Abstract
The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-β–binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.
Highlights
The extracellular protease A Disintegrin-like And Metalloproteinase with Thrombospondin Motif (ADAMTS)-7 has been strongly implicated in the aetiology of atherosclerosis and coronary artery disease (CAD)
With the aim to identify a suitable ADAMTS-7 Fluorescence resonance energy transfer (FRET) substrate, we tested initially if FRET substrates designed for ADAMTS-4 (5,6 fluorescein [FAM]-AE#LQGRPISIAK-carboxytetramethylrhodamine [Tamra])[18,23] and ADAMTS-5 (FAM-TESE#SRGAIYKK-TAMRA)[24] could be cleaved by ADAMTS-7 at low nM enzyme concentrations, which was not the case
As ADAMTS-7 is prone to autolysis[8], we had designed a FRET substrate based on autolytic cleavages in ADAMTS-7 (TAMRA-IRIQE $ VAE $ AANKFAM), which was not cleaved at low nM enzyme concentrations
Summary
The extracellular protease A Disintegrin-like And Metalloproteinase with Thrombospondin Motif (ADAMTS)-7 has been strongly implicated in the aetiology of atherosclerosis and coronary artery disease (CAD). A genomewide association study (GWAS) has demonstrated an inverse association between atherosclerosis and a non-synonymous single-nucleotide polymorphism (SNP), leading to a Ser-to Pro substitution in the prodomain of ADAMTS-71. This aminoacid substitution interferes with ADAMTS-7 secretion and activation by proprotein convertases such as furin, suggesting that decreased ADAMTS-7 activity may be beneficial for the treatment of atherosclerosis[1,2]. ADAMTS-7 catalytic activity is required for optimal vascular smooth muscle cell migration[2]. These findings suggest that pharmacological inhibition of ADAMTS-7 activity could slow down the progression of atherosclerosis and associated CAD. FRET substrates designed for ADAMTS-4 and ADAMTS-5 were not efficiently cleaved by ADAMTS-7 so the design of a new FRET substrate was required
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