Tucatinib alleviates postmenopausal osteoporosis by suppressing osteoclast differentiation via regulating the DRP1/NFATc1/CTSK signaling pathway.

Postmenopausal osteoporosis (PMOP) represents the most prevalent metabolic bone disease among postmenopausal women worldwide. Cathepsin K (CTSK), a key mediator of osteoclastic bone resorption, serves as a critical therapeutic target for PMOP. Through structure-based virtual screening coupled with functional validation, we identified Tucatinib as a potent CTSK inhibitor. Microscale thermophoresis (MST), molecular docking, and CTSK activity assays confirmed that Tucatinib directly binds to and inhibits CTSK, effectively suppressing osteoclast-mediated bone resorption. Notably, Tucatinib attenuated NFATc1-driven osteoclast differentiation in bone marrow-derived monocytes/macrophages (BMMs). In ovariectomized mouse models, Tucatinib significantly prevented estrogen deficiency-induced bone loss. Mechanistic investigations revealed that Tucatinib maintains mitochondrial homeostasis by inhibiting dynamin-related protein 1 (DRP1) phosphorylation at Ser616 during early-stage osteoclast differentiation and reducing mitochondrial reactive oxygen species (mtROS) production, thereby stabilizing mitochondrial fission/fusion dynamics and suppressing NFATc1 activation. Our findings demonstrate that Tucatinib alleviates osteoporotic bone loss by simultaneously modulating the DRP1/NFATc1 axis and CTSK enzymatic activity, offering a promising dual-action therapeutic strategy for PMOP.