Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Genomics and proteomics offer indirect protein function insights, as transcription/translation levels don't always correlate with enzymatic activity.
• A direct, genome-wide method to assess enzyme activities is highly desirable for functional studies.

Purpose of the Study:

• To develop a direct method for broadly determining cysteine protease activities on a genome-wide scale.
• To create chemical probes for tracking cysteine protease activity and profiling them during cancer progression.

Main Methods:

• Engineered chemical probes based on the E-64 inhibitor scaffold, incorporating affinity tags (biotin, radio-iodine).
• Synthesized probe analogs (DCG-03, DCG-04) with varying peptide recognition portions to target cysteine proteases.
• Utilized a library of probe derivatives to profile protease activity in crude cellular extracts and identify targets via mass spectrometry.

Main Results:

• Developed probes (DCG-03, DCG-04) that effectively target a broad range of cysteine proteases.
• Successfully profiled cysteine protease activities during the progression of normal skin cells to carcinoma.
• Demonstrated the ability to purify and identify modified proteases using the probes' affinity tags.

Conclusions:

• Created a simple, flexible method for functional identification and activity tracking of cysteine proteases in crude mixtures.
• Applied the probes to determine relative protease activities in a cancer progression model system.
• Showcased the utility of affinity probe libraries for rapid functional profiling without prior target purification.