RANKL-IN-2

RANKL-IN-2 is an orally active RANKL inhibitor with Kd values of 3.21 μM in SPR and 4.625 μM in MST experiments. It disrupts the RANKL-RANK interaction by binding to RANKL and inhibits osteoclastogenesis through suppressing ROS, MAPK, and NF-κB pathways. In vitro, RANKL-IN-2 suppresses osteoclastogenesis by inhibiting RANKL-induced osteoclast formation, bone resorption, and osteoclast-specific gene and protein expression. Additionally, RANKL-IN-2 prevents bone loss in ovariectomized osteoporosis mouse models, making it useful for osteoporosis research.

RANKL-IN-2 (compound 3w) effectively inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells at a concentration of 1 μM for 4 days. It also suppresses RANKL-induced osteoclastogenesis in bone marrow macrophages (BMMs) in a dose-dependent manner over 5 days, with an IC50 of 0.577 μM when used at concentrations of 0.0625-2 μM. Additionally, at 0.5-2 μM for 7 days, it inhibits the formation of F-actin rings and bone resorption in BMMs in a dose-dependent manner. The compound further reduces the expression of osteoclast marker genes and proteins in BMMs at the same concentrations over 5 days. RANKL-IN-2 also modulates the ROS signaling pathway by inhibiting ROS production and enhancing antioxidant enzyme expression in BMMs. It reduces both intracellular and mitochondrial ROS generation when used at 0.5-2 μM for 48 hours. The compound inhibits the MAPK and NF-κB signaling pathways in BMMs at 1 μM following pretreatment for 6 hours and subsequent RANKL stimulation for 0-60 minutes. RANKL-IN-2 does not affect the differentiation or mineralization of MC3T3-E1 cells at 0.5-2 μM over 7 or 21 days. The compound remains stable in PBS and rat plasma for at least 12 hours.

RANKL-IN-2 (compound 3w) administered at 10 mg/kg via oral gavage every other day for 8 weeks can prevent bone loss induced by ovariectomy in mice by enhancing bone structural parameters and reducing osteoclast formation.