TARGETING SLC11A2 PALMITOYLATION FOR TREATMENT OF IRON OVERLOAD AND OSTEOPOROSIS

Osteoclast-mediated bone resorption plays a crucial role in maintaining skeletal homeostasis and serves as an important target for clinical treatment of bone metabolism-related diseases. Lipids are key metabolic signals regulating osteoclast differentiation, though their mechanisms remain to be elucidated. In this study: We screened a library of lipid metabolism-related compounds and found that multiple palmitoylation inhibitors significantly inhibited osteoclast differentiation. These inhibitors effectively prevented bone loss in mice subjected to a high-fat diet and ovariectomy without impacting bone formation. Through transcriptomic and proteomic analysis, we identified that the palmitoyltransferase ZDHHC13 is significantly upregulated during osteoclast differentiation and in patients with osteoporosis. Conditional knockout of ZDHHC13 in mice led to an increase in bone mass and a reduction in bone resorption. Combining palmitoyl-proteomics and IP-MS, we discovered that the Fe 2+ transporter SLC11A2 is a substrate of ZDHHC13. Conditional knockout of SLC11A2 also resulted in increased bone mass and decreased bone resorption in mice. GWAS analysis indicated that SLC11A2 expression is negatively correlated with bone density in humans. Further studies revealed the dynamic palmitoylation process of SLC11A2, where ZDHHC13 mediates its palmitoylation, promoting its correct localization on the cell membrane. We then identified that palmitoylation of SLC11A2 occurs at the cysteine residue C64 and confirmed in vivo that this modification regulates Fe 2+ intake and osteoclast differentiation. We also found that natural mutants of SLC11A2 with significantly reduced palmitoylation levels provide a partial mechanistic explanation for certain hereditary anemias in humans. Subsequently, we mapped the dynamics of Fe 2+ and mitochondrial homeostasis during osteoclast differentiation, elucidating the mechanism by which SLC11A2 knockout or palmitoylation defects lead to cellular Fe 2+ deprivation and subsequent mitochondrial energy metabolism imbalance. Using high-content imaging technology for high-throughput screening of SLC11A2 inhibitors, we discovered that the FDA-approved drugs Azelnidipine and Efonidipine (dihydropyridine class antihypertensive drugs) can target and inhibit SLC11A2 palmitoylation and Fe2+ transport. Molecular dynamics simulations and structure-activity relationship analysis indicated that Azelnidipine and Efonidipine bind to SLC11A2 through their unique functional groups, independent of their dihydropyridine structure. Further confirmation showed that Azelnidipine and Efonidipine effectively inhibited osteoclast differentiation and prevented bone loss in mice, while also reducing tissue iron accumulation. Therefore, our study systematically elucidates the mechanism by which palmitoylation modifications regulate osteoclast differentiation; reveals the critical role of SLC11A2 palmitoylation-mediated iron homeostasis in human diseases; and identifies the clinical potential of Azelnidipine and Efonidipine in treating iron overload-related diseases.