The dinitrothiophenol SN-2 reduces osteoclastogenesis and estrogen deficiency-induced bone loss by inhibiting TRAF6 auto-ubiquitination.

Post-menopausal osteoporosis is characterized by overactive bone resorption in response to estrogen withdrawal. As Receptor Activator of NF-κB ligand (RANKL)-induced auto-ubiquitination of TNF receptor-associated factor 6 (TRAF6) on its K124 residue promotes osteoclastogenesis, inhibiting TRAF6K124 auto-ubiquitination may be a promising therapeutic strategy for post-menopausal osteoporosis. We investigated the impact of the synthetic dinitrothiophenol SN-2 (4-(Dimethylamino)-2,6-bis[[N-[2-[(2-nitrophenyl)dithio]-ethyl]amino]methyl]pyridine) on TRAF6K124 auto-ubiquitination, osteoclast generation, and osteoporotic bone loss. In vitro, SN-2 inhibited RANKL-induced osteoclast differentiation and osteoclast marker expression in a dose-dependent manner. SN-2 also inhibited RANKL-induced F-actin ring formation and bone resorption in vitro. SN-2 suppressed RANKL-induced TRAF6K124 auto-ubiquitination (TRAF6-Ub), TRAF6's binding to Transforming Growth Factor Beta-Activated Kinase 1 (TAK1), and TAK1 phosphorylation, thereby inhibiting downstream NF-κB and c-Jun N-terminal Kinase (JNK) signaling pathways critical to osteoclast differentiation. In an ovariectomized mouse model of osteoporosis, SN-2 ameliorated estrogen-deficiency-induced bone loss, improved femoral biomechanical parameters, and inhibited osteoclastogenesis in vivo. SN-2 may show promise as a therapeutic agent in treating bone disorders characterized by RANKL signaling over-activation and excessive bone resorption.