EGF receptor-targeting peptide conjugate incorporating a near-IR fluorescent dye and a novel 1,4,7-triazacyclononane-based 64Cu(II) chelator assembled via click chemistry

A new Boc-protected 1,4,7-triazacyclononane (TACN)-based pro-chelator compound featuring a “clickable” azidomethylpyridine pendant has been developed as a building block for the construction of multimodal imaging agents. Conjugation to a model alkyne (propargyl alcohol), followed by deprotection, generates a pentadentate ligand, as confirmed by X-ray crystallographic analysis of the corresponding distorted square-pyramidal Cu(II) complex. The ligand exhibits rapid 64Cu2+-binding kinetics (> 95% radiochemical yield in < 1 min) and a high resistance to demetallation. It may thus prove suitable for use in 64Cu-based in vivo positron emission tomography (PET). The new chelating building block has been applied to the construction of a bimodal (PET/fluorescence) peptide-based imaging probe targeting the epidermal growth factor (EGF) receptor, which is highly over-expressed on the surface of several types of cancer cells. The probe consists of a hexapeptide sequence, Leu-Ala-Arg-Leu-Leu-Thr (designated “D4”), coupled to a β-homopropargylglycine residue with the TACN-based chelator “clicked” to its side chain, followed by a Cys-β-Ala-β-Ala spacer. A sulfonated near-infrared (NIR) fluorescent cyanine dye (sulfo-Cy5) was introduced at the N-terminus to study the EGF receptor-binding ability of the probe by laser-fluorescence spectroscopy. Binding was also confirmed by co-immunoprecipitation methods, and an apparent dissociation constant (Kd) of ca. 10 nM was determined from radioactivity-based measurements of probe binding to two EGF receptor-expressing cell lines (FaDu and A431). The probe is shown to be a biased or partial allosteric agonist of the EGF receptor, inducing phosphorylation of Thr669 and Tyr992, but not the Tyr845, Tyr998, Tyr1045, Tyr1068 or Tyr1148 residues of the receptor, in the absence of the orthosteric EGF ligand. Additionally, the probe was found to suppress the EGF-stimulated autophosphorylation of these latter residues, indicating that it is also a non-competitive antagonist.