Fig. 2: Small-molecule ligands bias specificity of human POR to reduce diverse electron acceptors.

A Human POR proteoliposome activity to reduce diverse electron acceptors was assessed using 100 μM NADPH and 10 μM RS (left), 500 μM MTT (middle) or 40 μM Cytc (right) by monitoring changes in absorbance (550 nm for Cytc, 610 nm for MTT) or fluorescence (582 nm for RS). Note the increased noise due to less sensitive UV–VIS readout for Cytc. All activity traces depict the mean ± SD of at least three independent measurements. POR activity was extracted by fitting the linear region of the traces. B Ligands affect the electron donating capacity of human POR differentially dependent on the electron acceptor indicating biased specificity. Rifampicin reduces POR activity towards Cytc, has a small effect on MTT reduction and enhances POR activity to reduce the electron acceptor resazurin by 3-fold. Cyclophosphamide results in minute increased activity towards Cytc, while dhurrin reduces activity towards Cytc. The bar plot represents the mean ± SD of independent replicates normalized to DMSO controls with propagated error (n = 2–6; see Supplementary Methods and Supplementary Table 3 for exact value of n for each experimental condition). Note, overlapping data points appear shaded. Data for Cytc from Fig. 1 are included for comparison. All data are corrected for potential ligand photophysical effect (see Supplementary Fig. 7). Level of significance is determined by one-way ANOVA and Tukey’s HSD test correcting for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.005; see Supplementary Material for details). Source data are provided as a Source Data file.