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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Target validation uncouples TSPO from 19-Atriol-mediated inhibition of steroidogenesis and reveals true enzymatic
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 Bt 2 -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.
Insights
The mitochondrial translocator protein (TSPO) does not mediate 19-Atriol's effects on steroidogenesis. Instead, 19-Atriol directly inhibits 3β-hydroxysteroid dehydrogenase (3β-HSD), revealing new targets in steroid hormone biosynthesis.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- The mitochondrial translocator protein (TSPO) was previously thought to regulate steroid hormone biosynthesis by mediating cholesterol import.
- Pharmacological ligands targeting TSPO are still believed to influence steroid output, despite genetic evidence refuting TSPO's role.
- 19-Atriol was reported to inhibit progesterone synthesis in MA-10 Leydig cells by binding to TSPO.
Purpose of the Study:
- To investigate the mechanism of action for 19-Atriol in steroidogenesis.
- To determine if TSPO is the functional target of 19-Atriol in MA-10 Leydig cells.
- To identify the direct enzymatic targets of 19-Atriol within the steroidogenic pathway.
Main Methods:
- Utilized CRISPR/Cas9 technology to generate Tspo-deleted MA-10 Leydig cells.
- Assessed 19-Atriol's impact on steroid output in both wild-type and Tspo-deleted cells.
- Employed mass spectrometry to identify metabolites and elucidate enzymatic interactions.
Main Results:
- 19-Atriol inhibited steroid production independently of TSPO expression in MA-10 cells.
- 19-Atriol competitively inhibited 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking pregnenolone to progesterone conversion.
- 19-Atriol is a substrate for 3β-HSD, producing 19-hydroxytestosterone (19-OHT), which also inhibits 3β-HSD; both compounds affected cholesterol conversion upstream.
Conclusions:
- TSPO is not the functional mediator of 19-Atriol's steroidogenic effects.
- 19-Atriol directly targets 3β-HSD and potentially upstream enzymes like CYP11A1 and STAR.
- This study highlights the necessity of rigorous target validation for compounds, especially those previously linked to TSPO.
