RAF265, a dual BRAF and VEGFR2 inhibitor, prevents osteoclast formation and resorption. Therapeutic implications

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.

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.

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.

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.