EGF receptor-targeting peptide conjugate incorporating a near-IR fluorescent dye and a novel 1,4,7-triazacyclononane-based (64)Cu(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 (64)Cu(II)-binding kinetics (>95% radiochemical yield in <5 min) and a high resistance to demetalation. It may thus prove suitable for use in (64)Cu(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 "D4"), followed by a Cys-β-Ala-β-Ala spacer, then a β-homopropargylglycine residue with the TACN-based chelator "clicked" 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 (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 noncompetitive antagonist.
Institute of Radiopharmaceutical Cancer Research and ‡Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf eV , P.O. Box 510119, D-01314 Dresden, Germany.
Katrin
K
Viehweger
Lisa
L
Barbaro
Karina Pombo
KP
García
Tanmaya
T
Joshi
Gerhard
G
Geipel
Jörg
J
Steinbach
Holger
H
Stephan
Leone
L
Spiccia
Bim
B
Graham
D002614:Chelating Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D054333:Drug Partial Agonism; D066246:ErbB Receptors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry*; D019265:Spectroscopy, Near-Infrared; D013329:Structure-Activity Relationship
D002614:Chelating Agents; D005456:Fluorescent Dyes; D006571:Heterocyclic Compounds; D009942:Organometallic Compounds; D010455:Peptides; C075712:1,4,7-triazacyclononane; D003300:Copper; D066246:ErbB Receptors