Development of a fluorogenic ADAMTS-7 substrate.

Area of Science:

• Biochemistry and Molecular Biology
• Cardiovascular Research
• Drug Discovery

Background:

• ADAMTS-7, an extracellular protease, is a potential therapeutic target for atherosclerosis and coronary artery disease (CAD).
• Development of ADAMTS-7 inhibitors has been limited by the absence of suitable peptide substrates and activity assays.
• A convenient assay is crucial for high-throughput screening (HTS) of potential ADAMTS-7 inhibitors.

Purpose of the Study:

• To develop the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7.
• To utilize the novel substrate for measuring inhibition constants of known inhibitors.
• To enable high-throughput screening for novel ADAMTS-7 inhibitors to treat cardiovascular diseases.

Main Methods:

• Development and characterization of a novel FRET peptide substrate (ATS7FP7) for ADAMTS-7.
• Measurement of inhibition constants (Ki) for TIMP-4 and hydroxamate-based inhibitors using ATS7FP7.
• Comparison of FRET assay results with IC50 values from an established SDS-PAGE assay using LTBP4S-A substrate.

Main Results:

• The novel FRET substrate, ATS7FP7, was successfully developed for ADAMTS-7 activity.
• Inhibition constants measured with ATS7FP7 correlated well with IC50 values from the SDS-PAGE assay.
• ATS7FP7 demonstrated suitability for measuring inhibition of ADAMTS-7 by endogenous and synthetic inhibitors.

Conclusions:

• The fluorogenic FRET substrate ATS7FP7 is a valuable tool for quantifying ADAMTS-7 activity and inhibition.
• ATS7FP7 facilitates high-throughput screening, paving the way for discovering novel ADAMTS-7 inhibitors.
• This advancement is expected to accelerate translational research for new cardiovascular disease treatments targeting ADAMTS-7.