Abstract
AimThe 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 MethodsCell 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.ResultsTreatments 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.
Highlights
Osteoporosis is a common medical and socioeconomic threat characterized by a systemic loss of bone mass, strength, and microarchitecture, which increases the risk of fragility fractures [1, 2]
Treatments with two CatK inhibitors (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
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
Summary
Osteoporosis is a common medical and socioeconomic threat characterized by a systemic loss of bone mass, strength, and microarchitecture, which increases the risk of fragility fractures [1, 2]. Drug treatment strategies have been developed, aimed at inhibiting excessive bone resorption and at increasing bone formation. One of the most promising drug treatments is based on the specific inhibition of the osteoclast protease cathepsin K (CatK) to slow down bone resorption [5]. The inhibition of bone resorption observed in human and animal models deficient for CatK indicated that this enzyme is a suitable target for intervention by small molecules that might be used as therapeutic agents in osteoporosis. Four CKIs, Relacatib, Balicatib, MIV-711 and Odanacatib (ODN) have been evaluated as possible drug therapies to prevent bone resorption [11,12,13]. Developed as antiresorptive agents, several compounds show lysosomotropic effects [16], cutaneous adverse effects and anabolic activity [18], which are intrinsically related to the selectivity of inhibitors toward CatK. The other inhibitor (CKI-8) from the second group, led to further improvement in the CatK selectivity by shortening the length of P3-P2 linker (Fig 1)
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