Fluorescence Probes for Imaging Basic Carboxypeptidase Activity in Living Cells with High Intracellular Retention

• Published in: Analytical chemistry (Washington) Vol. 93; no. 7; pp. 3470 - 3476
• Main Authors: Iwaki, Hirohisa, Kamiya, Mako, Kawatani, Minoru, Kojima, Ryosuke, Yamasoba, Tatsuya, Urano, Yasuteru
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.02.2021
• Subjects: Amino acids; Arginine; Benzene; Biotechnology; Breast cancer; Carboxypeptidase; Cells (biology); Chemistry; Fluorescein; Fluorescein diacetate; Fluorescence; Fluorescent indicators; Imaging; Intracellular; Lysine; Peptides; Probes; Retention; Substrates; Tumor cell lines
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.0c04793

Basic carboxypeptidases (basic CPs) cleave the C-terminal basic amino acid of peptides, and their activity is upregulated in some types of cancers. Therefore, detecting the activity of basic CPs in living cells would be important not only for studying the physiological functions of these enzymes but also for visualization of cancerous tissues. Here, we report two fluorescein diacetate (FDA)-based activatable fluorescence probes, named 5ArgAF-FDA and 5LysAF-FDA, in which the substrate amino acid arginine or lysine is conjugated to the benzene moiety via an azoformyl linker. In live-cell fluorescence imaging of CPM, one of the seven basic CPs, 5ArgAF-FDA showed a larger intracellular fluorescence increase than did 5LysAF-FDA within a few minutes. This increase was inhibited by coincubation with 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), an inhibitor of basic CPs. When 5ArgAF-FDA was applied to a coculture of two breast cancer cell lines with different CPM activities, the fluorescence increase in individual cells was correlated with the expression level of CPM, suggesting that 5ArgAF-FDA has the ability to distinguish cell lines having different levels of CPM activity, owing to its high intracellular retention. We believe these probes will be useful for imaging cancers with upregulated basic CP activity.