Synthesis, molecular dynamics, and functional analysis of mitochondria-targeted estrogens through estrogen receptors

Estrogens play an important role in regulating mitochondrial function and cellular metabolism. Most of the effects of estrogens are mediated by estrogen receptors α and β (ERα and ERβ), located in various cellular compartments, including the mitochondria. The aim of this study was to validate the mitochondrial accumulation of mitochondria-targeted estrogens and determine whether their biological effects are mediated by ERs. Two mitochondria-targeted estrogen derivatives, mitoE2 and mitoEE2, were synthesized by covalently coupling a triphenylphosphonium (TPP) moiety to 17β-estradiol (E2) and 17α-ethynylestradiol (EE2), respectively, and were structurally characterized. Mitochondrial and cytosolic accumulation in MCF-7 cells was quantified by high resolution mass spectrometry. Cell viability assays were performed to assess the cytocompatibility of TPP. To investigate the binding of mitochondria-targeted estrogens to ERs molecular dynamics simulations were performed. Functional assays were assessed in vitro in the MCF-7 cell line through Manganese-dependent Superoxide Dismutase (MnSOD) activity assays using selective ER antagonists, and by gene expression analyses of mitochondrial-encoded cytochrome c oxidase COX1 and nuclear-encoded COX4. Both mitoE2 and mitoEE2 preferentially accumulated in mitochondria compared with the cytosol. The molecular dynamic simulations showed a thermodynamically stable binding to the ligand-binding domain of ERα and ERβ. In vitro analyses revealed that ERβ selectively mediates the inhibition of MnSOD activity induced by mitoE2 and that mitochondria-targeted estrogens differentially modulate COX1 and COX4 expression in a time-dependent manner. In conclusion, this work contributes to understanding the molecular mechanisms underlying the effects of mitochondria-targeted estrogens on mitochondrial function. Our findings support the need for further investigation to determine their role as modulators of mitochondrial processes and provide a mechanistic basis for future studies in additional cellular and disease-specific models.