Fluorescent substrates useful as high-throughput screening tools for ADAM9.

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Drug Discovery

Background:

• ADAM9 is a metalloproteinase implicated in various biological processes.
• Development of specific substrates is crucial for studying ADAM9 activity and for drug screening.
• Fluorescence resonance energy transfer (FRET) assays offer sensitive detection of protease activity.

Purpose of the Study:

• To design and validate novel FRET-based substrates for ADAM9.
• To utilize these substrates for characterizing ADAM9 enzymatic activity.
• To employ the most efficient substrate in screening for ADAM9 inhibitors.

Main Methods:

• Synthesis of FRET peptides incorporating donor (5-carboxy fluorescein) and quencher (Dabcyl) pairs.
• Peptide design based on known ADAM9 cleavage sites (TNF-alpha, TGF-alpha, CD23, betacellulin).
• Enzymatic assays to determine substrate processing efficiency and IC50 values of inhibitors.

Main Results:

• Several FRET substrates based on TNF-alpha and TGF-alpha precursors were efficiently processed by ADAM9.
• A substrate based on the betacellulin cleavage site showed higher selectivity for ADAM9.
• The TNF-alpha-based substrate was optimal for inhibitor screening, identifying TMI-1 (IC50 = 2.1 nM) and GI254023 (IC50 = 280 nM) as inhibitors.

Conclusions:

• Novel FRET substrates are effective tools for studying ADAM9 activity.
• These substrates facilitate high-throughput screening for potent ADAM9 inhibitors.
• The developed assay platform is valuable for drug discovery targeting ADAM9.