Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Authors

  • Peter A. Hill,

    1. Department of Cell and Moleculer Biology, Strngeways Reserch Laboratory, Cambridge CB1 4RN, United Kingdom
    2. Department of Orthodonitcs and Paediatric Density, UMDS of Guy's and St. Thomas's Hospitals, University of London, London SE1 9RT, United Kingdom
    Current affiliation:
    1. Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, United Kingsom
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  • David J. Buttle,

    Corresponding author
    1. Department of Biochemistry, Strngeways Reserch Laboratory, Cambridge CB1 4RN, United Kingdom
    • Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom
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  • Sheila J. Jones,

    1. Department of Anatomy and Embryology, University College London, London WC1E 6BT, United Kingdom
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  • Alan Boyde,

    1. Department of Anatomy and Embryology, University College London, London WC1E 6BT, United Kingdom
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  • Mitsuo Murata,

    1. Department of Research Centre, Taisho Pharmaceutical Co., Ltd., Saitama 330, Japan
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  • John J. Reynolds,

    1. Department of Cell and Moleculer Biology, Strngeways Reserch Laboratory, Cambridge CB1 4RN, United Kingdom
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  • Murary C. Meikle

    1. Department of Cell and Moleculer Biology, Strngeways Reserch Laboratory, Cambridge CB1 4RN, United Kingdom
    2. Department of Orthodonitcs and Paediatric Density, UMDS of Guy's and St. Thomas's Hospitals, University of London, London SE1 9RT, United Kingdom
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Abstract

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane-permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane-permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of β-glucuronised, and N-acetyle-β-glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does-dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.

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