Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Cathepsin K (CatK) is a cysteine protease implicated in skeletal disorders due to its collagenase and elastase activity.
• Oligomeric complex formation of CatK with glycosaminoglycans enhances its collagenase activity.
• Inhibitors disrupting these complexes offer selective collagenase inhibition.

Purpose of the Study:

• To develop a fluorescence polarization (FP) assay for screening substrate-specific ectosteric CatK collagenase inhibitors.
• To identify novel compounds that selectively inhibit CatK's collagenase activity.

Main Methods:

• Screening of 4761 compounds using a developed fluorescence polarization (FP) assay.
• Characterization of inhibitor selectivity against collagenase and active site substrate hydrolysis.
• Evaluation of inhibitor efficacy in preventing osteoclast bone resorption.

Main Results:

• Identified 38 compounds inhibiting CatK collagenase activity without affecting active site substrate hydrolysis.
• Classified inhibitors into negatively charged and polyaromatic groups, suggesting distinct ectosteric binding sites.
• Demonstrated that two inhibitors effectively prevented CatK-mediated bone resorption in osteoclasts.
• Observed that some ectosteric inhibitors could differentiate between CatK's collagenase and elastase activities.

Conclusions:

• Ectosteric inhibitors provide a selective approach to targeting CatK's collagenase activity.
• These inhibitors represent a promising alternative to active site-directed drugs, potentially reducing side effects.
• The identified compounds offer a foundation for developing novel therapeutics for skeletal disorders.