Dual-functional molecule 3c targeting CYP24A1-mediated 25-hydroxyvitamin D metabolism and microbial biofilms overcomes mixed vaginal infections

Abstract

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.