Fig. 5. Biased metabolism: small-molecule ligands bias steroidogenic CYP-activities in human cells and microsomes.

A A human adrenocortical cell line (NCI-H295R) was used to assess the effect of small-molecule ligands on steroidogenic CYP17A1 and CYP21A2 hydroxylase activity, and CYP17A1 lyase activity, using radiolabeled substrates (see Supplementary Methods and Supplementary Table 4). Cell viability was assessed based on MTT reduction. Rifampicin shows a small inhibiting effect towards CYP21A2. The cells display increased MTT reduction indicating increased reductase activity. No significant effects on CYP17A1 activities are observed. Cyclophosphamide causes a significance increase in CYP17A1 lyase and less significant towards 17-OHase and 21A2 activities, while MTT reduction decreases slightly. Dhurrin causes inhibition of both CYP17A1 and CYP21A2 activities. B Abiraterone was used as a control inhibitor of CYP17A1 and CYP21A2 in H295R cells. C The effect of ligands on CYP19A1 activity was assessed on microsomes from a human choriocarcinoma cell line (JEG3). Rifampicin shows a concentration dependent inhibitory effect on CYP19A1 activity (32 ± 11% of control). Cyclophosphamide and dhurrin display no significant effect. A, C Error bars represent mean ± SD of 3–4 biological replicates normalized to DMSO controls with propagated error. See Supplementary Fig. 14 for raw data and Supplementary Table 4 for exact value of n for each experimental condition. Note, overlapping data points appear shaded. 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 Methods for details). Source data are provided as a Source Data file.