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ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.

ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation through removal of its prodomain before it can exert catalytic activity. We examined various proteinases as well as auto-activation under a wide range of conditions for removal of the prodomain and induction of enzymatic activity. The proprotein convertases, furin, PACE4, and PC5/6 efficiently removed the prodomain through cleavage at Arg(212)/Phe(213), generating an active enzyme. Of a broad range of proteases evaluated, only MMP-9 and trypsin were capable of removing the prodomain. In the presence of mercuric compounds, removal of the prodomain through autocatalysis was not observed, nor was it observed at temperatures from 22 to 65 degrees C, at ionic strengths from 0.1 to 1M, or at acidic/neutral pH. At basic pH 8-10, removal of the prodomain by autocatalysis occurred, generating an active enzyme. In conclusion, the pro-form of ADAMTS-4 is not catalytically active and only a limited number of mechanisms mediate its N-terminal activation.

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s≤150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.

The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be ~1 × 10[superscript −6] cm[superscript 2]/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery., National Institutes of Health (U.S.) (Grant AR45779), National Institutes of Health (U.S.) (Grant AR33236), Pfizer Inc.

A rapid HPLC assay was developed for monitoring the activity of the two proprotein convertases, PACE-4 and furin. Six novel peptide substrates were synthesized containing the minimal PC recognition sequence (Arg-X-X-Arg), as well as tryptophan residue(s) for easy detection. Four of the peptides were cleaved by both PCs and their kinetic parameters determined. Two peptides were not cleaved but were shown to be good negative controls although not inhibitors of either PC. In addition, inhibition curves were plotted and IC50 values calculated for PACE-4 and furin in the presence of two polyarginine peptides, hexa and deca-D-arginine.