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Inhibition of bone resorption by selective inactivators of cysteine proteinases
P A Hill1, D J Buttle, S J Jones
1Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom.
Abstract:
Inactivators of cysteine proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep475, a compound with low membrane permeability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectively inactivates cathepsin B; and CA074Me, the membrane-permeant prodrug of CA074. The test systems consisted of 1) monitoring the release of radioisotope from prelabelled mouse calvarial explants and 2) assessing the extent of bone resorption in an isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhibited both stimulated and basal bone resorption in vitro while CA074 was without effect; the inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of beta-glucuronidase, and N-acetyl-beta-glucosaminidase, or the spontaneous release of lactate dehydrogenase. Ep453, Ep475, and CA074Me dose-dependently inhibited the resorptive activity of isolated rat osteoclasts cultured on bone slices with a maximal effect at 50 microM. The number of resorption pits and their mean volume was reduced, whilst the mean surface area remained unaffected. Again, CA074 was without effect. Ep453, Ep475, and CA074Me, but not CA074, when administered subcutaneously at a dose of 60 micrograms/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonstrates that cathepsins B, L, and/or S are involved in bone resorption in vitro and in vivo. Whilst cathepsin L and/or S act extracellularly, and possibly intracellularly, cathepsin B mediates its effects intracellularly perhaps through the activation of other proteinases involved in subosteoclastic collagen degradation.
Insights
Cysteine proteinase (CP) inhibitors, specifically Ep453, Ep475, and CA074Me, effectively reduced bone resorption in vitro and in vivo. These compounds target cathepsins B, L, and/or S, indicating their crucial role in bone remodeling processes.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Bone resorption is a complex process regulated by various enzymes.
- Cysteine proteinases (CPs) are implicated in bone remodeling, but their specific roles are not fully elucidated.
- Inhibitors of CPs offer potential therapeutic targets for bone diseases.
Purpose of the Study:
- To investigate the efficacy of CP inactivators as inhibitors of bone resorption.
- To determine the involvement of specific cathepsins (B, L, S) in bone resorption processes.
- To evaluate the effects of CP inhibitors in vitro and in vivo models.
Main Methods:
- Tested four CP inactivators: Ep475, Ep453, CA074, and CA074Me.
- Assessed bone resorption using radioisotope release from mouse calvarial explants.
- Evaluated osteoclast activity on bone slices using confocal microscopy.
- Administered inhibitors subcutaneously to assess in vivo effects.
Main Results:
- Ep453, Ep475, and CA074Me significantly inhibited both basal and stimulated bone resorption in vitro.
- CA074 showed no effect on bone resorption.
- Inhibitors reduced resorptive activity of isolated osteoclasts and inhibited bone resorption in vivo by approximately 20%.
- No adverse effects on protein synthesis, DNA synthesis, or enzyme secretion were observed.
Conclusions:
- Cathepsins B, L, and/or S are critically involved in bone resorption.
- Extracellular cathepsin L and/or S, and intracellular cathepsin B, play distinct roles in this process.
- CP inhibitors demonstrate therapeutic potential for modulating bone resorption.