Tioconazole

Sterol 12α-hydroxylase (CYP8B1) is a key regulator of bile acid (BA) homeostasis and an emerging therapeutic target for metabolic disorders. To address the challenge of cellular CYP8B1 inhibition characterization, this work developed a pharmacologically optimized HepG2 cells model using triiodothyronine-dexamethasone-bezafibrate (TDB) induction, which significantly enhances the 12α-hydroxylation activity along the acidic pathway of BA biosynthesis in HepG2 cells. Employing stable isotope tracing with apolipoprotein A1-solubilized 2,3,4-¹³C₃-cholesterol, we established a liquid chromatography-mass spectrometry-based flux analysis platform to track de novo BA synthesis. Combined with a recombinant CYP8B1 assay, flux analysis revealed that CYP8B1 participates in cholic acid synthesis in HepG2 cells, typically via 12α-hydroxylation of 7α-hydroxy-3-oxo-4-cholestenoic acid and dihydroxycholestanoic acid. In TDB-HepG2 cells, azole antifungals exhibited differentiated inhibition of 12α-hydroxylation activity, generally mirroring the enzymatic data. Econazole acted as a relatively selective CYP8B1 inhibitor with a cellular half-maximal inhibitory concentration of 0.31-0.45 μM, tioconazole and posaconazole dually inhibited CYP8B1 and sterol 27-hydroxylase (CYP27A1), itraconazole and voriconazole primarily inhibited CYP27A1, and fluconazole showed no activity toward either enzyme. This study provides the first direct evidence that CYP8B1 participates in cholic acid synthesis via the acidic pathway and establishes a high-throughput cellular platform for screening CYP8B1 inhibitors, revealing azoles as effective modulators of this pathway. SIGNIFICANCE STATEMENT: Optimized HepG2 model using a ¹³C₃-cholesterol flux assay provides direct evidence that CYP8B1 participates in cholic acid biosynthesis via the acidic pathway and establishes a high-throughput cellular platform for screening CYP8B1 inhibitors, revealing azoles as effective modulators of this pathway.