Peptide-protein and protein-protein interactions play vital roles in various biological processes. Therefore, covalent trapping of these interactions becomes a useful analytical tool and paves the way towards the development of covalent therapeutics. Within our research group a cross-link technology (Figure 1), based on the introduction of a furan containing unnatural amino acid (2-furyl-L-alanine or furan-modified L-lysine) was developed and applied for covalent trapping of peptide-protein¹ and protein-protein interactions.² Crosslinking with target receptors can be initiated in vitro upon visible light irradiation of a photosensitizer (PS) leading to singlet oxygen generation and oxidation of the furan (Fur) to a flexible reactive keto-enal moiety, which can subsequently scan its immediate surroundings for proximal nucleophiles (lysine (K), cysteine and tyrosine) in the bound partner.³

On live cells, a furan-modified kisspeptin-10 peptide ligand was crosslinked to its membrane receptor GPR54 due the presence of spontaneously generated ROS species (originating from NOX enzymes on the cell membrane), as proven by western blotting.¹ To further explore scope and limitations of the furan-based crosslinking methodology, an elaborate investigation on the covalent trapping of protein-protein interactions in a more therapeutically relevant model system, was performed. Furan-equipped nanobodies were recombinantly produced upon genetic code expansion in E. Coli ⁴ and further tested for their crosslinking behaviour towards target receptors that recently gained more attention as potential cancer biomarkers.

Figure 1. Furan-oxidation mediated crosslinking of a ligand to its target receptor.