Inhibition of Cathepsin K with Lysosomotropic Macromolecular Inhibitors † Dong Wang, ‡ Michal Pechar, ‡,§,| Weijie Li, §,⊥ Pavla Kopec ˇkova ´, ‡ Dieter Bro ¨mme, ⊥ and Jindrˇich Kopec ˇek* ,‡,# Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD and Department of Bioengineering, UniVersity of Utah, Salt Lake City, Utah 84112, and Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029 ReceiVed February 20, 2002; ReVised Manuscript ReceiVed May 6, 2002 ABSTRACT: Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonyl- leucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohy- drazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water- soluble polymers {R-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2- hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I}. The conjugation of inhibitor I to N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K(k obs /[I] ) 1.3 × 10 6 M -1 s -1 ) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 × 10 5 M -1 s -1 ). The k obs /[I] values for I attached to the side chains of HPMA copolymers (PHPMA- GG-I and PHPMA-I) were about 3 × 10 4 M -1 s -1 . When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k obs /[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization. Cathepsin K, a 24 kDa cysteine protease of the papain superfamily, was first discovered in a rabbit osteoclast cDNA library and named OC-2 (1). The human equivalent of the protein was subsequently cloned by several groups and named cathepsin O (2), K (3), X (4), and O2 (5). By NCIUB recommendation, the protease is now designated cathepsin K. It shares the highest homology in DNA and amino acid sequence to cathepsins L and S (5). In osteoclasts, cathepsin K is expressed at high level, while cathepsins B, L, and S are expressed at relatively low or undetectable levels (1, 5-7). Deficiency of cathepsin K activity in osteoclasts induces pycnodysostosis, a rare inherited disorder with an osteopetrotic phenotype (8, 9). Specific inhibition of cathepsin K expression with antisense olignucleotides has been shown to produce a 40-50% reduction in bone resorption (10). Cathepsin K is the only known mammalian cysteine protease capable of cleaving native type I collagen in its triple helix region (11, 12). These findings clearly demonstrate the unique physiological role of this enzyme in organic matrix degradation during the bone resorption process. Recently, cathepsin K has also been shown to be able to cleave native type II collagen (11, 13). The expression of cathepsin K in rheumatoid arthritis-derived synovial fibroblasts was observed and correlated with the severity of the disease (14). Such reports suggested the involvement of cathepsin K in cartilage breakdown in rheumatoid arthritis patients. As a potential therapeutic target for the treatment of osteoporosis and rheumatoid arthritis, successful inhibition of cathepsin K may contribute to an understanding and final cure of these diseases. Natural inhibitors of cysteine proteases (including cathepsin K), such as cystatins (15) and pro- domains of the zymogens (16, 17), have been well studied and understood. Many low-molecular-weight cathepsin K † This research was supported in part by NIH Grant EB00251 (to D.W., M.P., P.K., and J.K.) and by NIH Grant AR46182 and the Biomedical Science Grant from the Arthritis Foundation (to W.L. and D.B.). * Corresponding author. Address: University of Utah, Department of Pharmaceutics and Pharmaceutical Chemistry, 30 S 2000 E Rm. 301, Salt Lake City UT 84112. Phone: + 801-581-4532. Fax: + 801- 581-3674. E-mail: Jindrich.Kopecek@m.cc.utah.edu. ‡ Department of Pharmaceutics and Pharmaceutical Chemistry/ CCCD, University of Utah. § These authors contributed equally to this work. | Permanent address: Institute of Macromolecular Chemistry, Acad- emy of Sciences of the Czech Republic, Prague. ⊥ Department of Human Genetics, Mount Sinai School of Medicine. # Department of Bioengineering, University of Utah. 8849 Biochemistry 2002, 41, 8849-8859 10.1021/bi0257080 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/20/2002