(259) TSPO Link Unfounded in 19-Atriol (Androst-5-ene-3,17,19-triol) Inhibition of Steroidogenesis

Amy H. Zhao¹, Prasanthi P. Koganti¹, Mingxing Qian², Anthony Garcia³, Patrick O’Day³, Richard J. Auchus³, Douglas F. Covey², and Vimal Selvaraj¹

1. Cornell University, Ithaca NY, USA

2. Washington University School of Medicine, St. Louis, MO, USA

3. University of Michigan Medical School, Ann Arbor, MI, USA


Abstract Text:

Both in vivo and in vitro genetic deletion of the mitochondrial translocator protein (TSPO), using the same model systems that originally fashioned its function in mitochondrial cholesterol import for steroidogenesis, have veritably refuted this claimed role. Nevertheless, there have been arguments that pharmacological targeting of TSPO could still exhibit agonistic or antagonistic effects on steroidogenesis. Using CRISPR/Cas9-targeted TSPO knockout MA-10 cells, we previously demonstrated that the claimed agonistic function of PK11195, a prototypical TSPO binding drug was unsubstantiated. In this study, we scrutinized the claim from Vassilios Papadopoulos and colleagues that 19-Atriol (Androst-5-ene-3,17,19-triol) is a TSPO antagonist that could bind TSPO and inhibit steroidogenesis in MA-10 Leydig cells [Midzak, A., et al. (2011), J. Biol. Chem. 286: 9875-87].

Using CRISPR/Cas9-mediated Tspo gene deleted MA-10:Tspo^Δ/Δ cells, we performed target validation for 19-Atriol. Our results revealed that TSPO is not the target of 19-Atriol. However, 19-Atriol could clearly inhibit Bt2-cAMP-induced steroidogenesis measured by progesterone production in MA-10:Tspo^Δ/Δ cells. Investigating the possible enzymatic step involved, we observed specific competitive inhibitory effect of 19-Atriol in the conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Using mass spectrometry, we identified that 19-Atriol acts as a substrate of 3β-HSD and is converted to 19-hydroxytestosterone (19-OHT), providing the basis for inhibition. However, we also made an unexpected observation that cholesterol to pregnenolone conversion was also decreased with 19-Atriol treatment during Bt2-cAMP-induced steroidogenesis. Conversion of CYP11A1 intermediate, 22R-Hydroxycholesterol to pregnenolone was partially inhibited by 19-Atriol, but not by its metabolite 19-OHT. Structural features of 19-Atriol and 19-OHT show strong similarities to endogenous substrate binding to both CYP11A1 and 3β-HSD. These findings demonstrate that 19-Atriol is a competitive inhibitor of 3β-HSD and could also partially inhibit CYP11A1 or impact upstream steps in cholesterol delivery. Therefore, our results are conclusive in that the previously claimed 19-Atriol inhibition of steroidogenesis by targeting TSPO is baseless. In rectifying this core error in describing the mechanism of 19-Atriol pharmacology, we highlight the need for rigorous target validation for all putative TSPO-binding drugs using gene-deleted models.