Genetically Engineered Mouse Models Reveal the Importance of Proteases as Osteoarthritis Drug Targets

Abstract

More than two decades of research has revealed a combination of proteases that determine cartilage degradation in osteoarthritis. These include metalloproteinases, which degrade the major macromolecules in cartilage, aggrecan and type II collagen, serine proteases, and cysteine proteases, for example cathepsin K. This review summarizes the function of proteases in osteoarthritis progression, as revealed by studies of genetically engineered mouse models. A brief overview of the biochemical characteristics and features of several important proteases is provided, with the objective of increasing understanding of their function. Published data reveal at least three enzymes to be major targets for osteoarthritis drug development: ADAMTS-5, MMP-13, and cathepsin K. In surgical models of osteoarthritis, mice lacking these enzymes are protected from cartilage damage and, to varying degrees, from bone changes. In-vivo studies targeting these proteases with selective small-molecule inhibitors have been performed for a variety of animal models. Mouse models will provide opportunities for future tests of the therapeutic effect of protease inhibitors, both on progression of structural damage to the joint and on associated pain.

Figure 1. Network of proteases in OA cartilage degradation

Key proteases from different classes are shown, mainly based on ex vivo human and bovine cartilage explant studies. ADAMTS-4/5 and MMP-13 are the main metalloproteases responsible for degrading aggrecan and type II collagen, respectively [79, 80]. These enzymes can also act on other putative substrates, the degradation of which may contribute to the weakening of the cartilage matrix [81]. These metalloproteases are synthesized as catalytically inactive zymogens, and it has been shown that the proforms of ADAMTS-4/5 are activated by proprotein convertases (PC) [–84], with PACE4 identified as the major PC in human OA cartilage [78]. Tissue inhibitor of metalloprotease (TIMP)-3 is an endogenous inhibitor of MMP-13 [85] and ADAMTS-4/5 [86]. The cysteine protease, cathepsin K (catK), can also degrade collagen and aggrecan [87, 88]. ADAM8 can cleave fibronectin [89], as can HtrA1 (high temperature requirement A1) [90], and fibronectin fragments further induce the release of catabolic enzymes by chondrocytes [–93]. HtrA1 can cleave many substrates in vitro, including aggrecan, fibromodulin, and decorin [94, 95]. To date, a role for aspartyl proteases has not been reported, although it was recently shown that selective inhibition of the membrane-anchored aspartyl protease, BACE-1 (beta-secretase), primarily known for its role in Alzheimer’s disease through cleavage of amyloid precursor protein, blocked cytokine-induced aggrecan loss from bovine and human cartilage explants. The mechanism of action is not yet understood [96].

Figure 2. Domain structures of ADAMTS-4, ADAMTS-5, ADAM-8, MMP-13, cathepsin K and PACE4

Pro, DG, Cys, EGF, and GBD indicate the propeptide, blood coagulation inhibitor/disintegrin, ADAM/cysteine-rich, epidermal growth factor-like domain and galactose-binding domain-like, respectively. The histidine-sites chelating zinc ion observed for ADAMTS-4, ADAMTS-5, ADAM-8, MMP-13 and the catalytic triad residues for cathepsin K and PACE4 are marked in blue. The sequence features were retrieved from the InterPro database of EMBL-EBI, and the accessions used included P29122 (PACE-4), P43235 (Cathepsin K), P78325 (ADAM-8), P45452 (MMP-13), Q9UNA0 (ADAMTS-5) and O75173 (ADAMTS-4). This graph was prepared using DOG [97].

Figure 3. Schematic diagrams of the catalytic mechanism for serine-, cysteine- and metallo-proteinases

The numbering of the residues involved in a catalytic reaction is in accord with PACE4 (serine proteinase, A and D), cathepsin K (cysteine proteinase, B and E) and ADAMTS-5 (metalloproteinase, C and F), respectively.

Figure 4. 3D crystal structures of the catalytic domains of ADAMTS-4, ADAMTS-5, ADAM-8, MMP-13, Cathepsin K and PACE-4

Proteins are displayed as ribbons, ligands and zinc ions are represented as sticks and gray spheres, respectively. The model of PACE4 is built using SWISS-MODEL [98]. PACE4A-I was chosen as the target sequence and the crystal structure of furin (PDB code: 1P8J) [99] was used to model PACE4. A-F display the structures of ADAM-8, ADAMTS-5, ADAMTS-4, MMP-13, cathepsin K and PACE4A-I, respectively. The catalytic triad residues of PACE4A-I are shown as sticks. The PDB codes used were 4DD8 (ADAM-8) [100], 3C9E (Cathepsin K) [101], 3ELM (MMP-13) [12], 2RJP (ADAMTS-4) [102], 3B8Z (ADAMTS-5) [103]. The graphs were prepared using PyMOL [104].