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Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and
Zhong-Yuan Ren1, Irma Machuca-Gayet2, Chantal Domenget2
1State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130012, China; Université de Lyon, Villeurbanne F-69622, France; Université Lyon 1, Villeurbanne F-69622, France; CPE Lyon, Villeurbanne F-69622, France; INSA-Lyon, Villeurbanne F-69622, France; Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, Villeurbanne F-69622, France; CNRS UMR 5246, Villeurbanne F-69622, France.
Aim:
The cysteine protease cathepsin K (CatK), abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs) to test their effects on osteoblasts and osteoclasts.
Research Design And Methods:
Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices.
Results:
Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10-100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed down by the addition of CKI-8 and CKI-13.
Conclusion:
CKI-8 and CKI-13 screened here show promise as antiresorptive osteoporosis therapeutics but some off target effects on osteoblasts were found with CKI-13.
Insights
Two selective cathepsin K inhibitors (CKIs) show potential for osteoporosis treatment by reducing bone resorption. However, CKI-13 exhibited some off-target effects on osteoblast mineralization, indicating a need for further evaluation.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Cathepsin K (CatK) is crucial for bone matrix degradation by osteoclasts.
- Targeting CatK offers a potential therapeutic strategy for osteoporosis.
- Evaluating CatK inhibitors' effects on osteoblasts and osteoclasts is essential.
Purpose of the Study:
- To assess the effects of two selective CatK inhibitors (CKIs), CKI-8 and CKI-13, on osteoblasts and osteoclasts.
- To determine the therapeutic potential of these CKIs as anti-resorptive agents for osteoporosis.
Main Methods:
- Cell viability assays were performed on Saos-2 cells, RAW 264.7 macrophages, and mature osteoclasts.
- Osteoblast mineralization was monitored using Alizarin Red staining and alkaline phosphatase activity.
- Osteoclast-induced bone resorption was evaluated on bovine bone slices.
Main Results:
- CKI-8 and CKI-13 were non-toxic to tested bone cells at concentrations up to 100 nM.
- CKI-8 did not affect osteoblast mineralization, while CKI-13 showed a ~40% reduction in Saos-2 cells.
- Both CKIs effectively reduced bone resorption, with CKI-13 being more potent than E-64, and appeared to slow osteoclast mobility.
Conclusions:
- CKI-8 and CKI-13 demonstrate promise as anti-resorptive osteoporosis therapeutics.
- CKI-13 displayed some off-target effects on osteoblast mineralization, warranting further investigation.
- These selective CatK inhibitors warrant further study for osteoporosis treatment development.
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