A fluorescent photoprobe for the imaging of endothelin receptors

A novel fluorescent photoprobe for the imaging of endothelin A receptors (ETAR) was developed. Based on the nonpeptidyl, high-affinity, and selective ETAR antagonist 3-benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one (PD 156707), a modification of the lead structure with a PEG-spacer containing an amino moiety was performed. Labeling of this precursor with the fluorescent marker Cy 5.5 NHS-ester was accomplished by adaption of common peptide labeling procedures. The affinity of the Cy 5.5-labeled receptor antagonist was evaluated using human carcinoma cell lines with different degrees of ETAR expression. Fluorescence microscopy revealed that ETAR-positive MCF-7 human breast adenocarcinoma and HT-1080 human fibrosarcoma cells effectively bind the photoprobe at very low doses (nM), while ETAR-negative MDA-MB-435 human breast cancer cells showed no fluorescence signal. Binding specificity of the probe could be demonstrated by predosing with a specific ETAR antibody or the parent antagonist PD 156707 as a competing inhibitor. The results suggest that the modified photoprobe tightly binds to ETA receptors and thus may be a possible candidate for the imaging of ETAR-overexpressing tissues in vivo.