Highly Selective Aza-nitrile Inhibitors For Cathepsin K: Synthesis And The Effect Detection

Osteoarthritis(OA) is an inflammatory disease which is caused by the interaction of mechanical injury and biological factors. As a degenerative joint disorder, OA is characterized by destruction of the articular cartilage, subchondral bone alterations and synovitis, and seriously affects the daily lives of their patients. This joint disease will get attention increasingly in the future coming years.Emerging evidence has suggested that during OA there is anincreased catabolic activity related to enhanced expression of proteinases associated with loss of proteoglycans and with a degradation of type II collagen. Cathepsin K(CatK) belongs to the family of papain-like cysteine peptidases, and it is unique due to its ability to cleave the triple helix of collagen molecules at multiple locations. So CatK is an important target enzyme for the treatment of osteoporosis and has been the focus of considerable attention because of its significant importance in bone biology.Based on the advantages ofnitrile inhibitors, our team has been researching the design, synthesis and mechanism of the inhibitors towards Cat K for many years. In this thesis a series of higly selective aza-peptide nitrile inhibitors have been synthesized after structure optimization. Then the inhibition and selectivity of Cat K inhibitors can be detected through enzyme activity assay; The structure-activity relationship and molecular mechanism can also be explained by covalent docking; Afterwards the cell viability assay was done to test the toxicity of inhibitors to cells, gelatin zymgraphyand quantitative fluorescence measurement were used to detect the activity of cathepsin K at cell level. The main contents are as follows.1. Based on the previous aza-dipeptide nitriles inhibitors towards Cat K, we designed a novel backbone structure, which lacked the P2-P3 amide linker to optimize its orientation and distance with the S3 pocket and had different P3 groups to optimize the selection over other cathepsins. Phenyl derivatives with an extension at either the para- or meta-position of the phenyl ring were prepared. After purification and characterization by 1H-NMR, 13C-NMR and HPLC-MS, the compounds were tested in the active assays of human cathepsins K, L, S and B. They all exhibited sub- to nanomolar Ki values and displayed obvious improvements in the selectivity profiles for cathepsin K over the other three enzymes, especially over cathepsin L. Both cathepsin K and L were endopeptidases,with high sequence homology(60% identity, 76% similarity) between them which made it very difficult to be inhibited selectively. It was particularly gratifying to note that the triaryl meta-derivative 13’ displayed a favorable balance between cathepsin K potency(Ki = 0.3 nM) and selectivity over cathepsin L(~320-fold), S(~1784-fold) and B(~8566-fold) among all the reported inhibitors.2. To provide insight into the structure-activity relationship and inhibition mechanism, we applied a docking program GOLD 5.1 for covalent docking. Take 6’ and 13’ for example, their P2, P3 group resided well in the pockets of cathepsin K, and the binding affinities between inhibitors and amino acid residues of Cat K were pleasing. On the contrary, it was very different when they were modeled in cathepsin L. P2, P3 groups extended into the solvent or distorted in the pocket, or interacted little with amino acid residues of Cat L. This further explained the experimental results of better selectivity profiles for inhibitors with respect to cathepsin K against L. But the GOLD 5.1 could not predict the selection by the values of Golscore Fitness; In view of this deficiency, improvements also be performed to achieve good theoretical simulation in matching the experimental results.3. To provide a proof of concept of the biological evaluation of CKI-E(14’) and CKI-F(15’) with those of the commercial inhibitor Odanacatib. In this part, the primary chondrocytes were obtained from the 5-6 day old mice(C57BL/6J strain), and cultured by different ways in order to express more cathepsin K. Then we tested their effects on cell viability towards 3 types of cells(Mouse smooth muscle cell line—MOVAS, Human osteosarcoma cell line—MG-63, and primary chondrocytes) using MTT test, the results showed that they did not induce significant toxicity to the cells even up to 1 ?M. In order to express more Cathepsin K, the primary chondrocytes were after passages or stimulated with AA and β-GP, finally the Cat K activity or the effect of inhibitors on Cat K activity had been examined by zymography and by quantitative fluorescent assay. The result showed that Cat K was expressed increasingly, and CKI-E, CKI-F had an obvious inhibition to Cat K which was expressed by the chondrocytes after passages, which proved them the good candidates for further drug development.In this thesis, we used kinds of technologies and methods of organic synthesis in chemistry, theoretical simulation in computer science, enzymology and cytology in biology. The systematic research has been done including structure optimization, organic synthesis, enzyme activity assay, structure-activity relationship, mechanism study, cell viability test and effect on Cat K activity at cell level. Happily, the good results were obtained which provided a theoretical basis for the design of more potent and selective Cat K inhibitors, and the clinical treatments of osteoarthritis.