Research

The group has been involved in developing ground-breaking new methodologies, with the Baker and Caddick research groups, that allow for reversible and irreversible homogeneous protein modification. For example, dihalomaleimides and dihalopyridazinediones can be used for the reversible, or irreversible, conjugation of biomolecules in high yield on various proteins. Reversible constructs have been shown to disassemble in the reducing-environment of the cytoplasm of mammalian cells. The methodology is currently being exploited in a variety of applications such as the homogeneous modification of antibodies and antibody fragments, and in reversible-affinity labelling for pull-down experiments.

In recent years, the group has been involved in the development of a fundamentally novel approach to radical C-H bond activation that has been published in high impact journals such as Nature Chemistry and Chemical Science. This work is based on the discovery that acyl radicals generated from aldehydes in the presence of air can undergo hydroacylation with certain alkenes. To date, the hydroacylation of vinyl sulfonates, vinyl sulfones, unsaturated esters and vinyl phosphonates has been reported, to generate a variety of unsymmetrical ketones. The group has also explored the aerobic hydroacylation of azo-dicarboxylates with various aldehydes to generate hydrazide dicarboxylates in high yields, even when employing aldehyde as the limiting reagent. The use of stoichiometric amounts of aldehyde in hydroacylation processes offers the opportunity to employ high value aldehydes, which has been a major limitation of previous protocols. Most recently, we have unearthed a method for the functionalisation of ethereal-based saturated heterocycles with concomitant aerobic C–H activation and C–C bond formation.

The group has made a major contribution to the establishment of a new technology that allows for the site‑selective dual labelling of proteins guided by local protein microenvironment. This is a fundamentally novel approach to achieve chemoselectivity on proteins that exploits the differential solvent accessibility of certain hydrogen atoms to selectivity promote or halt reaction. The methodology can introduce orthogonal handles which can be functionalised in a sequential manner, allowing for facile introduction of various cargos selectivity, e.g. drugs, radio labels or fluorophores. We have also, more recently, developed a tri-click method for the tris-modification of various proteins in a facile manner.

Although recent methods for the engineering of antibody-drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. The group, in collaboration with the Baker, Caddick, Pedley and Chester research groups, have made a significant step towards the ideal platform for next generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification is delivered. The relevance of the work in a biological context, on full antibody and bispecific constructs, in cytotoxicity assays and cell internalisation studies with HER2-positive and negative breast cancer cell lines has also been demonstrated.

The group, in collaboration with Dr Liam Browne at Harvard University, is focused on bridging a thiol-reactive, light-sensitive azobenzene molecule across cysteines (introduced by mutagenesis) into ion channels. Such chemically modified channels can then be opened by irradiation at 440 nm and closed by light at 360 nm, in the absence of any extracellular ligand, by a pushing and pulling force created by switching between the cis– and trans-states of the azobenzene. This has been exemplified to a great degree of success on various P2X receptors and an acid-sensing ion channel. This overall work will extend our understanding of the molecular mechanism of gating and provides a tool to investigate the role of various receptors in intact cells and tissues. Moreover, a collaboration with Prof. Trevor Smart at UCL to appraise the methodology on the highly significant GABA-A receptor has recently been established. The appropriate cysteine mutations have been identified and the technology will be applied on this important receptor in the near future.

The ability of nature to engineer functional and dynamic structures with tuneable size has been mimicked by synthetic amphiphiles for various applications. Of particular note are new advances in controlled polymerisation techniques that have allowed the design of a new class of polymer-based vesicles, polymersomes, which are based on the self-assembly of block-copolymers. In comparison to their lipid counterparts, polymersomes are significantly more stable in terms of mechanical and chemical properties and can also be functionalised in various ways. It is of great interest to design polymersomes for drug-delivery, and an important feature to ensure effective cellular entry is the polymersome’s surface topology. In analogy with natural viruses (the most effective systems in entering cells), the ideal surface topology has to display clusters of ligands dispersed within cell inert materials. With Prof. Giuseppe Battaglia at UCL, the group will be developing constructs that will allow for the creation of a variety of polymersomes, with our recent work already showing great promise for the construction of patchy polymersomes. Such a control of ligand-receptor interaction will enable us to match closely the cell membrane topology and thus create more favourable energy landscapes for deforming the membrane.

The modification of lysine residues with acylating agents has represented a ubiquitous approach to the construction of antibody conjugates, with the resulting amide bonds being robustly stable and clinically validated. However, the conjugates are highly heterogeneous, due to the presence of numerous lysines on the surface of the protein, and greater control of the sites of conjugation are keenly sought. We developed a novel approach to achieve the targeted modification of lysines distal to an antibody fragment’s binding site, using a disulfide bond as a temporary ‘hook’ to deliver the acylating agent. This cysteine-to-lysine transfer (CLT) methodology offers greatly improved homogeneity of lysine conjugates, whilst retaining the advantages offered by the formation of amide linkages.