Dual-functional molecule 3c targeting CYP24A1-mediated 25-hydroxyvitamin D metabolism and microbial biofilms...

In the context of clinical treatment for mixed vaginal infections, which pose dual challenges of biofilm resistance and immune microenvironment imbalance, this study synthesized 12 "triazole-Schiff base" hybrid compounds using previously reported synthetic routes. This design was guided by the core mechanism in which CYP24A1 enzyme overexpression in the vitamin D-vitamin D receptor immunoregulatory pathway leads to degradation of active vitamin D. Among them, compound 3c exhibited excellent broad-spectrum antibacterial and antifungal activity (MIC = 16 μg mL^-1), low cytotoxicity, and significant inhibition and eradication of Candida albicans biofilms. Mechanistic studies revealed that the compound possesses both membrane-targeting disruptive effects and CYP24A1 inhibitory activity. At the enzymatic level, compound 3c achieved 32% inhibition of CYP24A1 at 100 μM. In cellular models, it did not affect basal CYP24A1 mRNA expression at 10^-7 M, but when combined with 1,25(OH)₂D₃, it up-regulated CYP24A1 mRNA levels approximately twofold. Further LC-MS/MS analysis confirmed that the addition of 10^-7 M of compound 3c significantly slowed the metabolic clearance of 1,25(OH)₂D₃ in HEK293T cells, maintaining an average concentration of 495 pM after 24 h. In summary, compound 3c employs a multi-target synergistic mechanism of "direct antimicrobial activity-immunomodulation-biofilm penetration" to effectively overcome biofilm-mediated resistance and improve the local immune microenvironment, thus offering a promising lead compound with clinical translational potential for addressing the therapeutic challenges in mixed infections.