Development and Application of Small Molecule-Peptide Conjugates as Cathepsin K-Specific Covalent Irreversible Inhibitors in Human Osteoclast and Lung Cancer.

Cathepsin K (CTSK), a proteolytic enzyme that degrades the extracellular matrix, is recognized as a significant therapeutic target for osteoporosis, osteoarthritis, and rheumatoid arthritis. Due to adverse effects, no clinically approved drugs exist for CTSK. In order to develop safer therapeutics, highly selective CTSK inhibitors are required to elucidate the origins of side effects. Here, we developed various hybrid inhibitors by combining peptide sequences with small organic molecules. An acyloxymethyl ketone electrophile was incorporated as a bioisostere of the glycine-glycine cleavage site and inverse peptide sequences to enhance prime site interactions, as seen in the crystal structure. Additionally, a diphenyl group was incorporated to improve nonprime site interactions, culminating in highly selective and potent irreversible CTSK inhibitors with negligible off-target binding by closely related cathepsins. These novel inhibitors were also designed to attach to targeting moieties, further reducing off-target effects in vivo. Our findings demonstrate that these highly selective inhibitors are nontoxic, effectively inhibit bone resorption by human osteoclasts, block CTSK activity in cells and their nuclei, and inhibit activity in human lung cancer tissue. This study highlights significant advancements in designing CTSK inhibitors with potential clinical applications for lung cancer and osteoclast-related conditions.