Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption

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.

Rosmarinic acid alleviates diabetic osteoporosis by suppressing the activation of NLRP3 inflammasome in rats

“…3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Cell viability was measured using the MTT colorimetric assay as previously described [10]. In brief, treated OCs were stained with MTT reagent (0.5 mg ml À1 ) for 4 h, followed by the addition of 10% sodium dodecyl sulfate in 0.01 M HCl at 37 8C overnight.…”

“…Bone resorption pit assay was performed as previously described [10]. In brief, BMMs-differentiated osteoclasts were detached from the flask using 25 mM EDTA solution to seed on bovine bone slices at a density of 50,000 cells per well in 0.5 ml medium in a 96-well plate, then treated with 35 mM high glucose in the presence or absence of 0.5 ml RA for 72 h as mentioned above.…”

Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity

“…This cell line can support osteoclast formation in vitro , by secreting RANKL, OPG, and M-CSF, without supplementation from any other regulatory factors [ 20 ]. The RAW 264.7 monocyte cell line is the most commonly used osteoclast precursor; it is widely used to examine osteoclast formation in vitro [ 21 , 22 , 23 , 24 , 25 ].…”

Wirkung von Ostarine – ein selektiver Androgenrezeptormodulator (SARM) – auf das osteoporotische Femur der orchiektomierten Ratte

“…Bisphosphonate, Denosumab oder andere Antiresorptiva unterscheiden sich durch den Wirkmechanismus, als dass die Hemmung der Cathepsin K-Aktivität die Osteoklastenfunktion reversibel supprimiert. Zu einer Apoptose oder Funktionseinschränkung von Osteoklasten führt es allerdings nicht(Gamsjäger und Resch 2015;Ren et al 2015).Die Firma Merck (MSD, Darmstadt, Deutschland) hatte einen Cathepsin-K-Hemmer, Odanacatib, in klinischen Phase-III-Studien zur Behandlung von Osteoporose. Im September 2016 gab Merck bekannt, dass die Entwicklung von Odanacatib nach eigener Einschätzung unerwünschter Ereignisse eingestellt wird.…”

“…Eine unabhängige Einschätzung ergab ein erhöhtes Schlaganfallrisiko(Brömme und Lecaille 2009; Schatz 2017). Andere Cathepsin-K-Inhibitoren befinden sich in verschiedenen Entwicklungsstadien(Asagiri et al 2014;Hussein et al 2014;Ren et al 2015). Medivir hat einen Cathepsin-K-Hemmer, MIV-711 Thousand Oaks, USA) und die anschließende Weiterbehandlung mit Alendronsäure, welche die Reduzierung von Wirbelfrakturen und Frakturen insgesamt gegenüber einer alleinigen Behandlung mit Alendronsäure aufwies.…”