title: Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: State 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.
authors.forename: Zhong-Yuan
authors.initials: ZY
authors.lastname: Ren
authors.affiliation: Université de Lyon, Villeurbanne F-69622, France; Université Lyon 1, Villeurbanne F-69622, France; Ecole Normale Supérieure de Lyon, Lyon F-69634 France; Institut de Génomique Fonctionnelle de Lyon, Lyon F-69634 France; CNRS UMR3444, Lyon F-69634 France.
authors.forename: Irma
authors.initials: I
authors.lastname: Machuca-Gayet
authors.affiliation: Université de Lyon, Villeurbanne F-69622, France; Université Lyon 1, Villeurbanne F-69622, France; Ecole Normale Supérieure de Lyon, Lyon F-69634 France; Institut de Génomique Fonctionnelle de Lyon, Lyon F-69634 France; CNRS UMR3444, Lyon F-69634 France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Domenget
authors.affiliation: 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.
authors.forename: Rene
authors.initials: R
authors.lastname: Buchet
authors.affiliation: State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130012, China.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Université de Lyon, Villeurbanne F-69622, France; Université Lyon 1, Villeurbanne F-69622, France; Ecole Normale Supérieure de Lyon, Lyon F-69634 France; Institut de Génomique Fonctionnelle de Lyon, Lyon F-69634 France; CNRS UMR3444, Lyon F-69634 France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Jurdic
authors.affiliation: 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.
authors.forename: Saida
authors.initials: S
authors.lastname: Mebarek
mesh_terms: D000818:Animals; D001862:Bone Resorption / Q000188:drug therapy*; D002113:Calcification, Physiologic / Q000187:drug effects*; D056657:Cathepsin K / Q000037:antagonists & inhibitors*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology; D010010:Osteoclasts / Q000187:drug effects* / Q000502:physiology
chemical_list: D056657:Cathepsin K