title: RAF265, a dual BRAF and VEGFR2 inhibitor, prevents osteoclast formation and resorption. Therapeutic implications.
abstract: BACKGROUND
  The RAS/RAF/MEK/ERK signaling pathway plays an important role in osteoclast (OC) differentiation and survival mediated by macrophage-colony stimulating factor (M-CSF). Also, vascular endothelial growth factor (VEGF) may greatly influence OC formation and resorption through VEGFR1 and VEGFR2. RAF265 is a novel, orally bioavailable dual inhibitor of RAF kinase and VEGFR2.
  
  
  METHODS
  Effect of RAF265 on osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) and OC resorption on calcium-coated wells was assessed by appropriate in vitro assays. Immunoblotting, real-time RT-PCR and flow cytometry were used to evaluate RAF265 mechanism of action.
  
  
  RESULTS
  RAF265 significantly impaired in vitro differentiation of PBMCs to OCs induced by receptor activator of NF-kB ligand (RANKL) and M-CSF (IC(50) ≅ 160 nM). In parallel, RAF265 exerted a potent inhibition of OC resorptive capacity (IC(50) ≅ 20 nM). RAF265 treatment led to ERK inhibition and diminished expression of c-fos and NFATc1 (nuclear factor of activated T cells, calcineurin-dependent 1), which would likely account for inhibition of osteoclastogenesis. The reduced gene expression of aVb3 integrin, CCR1, cathepsin K, carbonic anhydrase II, matrix metalloproteinase 9, urokinase and tissue-type plasminogen activators, vacuolar H(+)-ATPase subunit (ATP6V1A) and Rab7 GTPase would probably mediate RAF265 hindered resorption. RAF265 inhibitory effect on VEGFR2 (noticeable at 10-50 nM) was also found to be implicated in the potent inhibition of this agent on OC function.
  
  
  CONCLUSIONS
  We have found a new therapeutic application for RAF265 as an inhibitory agent of osteoclastogenesis and OC function, which might be useful for the treatment of skeletal disorders associated with increased bone resorption.
authors: Centro de Investigación del Cáncer, IBMCC (Universidad de Salamanca-CSIC), Campus Miguel de Unamuno, Avda. Coimbra s/n, 37007 Salamanca, Spain.
authors: Antonio
authors: A
authors: Garcia-Gomez
authors: Enrique M
authors: EM
authors: Ocio
authors: Atanasio
authors: A
authors: Pandiella
authors: Jesús F
authors: JF
authors: San Miguel
authors: Mercedes
authors: M
authors: Garayoa
mesh_terms: D001862:Bone Resorption / Q000188:drug therapy; D002478:Cells, Cultured; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors*
chemical_list: D007093:Imidazoles; D011725:Pyridines; C559019:RAF265; D040301:Vascular Endothelial Growth Factor Receptor-2; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf