Fluorogenic Labeling Probe for the Imaging of Endogenous β-Galactosidase Activity in Cancer and Senescent Cells

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• Glycosidases play vital roles in cellular processes and disease.
• Sensitive detection of glycosidase activity in live cells is essential for biological research.
• Existing methods for detecting beta-galactosidase (β-Gal) activity have limitations.

Purpose of the Study:

• To develop a novel fluorogenic probe for sensitive and specific detection of β-Gal activity in live cells.
• To utilize the probe for imaging endogenous β-Gal activity in cancer and senescent cells.
• To investigate the covalent attachment of the probe to proteins after enzymatic cleavage.

Main Methods:

• Design and synthesis of a fluorescein-based fluorogenic probe incorporating a galactose moiety.
• Utilizing spirocyclization to achieve quenched fluorescence, which is restored upon β-Gal enzymatic activity.
• Validation of probe specificity against other glycosidases and hydrolases.
• Live-cell imaging of β-Gal activity in cancer and senescent cells.
• Confirmation of β-Gal expression levels using Western blotting and PCR.
• Demonstration of covalent protein labeling by the probe in vitro and in vivo.

Main Results:

• A novel fluorogenic probe was synthesized, exhibiting quenched fluorescence that is restored upon β-Gal action.
• The probe demonstrated an approximately 210-fold increase in fluorescence intensity post-cleavage.
• High specificity was observed, with no false-positive signals from other hydrolases.
• Successful imaging of endogenous β-Gal activity in cancer and senescent cells was achieved.
• Imaging results correlated well with β-Gal expression levels determined by molecular biology techniques.
• The probe was found to covalently label adjacent proteins after galactose hydrolysis.

Conclusions:

• A sensitive and specific small-molecule fluorogenic probe for β-Gal activity has been developed.
• The probe enables effective live-cell imaging of β-Gal, correlating with expression levels.
• The probe's ability to covalently label proteins offers a unique tool for biochemical studies.
• This probe represents a valuable advancement for monitoring glycosidase activity in biological systems and disease contexts.