Targeting Mitochondrial Uncoupling Protein 2: Evaluation of a Novel Inhibitor in Macrophages

Uncoupling protein 2 (UCP2) is a mitochondrial inner membrane carrier highly expressed in macrophages, where it regulates immunometabolic responses by modulating mitochondrial reactive oxygen species (ROS) production and inflammatory signalling. Pharmacological inhibition or genetic silencing of UCP2 enhances ROS generation and alters macrophage activation, underscoring its relevance as a therapeutic target. Genipin, a natural UCP2 inhibitor, is widely used for this purpose; however, limited mitochondrial accumulation and concentrationdependent off-target effects restrict its applicability. To improve mitochondrial delivery, mito-genipin was synthesized in the hosting laboratory by conjugating genipin to a triphenylphosphonium (TPP⁺) moiety. We characterized its biological activity in RAW 264.7 macrophages and bone marrow– derived murine macrophages (BMDMs). MTS assays and Annexin V/PI staining revealed dose-dependent cytotoxicity and predominant apoptosis, with greater potency than genipin. Measurements of mitochondrial membrane potential (TMRM) and mitochondrial ROS (MitoSOX) demonstrated rapid hyperpolarization and markedly increased ROS production following mito-genipin treatment, whereas genipin elicited minimal effects. Moreover, mito-genipin enhanced IL-6 expression under M1-polarizing conditions and tended to reduce Arg1 expression during M2 stimulation, indicating a shift toward a proinflammatory phenotype. Overall, mito-genipin emerges as a more effective modulator of mitochondrial function than genipin and represents a valuable tool to investigate UCP2-dependent immunometabolic regulation in macrophages.