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Development of a fluorogenic ADAMTS-7 substrate

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journal contribution
posted on 13.01.2022, 17:02 by Salvatore Santamaria, Frederic Buemi, Elisa Nuti, Doretta Cuffaro, Elena De Vita, Tiziano Tuccinardi, Armando Rossello, Steven Howell, Shahid Mehmood, Ambrosius P. Snijders, Rens de Groot

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.

Funding

This work was supported by a grant from the British Heart Foundation awarded to Rens de Groot [PG/18/19/33584] and by funding from the University of Pisa (Fondi di Ateneo 2020) to Armando Rossello and Elisa Nuti. Salvatore Santamaria is the recipient of an Intermediate Basic Science Research Fellowship from the British Heart Foundation [FS/IBSRF/20/25032].

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