Laia Miret Casals
Talk Session: SESSION 8: PEPTIDE- AND PROTEIN-BASED THERAPEUTICS
Date: Tuesday, June 14, 2022
Talk Time: 09:25 am - 09:40 am
Talk Title: Furan-Oxidation Mediated Cross-Link Technology: From in vitro Analysis of Protein-Protein Interactions to Covalent GPCR-Ligand Interactions on Live Cells
Our research group developed a new furan-oxidation mediated technology, that offers a site-selective and efficient covalent link only when biological binding partners are in close proximity. Furan moiety is incorporated in peptides as a 2-furyl-L-alanine and can be oxidized to a reactive aldehyde by using singlet oxygen.
This singlet oxygen can be produced in vitro by irradiation with a white light source in the presence of a photosensitizer, such as Rose Bengal or Rhodamine B, or in situ by ROS on life cells. A site-selective crosslink reaction occurs between the furan moiety and sulfhydryl and/or amine groups present in the peptide or protein if sufficiently proximate. We are using this Furan Crosslink Technology to investigate peptide-protein and protein-protein interactions on live cells.
Our research group has developed a novel furan cross-link technology for oligonucleotides, further applicable to the investigation of peptide-protein interactions. As the furan moiety is isosteric with histidine and isoelectronic with tyrosine, the incorporation in peptides is well tolerated.
Here, we describe the application of this fast and highly efficient furan-oxidation mediated technology to protein- protein, coiled-coil peptide dimer, and peptide-protein3 interactions.
Initially, we studied the weak and dynamic protein-protein interaction between actin, the major cytoskeletal protein of the cell that forms filaments, and Tß4 that regulates the polymerization of actin and keeps actin in the monomeric form. Furan-armed-Tß4 analogues were shown to efficiently cross-link to monomeric actin upon singlet oxygen generation. The cross-link in the Tß4-actin covalent complex was to involve Tß4-Fua24 and Actin-Lys61 and a plausible chemical structure of the covalent linkage between Lys and the activated Fua was proposed.
A coiled-coil peptide dimer was used as a model system to explore furan reactivity, we described for the first-time reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link technology by the possibility to target different amino acids.
The furan-oxidation mediated cross-link technology was further optimized to enable cross-linking of furan-modified peptide ligands to GPCR proteins on live cells relying on the spontaneous endogenous oxidation of the furan moiety. We studied the neuropeptide kisspeptin-10 and its G-protein coupled receptor GPR54, which play a role in breast cancer and in the regulation of mammalian reproduction. We described selective cross-linking of a furan-modified kisspeptin-10 analogue to its membrane receptor GPR54 in live cells, with no toxicity and high efficiency