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Aggrecanase 1

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Yasunori Okada – One of the best experts on this subject based on the ideXlab platform.

Elizabeth C. Arner – One of the best experts on this subject based on the ideXlab platform.

  • substrate dependent inhibition kinetics of an active site directed inhibitor of adamts 4 Aggrecanase 1
    Biochemistry, 2007
    Co-Authors: Arthur J Wittwer, Elizabeth C. Arner, Anne-marie Malfait, Robert L Hills, Grace E Munie, R H Keith, C P Anglin, Micky D. Tortorella
    Abstract:

    ADAMTS-4 (Aggrecanase1) is implicated in the breakdown of articular cartcartilage and is an attractive target for therapeutic intervention in arthritis. Cleavage of the native substrate, aggrecan, occurs through exosite interactions and peptide sequence recognition. Although expected to be competitive with aggrecan, the hydroxamic acid, SC81956, demonstrated noncompetitive inhibition kinetics with a K i of 23 nM. The IC 50 of SC81956 did not change when aggrecan was varied from 12.8 to 200 nM (0.2-3.3 times the apparent aggrecan K m of 61 nM) but was shifted as expected for a competitive inhiinhibitor when increasing levels of a low molemolecular weight peptide substrate were added to a fluorogenic peptide assay system. These observations are consistent with a model for aggrecan cleavage where substrate initially binds at an exosite, followed by binding of the appropriate peptide sequence at the active site. A peptidecompetitive inhiinhibitor could bind both free enzyme and initial substrate-enzyme exosite complex but would be excluded by the final Michaelis complex. Noncompetitive appearing kinetics for such inhibitors is predicted as long as the equilibrium between the two forms of enzyme-substrate complex significantly favors the initial exosite complex. In support, hydrolysis of a low molemolecular weight peptide substrate and its inhibition by SC81956 were unaffected by aggrecan concentrations substantially above the K m . These observations suggest that the apparent K m for aggrecan cleavage predominately reflects the exosite interaction. Consequently, the efficacy of active-site inhibitors of ADAMTS-4 will not be limited by competition with native substrate as predicted from the K m determined by traditional kinetic models.

  • adamts 4 Aggrecanase 1 n terminal activation mechanisms
    Archives of Biochemistry and Biophysics, 2005
    Co-Authors: Micky D. Tortorella, Elizabeth C. Arner, Robert L Hills, Jennifer A Gormley, Lyle E Pegg, Grace E Munie, Anne-marie Malfait
    Abstract:

    Abstract 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 °C, at ionic strengths from 0.1 to 1 M, 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.

  • proprotein convertase furin interacts with and cleaves pro adamts4 Aggrecanase 1 in the trans golgi network
    Journal of Biological Chemistry, 2004
    Co-Authors: Ping Wang, Micky D. Tortorella, Kristen England, Anne-marie Malfait, Gary Thomas, Elizabeth C. Arner
    Abstract:

    Abstract A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: 206RPRR209, 209RAKR212, or 211KR212. The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furinindependent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.

Micky D. Tortorella – One of the best experts on this subject based on the ideXlab platform.

  • substrate dependent inhibition kinetics of an active site directed inhibitor of adamts 4 Aggrecanase 1
    Biochemistry, 2007
    Co-Authors: Arthur J Wittwer, Elizabeth C. Arner, Anne-marie Malfait, Robert L Hills, Grace E Munie, R H Keith, C P Anglin, Micky D. Tortorella
    Abstract:

    ADAMTS-4 (Aggrecanase1) is implicated in the breakdown of articular cartilage and is an attractive target for therapeutic intervention in arthritis. Cleavage of the native substrate, aggrecan, occurs through exosite interactions and peptide sequence recognition. Although expected to be competitive with aggrecan, the hydroxamic acid, SC81956, demonstrated noncompetitive inhibition kinetics with a K i of 23 nM. The IC 50 of SC81956 did not change when aggrecan was varied from 12.8 to 200 nM (0.2-3.3 times the apparent aggrecan K m of 61 nM) but was shifted as expected for a competitive inhibitor when increasing levels of a low molecular weight peptide substrate were added to a fluorogenic peptide assay system. These observations are consistent with a model for aggrecan cleavage where substrate initially binds at an exosite, followed by binding of the appropriate peptide sequence at the active site. A peptide-competitive inhibitor could bind both free enzyme and initial substrate-enzyme exosite complex but would be excluded by the final Michaelis complex. Noncompetitive appearing kinetics for such inhibitors is predicted as long as the equilibrium between the two forms of enzyme-substrate complex significantly favors the initial exosite complex. In support, hydrolysis of a low molecular weight peptide substrate and its inhibition by SC81956 were unaffected by aggrecan concentrations substantially above the K m . These observations suggest that the apparent K m for aggrecan cleavage predominately reflects the exosite interaction. Consequently, the efficacy of active-site inhibitors of ADAMTS-4 will not be limited by competition with native substrate as predicted from the K m determined by traditional kinetic models.

  • adamts 4 Aggrecanase 1 n terminal activation mechanisms
    Archives of Biochemistry and Biophysics, 2005
    Co-Authors: Micky D. Tortorella, Elizabeth C. Arner, Robert L Hills, Jennifer A Gormley, Lyle E Pegg, Grace E Munie, Anne-marie Malfait
    Abstract:

    Abstract 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 °C, at ionic strengths from 0.1 to 1 M, 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.

  • proprotein convertase furin interacts with and cleaves pro adamts4 Aggrecanase 1 in the trans golgi network
    Journal of Biological Chemistry, 2004
    Co-Authors: Ping Wang, Micky D. Tortorella, Kristen England, Anne-marie Malfait, Gary Thomas, Elizabeth C. Arner
    Abstract:

    Abstract A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: 206RPRR209, 209RAKR212, or 211KR212. The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.

Katy E Georgiadis – One of the best experts on this subject based on the ideXlab platform.

Elisabeth A Morris – One of the best experts on this subject based on the ideXlab platform.

  • n 8 hydroxy 5 substituted quinolin 7 yl phenyl methyl 2 phenyloxy amino acetamide inhibitors of adamts 5 Aggrecanase 2
    Bioorganic & Medicinal Chemistry Letters, 2008
    Co-Authors: Adam M Gilbert, Katy E Georgiadis, Erica Reifenberg, Carl R Flannery, Matthew Gregory Bursavich, Sabrina Lombardi, Elisabeth A Morris
    Abstract:

    Abstract N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-μM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.

  • N-((8-hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2).
    Bioorganic & medicinal chemistry letters, 2008
    Co-Authors: Adam M Gilbert, Katy E Georgiadis, Erica Reifenberg, Carl R Flannery, Matthew Gregory Bursavich, Sabrina Lombardi, Elisabeth A Morris
    Abstract:

    Abstract N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-μM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.

  • 5 phenyl 3 h spiro indoline 3 2 1 3 4 thiadiazol 2 one inhibitors of adamts 5 Aggrecanase 2
    Bioorganic & Medicinal Chemistry Letters, 2007
    Co-Authors: Matthew Gregory Bursavich, Katy E Georgiadis, Erica Reifenberg, Carl R Flannery, Adam M Gilbert, Sabrina Lombardi, Elisabeth A Morris
    Abstract:

    Abstract 5′-Phenyl-3′ H -spiro[indoline-3,2′-[1,3,4]thiadiazol]-2-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. Selected compounds 23 , 33 – 35 show sub-micromolar ADAMTS-5 potency and strong SAR trends with selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase1), MMP12, and MMP13. This series of compounds represents progress toward a selective ADAMTS-5 inhibitor as a disease modifying osteoarthritis drug.