Figure 6 | Scientific Reports

Figure 6

From: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

Figure 6

Assessment of CypD functional activity and compound binding.

rhCypD protein was incubated with chymotrypsin and compound for 30 minutes. N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was added and absorbance (405 nm) measured for 60 seconds at 3 second intervals. (a) Kinetic traces in the presence of ER-000444793 demonstrated no inhibition of enzyme activity. (b,c) Dose-dependent inhibition of rhCypD enzymatic activity observed with CsA and SfA. (d) Area-under-the-curve from a-c was calculated from the first 15 seconds of the reaction and results expressed as % inhibition, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). (e) rhCypD TR-FRET assay demonstrates a dose-dependent decrease in FRET by both CsA and SfA but no effect of ER-000444793. Data are expressed as % binding, normalised to DMSO (0% binding) and CsA (5 μM; 100% binding). All data are expressed as means (±s.d.) of at least three independent experiments. Abbreviations: CsA; cyclosporin A, SfA; sanglifehrin A, O.D; optical density, Cmp; compound.

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