Steroid hormones are metabolically derived from multiple enzymatic transformations of
cholesterol. The controlling step in
steroid hormone biogenesis is the delivery of
cholesterol from intracellular stores to the
cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator
protein (TSPO) plays an integral part in this mitochondrial
cholesterol transport. Consistent with its role in intracellular
cholesterol movement, TSPO possesses a
cholesterol recognition/interaction
amino acid consensus (CRAC) motif that has been demonstrated to bind
cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel
ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits
cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic
CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig
tumor cells and constitutive steroidogenesis in R2C rat Leydig
tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the
CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting
PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the
steroidogenic acute regulatory protein without any effect on TSPO
protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive
steroid formation.