Fig. 5: Cy5-conjugated cyclophilin D inhibitors delay calcium induced opening of the mPTP in isolated mouse liver mitochondria and enter human cells as ester prodrugs.

The calcium retention capacity of mitochondria was determined in isolated mouse liver mitochondria (0.5 µg mL⁻¹) in response to pulses of 60 µM CaCl₂ in the presence of the indicated CypD inhibitors (or inactive enantiomers). Concentrations used were 2 µM CsA, 10 µM B52-Cy5, 10 µM *B52-Cy5, 20 µM B53-Cy5, 20 µM *B53-Cy5. Mitochondrial uptake of extramitochondrial Ca²⁺ was assessed by monitoring the fluorescence of Calcium green 5n, depicted in arbitrary units (AU). The rapid increase in fluorescence after several pulses of Ca²⁺ are taken up corresponds to mitochondrial Ca²⁺ release through mPTP opening. a–c, Traces are shown from a representative experiment, with all assays performed on the same mitochondrial preparation and day. d, Quantitation of calcium retention capacity (CRC) reported as the ratio of the number of Ca²⁺ pulses required to induce mPTP opening in the listed condition relative to DMSO control conditions on the same mitochondrial preparation and day. e, Structures of Cy5-conjugated CypD-selective inhibitors and prodrugs. f, Fluorescence microscopy of HeLa cells co-incubated with ester prodrugs B52-Et-Cy5 and B53-Et-Cy5 (red), co-stained with mitochondrial (green) and nuclear (blue) dyes (Mitotracker Green and Hoechst 33342, respectively). Both prodrugs show good plasma membrane permeability and co-localization with Mitotracker Green, quantified in Extended Data Fig. 8a–i. Calcium retention data are from three independent experiments/mitochondrial isolations. Data bars and error bars represent mean ± s.d. DMSO versus CsA, P = 0.0135; *B52-Cy5 versus B52-Cy5, P = 0.0011; *B53-Cy5 versus B53-Cy5, P = 0.0382. *P < 0.05, **P < 0.005 by one-sided Student’s t-test. Microscopy images are a representative image of three technical replicates. Scale bars, 200 µm.

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