Speaker
Description
Background: Sterol 12α-hydroxylase (CYP8B1) is a promising target for treating metabolic diseases. However, developing selective inhibitors is hindered by incomplete methodologies and the absence of structural guidance. To overcome this, this study adopts an alternative strategy of screening plant-derived triterpenoids and animal bile acids as candidate inhibitors, leveraging their structural similarity to native substrates and historical use in traditional medicine.
Methods: A dual-substrate screening platform was established in recombinant and microsomal CYP8B1 to compare the novel substrate dihydroxycholestanoic acid (DHCA) with the classic substrate 7α-hydroxy-4-cholesten-3-one (C4) for evaluating azole inhibitors. This platform was then applied to screen natural triterpenoids and bile acids. Hit compounds were assessed for potency and selectivity against recombinant CYP8B1, CYP7A1, and CYP3A4. Subsequently, cellular inhibitory activity and pathway selectivity were analyzed via 2,3,4-13C3-cholesterol flux analysis in the triiodothyronine-dexamethasone-bezafibrate induced HepG2 cells. Finally, in vivo efficacy was confirmed in bile duct-cannulated rats using a dose-escalation protocol.
Results: DHCA could efficiently evaluate the inhibition of CYP8B1 compared with C4, and econazole was identified as a potent CYP8B1 inhibitor with selectivity over CYP7A1. Screening of 70 natural products revealed oleanolic acid, oleanonic acid, and 20S-protopanaxadiol as moderate dual CYP8B1/7A1 inhibitors, confirmed by metabolic flux analysis which also indicated off-target effects on CYP7B1, particularly for oleanonic acid. In vivo studies in rats showed cholestatic risks for all compounds upon administration. Econazole demonstrated the first dose-dependent in vivo CYP8B1 inhibition, while oleanolic acid and 20S-protopanaxadiol also showed weaker dose-dependent effects, oleanonic acid paradoxically increased 12α-hydroxylation despite potent in vitro activity.
Conclusions: This study demonstrates that DHCA serves as an effective alternative substrate to the classical probe C4 for in vitro screening. And it establishes an integrated methodological framework for discovering CYP8B1 inhibitors, combining enzymatic assays, cellular flux analysis, and species-specific bile acid profiling. While no selective natural inhibitors were found, econazole is the first inhibitor with demonstrated in vivo efficacy, highlighting key challenges in selectivity and species differences for future drug discovery.
Acknowledgments
Supported by the National Natural Science Foundation of China, grant No. 82373945.
