Identification of Substrate-Specific Inhibitors of Cathepsin K Through High-Throughput Screening

Research Article | Biochemical Journal
Identification of Substrate-Specific Inhibitors of Cathepsin K Through High-Throughput Screening
Author's Information

Simon Law

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Xin Du

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Preety Panwar

Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Nicolette S. Honson

Centre for Drug Research and Development, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Tom Pfeifer

Centre for Drug Research and Development, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Michel Roberge

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Dieter Brömme

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Abstract

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Keywords

cathepsin K, collagen, exosite inhibitor, high-throughput screening, osteoporosis, protease inhibitor

DOI: 10.1042/BCJ20180851

ADLID: 95499-v4

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Keywords
cathepsin K collagen exosite inhibitor high-throughput screening osteoporosis protease inhibitor

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