Synthesis and Application of Activity-Based Probes for Serine Proteases

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Activity-based probes (ABPs) are essential chemical tools that covalently bind to the active sites of enzymes.
• They are widely used in target identification, biomarker discovery, and the development of novel therapeutics.
• Serine proteases represent a critical enzyme class with significant implications in various physiological and pathological processes.

Purpose of the Study:

• To design and synthesize a novel library of activity-based probes (ABPs) specifically targeting serine proteases.
• To incorporate a bioorthogonal tag for versatile labeling applications and diphenyl phosphonate warheads to enhance selectivity and reactivity.
• To establish a robust protocol for synthesizing substituted warheads to improve probe affinity.

Main Methods:

• Synthesis of a diverse library of diphenyl phosphonate-based activity-based probes (ABPs).
• Incorporation of a bioorthogonal tag for subsequent detection and analysis.
• Biochemical validation including determination of inhibitory potency (IC50) and kinetic parameters (k_inact/K_i).
• Application of the developed ABPs for labeling serine proteases in biochemical assays and detection via fluorescence scanning.

Main Results:

• Successful synthesis of a tailored ABP library targeting serine proteases.
• Demonstrated enhancement in probe selectivity and reactivity through optimized diphenyl phosphonate warheads.
• Biochemical assays confirmed the inhibitory potencies and kinetic profiles of the synthesized probes.
• Effective labeling of target serine proteases using the developed ABPs, visualized through fluorescence detection.

Conclusions:

• The developed activity-based probes (ABPs) offer a valuable tool for studying serine protease activity.
• The optimized probe design enhances selectivity and affinity, facilitating precise enzyme labeling.
• These probes hold significant potential for advancing serine protease research and accelerating drug discovery efforts.