Mitochondria-Targeted Quinoline-Based Fluorescent Probes for Imaging of Viscosity and MAO-A with High-Throughput Inhibitor Screening

Monoamine oxidase A (MAO-A) is a key enzyme in neurotransmitter metabolism and oxidative stress regulation, while mitochondrial viscosity serves as an important indicator of organelle health. However, simultaneous detection of MAO-A activity and viscosity using a single fluorescent probe remains challenging. Here, we report a series of quinoline-based probes incorporating an <i>N</i>-alkylated tetrahydropyridine unit as an MAO-A-responsive moiety. Among them, <b>CMTP-1</b> exhibited high viscosity sensitivity, generating a "turn-on" fluorescence response via restricted intramolecular rotation. Oxidation of the tetrahydropyridine by MAO-A produced a pyridinium group, triggering intramolecular charge transfer (ICT) and fluorescence quenching. <b>CMTP-1</b> selectively localized to mitochondria, enabled visualization of endogenous MAO-A in neuroblastoma cells and zebrafish, and monitored viscosity changes in lipopolysaccharide (LPS)-induced inflammatory models. Furthermore, a <b>CMTP-1</b>-based high-throughput screening platform identified harmine as a potent MAO-A inhibitor. These results highlight <b>CMTP-1</b> as a versatile tool for probing mitochondrial viscosity and MAO-A activity, with broad potential in biomedical research and drug discovery.