# Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets

## TL;DR

This study shows that a compound called 19-Atriol inhibits steroid production not by acting on a protein called TSPO, but by directly affecting enzymes involved in steroid synthesis.

## Contribution

The study identifies 3β-HSD and other enzymes in the steroidogenic pathway as the true targets of 19-Atriol, refuting the role of TSPO in its mechanism.

## Key findings

- 19-Atriol inhibits steroid production by competitively inhibiting 3β-HSD, regardless of TSPO presence.
- 19-Atriol and its metabolite 19-OHT inhibit cholesterol-to-pregnenolone conversion.
- 19-Atriol partially inhibits CYP11A1 activity, possibly via effects on the steroidogenic acute regulatory protein (STAR).

## Abstract

The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.