Cathepsin K, the main bone degrading protease, and chondroitin 4-sulfate (C4-S) form a complex with enhanced collagenase activity. In this report, we demonstrate the specific inhibition of the collagenase activity of cathepsin K by negatively charged polymers without affecting the overall proteolytic activity of the protease. Three different mechanisms to interfere with cathepsin-catalyzed collagen degradation are discussed: 1) inhibition of the formation of the cathepsin K/C4-S complex, 2) inhibition of the attachment of C4-S to collagen, and 3) masking of the collagenase cleavage sites in collagen. By targeting these interaction sites, collagen degradation can be modulated while the non-collagenolytic activities of cathepsin K remain intact. The main inhibitory effect on collagen degradation is due to the impeding effect on the active cathepsin K/C4-S complex. Essential structural elements in the inhibitor molecules are negative charges which compete with the sulfate groups of C4-S in the cathepsin K/C4-S complex. The inhibitory effect can be controlled by length and charge of the polymers. Longer negatively charged polymers (e.g. polyglutamates, oligonucleotides) tend to inhibit all three mechanisms, whereas shorter ones preferentially affect the cathepsin K/C4-S complex.An imbalance between bone formation and resorption can cause various bone diseases such as osteoporosis, certain forms of arthritis, and Paget disease. Type I collagen represents an essential part (90%) of the organic bone mass (1). It has been shown that cathepsin K, a cysteine protease predominantly expressed in osteoclasts (2-4), is an efficient collagenase that cleaves type I collagen at multiple sites in its helical domain (5, 6). Based on the physiological role of cathepsin K in bone resorption, cathepsin K inhibitors are being developed for the treatment of osteoporosis (7). Those inhibitors may also serve as drugs for rheumatoid arthritis (8). Presently, conventional cathepsin K inhibitors target the active site, thus causing a complete inhibition of enzymatic activity. An active site inhibition is associated with the loss of other physiological functions of cathepsin K activity which may result in undesirable side effects. Ablation of the matrix-degrading function in tandem with preservation of its non-collagenolytic protease activity would be greatly advantageous and will be addressed in this report.Collagen consists of three intertwining ␣-chains of ϳ1000 residues each (9). The three-dimensional structures of triplehelical collagen and cathepsin K suggest that the active cleft of cathepsin K does not provide sufficient space to accommodate intact triple-helical collagen. The entrance to the catalytic site of cathepsin K is only 5 Å wide (10), while triple-helical collagen has a diameter of 15 Å. This leads to the assumption that prior to hydrolysis by cathepsin K, the triple-helical collagen has to be unwound to expose its single chains. It was recently demonstrated that bone-and cartilage-resident glycosaminoglycans such as chondr...