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

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• 作者：Katrin Viehweger ; Lisa Barbaro ; Karina Pombo Garc铆a ; Tanmaya Joshi ; Gerhard Geipel ; J枚rg Steinbach ; Holger Stephan ; Leone Spiccia ; Bim Graham
• 刊名：Bioconjugate Chemistry
• 出版年：2014
• 出版时间：May 21, 2014
• 年：2014
• 卷：25
• 期：5
• 页码：1011-1022
• 全文大小：432K
• 年卷期：v.25,no.5(May 21, 2014)
• ISSN：1520-4812

文摘

A new Boc-protected 1,4,7-triazacyclononane (TACN)-based pro-chelator compound featuring a 鈥渃lickable鈥?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 ⁶⁴Cu(II)-binding kinetics (>95% radiochemical yield in <5 min) and a high resistance to demetalation. It may thus prove suitable for use in ⁶⁴Cu(II)-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 overexpressed on the surface of several types of cancer cells. The probe consists of a hexapeptide sequence, Leu-Ala-Arg-Leu-Leu-Thr (designated 鈥淒4鈥?, followed by a Cys-尾-Ala-尾-Ala spacer, then a 尾-homopropargylglycine residue with the TACN-based chelator 鈥渃licked鈥?to its side chain. 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 coimmunoprecipitation methods, and an apparent dissociation constant (K_d) 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 noncompetitive antagonist.