Chemical structure and mitochondrial uncoupling properties of MB1-47. (A) Schematic illustration of the ETC and the effects of uncoupling depolarizing the mitochondrial inner membrane. (B) Chemical structure of NEN and MB1-47, niclosamide-based first- and second-generation mitochondrial uncouplers, respectively. (C) Pharmacokinetic properties of NEN and MB1-47 in mice in vivo. (D) Representative flow cytometry histograms showing mitochondrial membrane potential measured by tetramethylrhodamine ethyl ester (TMRE) staining in live DND41 and JURKAT cells after 72 hours of treatment with MB1-47 (4 μM and 2 μM, respectively). (E) Geometric mean ± standard deviation (SD) quantification of TMRE staining from triplicates in 2 PTEN⁺ (DND41 and HPB-ALL) and 2 PTEN⁻ (JURKAT and MOLT-3) cell lines upon 72 hours of MB-47 treatment. (F) Oxygen consumption rate (OCR) in DND41 cells, under basal conditions or in response to the indicated mitochondrial inhibitors, measured in real time using a Seahorse XF24 instrument. Data are presented as mean ± SD of n = 4 wells. (G) Metabolite levels in FCCP-treated vs MB1-47–treated DND41 cells for 24 hours (mean; n = 3). (H) Relative quantification of ATP levels from DND41 triplicates treated with DMSO, FCCP, or MB1-47 for 24 hours (mean ± SD). The goodness of fit (R²) was determined by using a simple linear regression model. Statistical significance (P) was determined by using an unpaired, 2-tailed Student t test. *P < .05; ***P < .005. AUC, area under the curve; MFI, mean fluorescence intensity.