A Fluorescent-Labeled Phosphono Bisbenzguanidine As an Activity-Based Probe for Matriptase

Area of Science:

• Biochemistry
• Chemical Biology
• Enzymology

Background:

• Activity-based probes (ABPs) are crucial tools for profiling enzyme activity in biological systems.
• Matriptase, a type II transmembrane serine protease, plays significant roles in various physiological and pathological processes.
• Existing methods for matriptase activity assessment have limitations.

Purpose of the Study:

• To design and synthesize a novel activity-based probe for selective and irreversible inhibition of matriptase.
• To characterize the probe's mechanism of action and its utility in enzyme activity profiling.
• To establish a new fluorescence-based detection method for protease activity using HPLC.

Main Methods:

• Design and multi-step synthesis of a coumarin-labeled bis-benzguanidine derivative incorporating a phosphonate warhead.
• Evaluation of the probe's irreversible inhibition of matriptase.
• Detection of enzyme activity using in-gel fluorescence and fluorescence High-Performance Liquid Chromatography (HPLC).

Main Results:

• Successful synthesis of the coumarin-labeled bis-benzguanidine probe.
• Demonstration of irreversible matriptase inhibition and simultaneous fluorescence labeling.
• Establishment of fluorescence HPLC as a novel and effective method for activity-based protease profiling.

Conclusions:

• The developed activity-based probe is effective for irreversible inhibition and fluorescence labeling of matriptase.
• Fluorescence HPLC represents a significant advancement for detecting protease activity with ABPs.
• This probe and method offer new avenues for studying matriptase function and related biological processes.