Discovery of Niclosamide Analogs with Potent Mitochondrial Uncoupling Activity and Reduced Mitochondrial Inhibition–Associated Toxicity

Niclosamide, an FDA-approved anthelmintic, functions as a mitochondrial uncoupler with promising anticancer potential, yet its efficacy remains limited, often ascribed to poor bioavailability. We identify a more fundamental constraintits narrow therapeutic window arising from a biphasic mechanism that promotes uncoupling at low doses but inhibits respiration at higher doses. To overcome this limitation, we synthesized 30 niclosamide analogs, systematically profiled their mitochondrial responses using Seahorse MitoTox assay, and developed QSAR models to uncover structural determinants of efficacy and toxicity. Niclosamide exhibited a narrow uncoupling range (0.5-1 μM) beyond which respiration was suppressed. Several analogs, including Nic-2, Nic-8, Nic-40, and Nic-43, sustained uncoupling for up to 9 h at concentrations up to 10 μM, with some showing improved signal modulation and reduced cytotoxicity. QSAR analysis revealed that substitution electronic properties and ring-specific hydrophobicity are related to the therapeutic index. These findings expand niclosamide's therapeutic window through rational scaffold tuning, enabling safer mitochondrial reprogramming strategies for cancer therapy.