10 min Rapid Fire International Peptide Symposium 2023

Covalent ligands on the rise: Development of ‘furan warhead’- equipped nanobodies (#80)

Laure Tack 1 , Laia Miret Casals 1 , Olivier Zwaenepoel 2 , Marleen Van Troys 2 , Jan Gettemans 2 , Annemieke Madder 1
  1. Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium
  2. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

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.

64899b631060c-Furan-oxidation+mediated+crosslinking+of+a+ligand+to+target+receptor.png

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

  1. W. Vannecke, C. Ampe, M. Van Troys, M. Beltramo, and A. Madder, Cross-Linking Furan-Modified Kisspeptin-10 to the KISS Receptor. ACS Chem Biol 2017,12, 2191-2200.
  2. L. Miret-Casals, W Vannecke et al. Furan warheads for covalent trapping of weak protein–protein interactions: cross-linking of thymosin β4 to actin. Chem. Comm. 2021, 57, 6054-6057.
  3. L. Miret Casals, S. Van De Putte, D. Aerssens et al. Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners. Frontiers in chemistry 2022, vol. 9.
  4. M. J. Schmidt, A. Weber, M. Pott, W. Welte, D. Summerer, Structural basis of furan-amino acid recognition by a polyspecific aminoacyl-tRNA-synthetase and its genetic encoding in human cells. Chembiochem. 2014, 15 , 12, 1755-60.