Development of "Furan Warhead"-Equipped Antagonistic Nanobodies for Covalent Cross-Linking to the Epidermal Growth...

Covalent therapeutics have gained attention in drug design, as they have the potential to result in enhanced potency and prolonged duration of action. However, the safety concerns associated with off-target effects have long been a serious hurdle for covalent drug design. Proximity-enabled covalent bond formation combined with the use of caged warheads offers an attractive strategy in this context. We here report on the use of the proximity-dependent furan-oxidation-based cross-linking and a previously reported antagonistic nanobody (EgA1), to design new nanobodies that covalently trap the soluble fraction of the epidermal growth factor receptor (sEGFR). The furan-containing EgA1 nanobodies were validated for binding to serum(s)EGFR via biomolecular binding assays. Their singlet oxygen-induced cross-linking behavior toward the sEGFR protein was evaluated in vitro through photosensitizer irradiation. Initial experiments using an alternative biocompatible Rose Bengal derivative as photosensitizer further show proof of concept for the use of such furan-containing nanobodies for covalent trapping of the EGFR present on the plasma membrane of MDA-MB-231 breast cancer cells.