directed towards has been outlined.