Synergistic effects of Cyp51 isozyme-specific azole antifungal agents on fungi with multiple <i>cyp51</i> isozyme genes

Pathogenic fungi pose significant societal challenges due to limited therapeutic targets resulting from the eukaryotic nature of fungi. This limitation emphasizes the importance of enhancing susceptibility to inhibitors of Cyp51, a crucial enzyme in ergosterol biosynthesis targeted by azole antifungals. In Cyp51 isozyme deletion strains (Δ<i>cyp51A</i> and Δ<i>cyp51B</i>) of <i>Trichophyton rubrum</i>, the predominant dermatophyte species, we found that Cyp51B is essential for basal mycelial growth, while Cyp51A functions as an inducible isozyme associated with azole tolerance. Based on these differential functions, we hypothesized that each isozyme would show distinct susceptibility to azole antifungals. Our study demonstrated that most azoles exhibited increased antifungal activity against Δ<i>cyp51A</i>, while select agents demonstrated increased antifungal activity against Δ<i>cyp51B</i>. Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against Δ<i>cyp51A</i>, whereas prochloraz demonstrated increased activity against Δ<i>cyp51B</i>. Combining these isozyme-selective agents exerted synergistic effects against the wild-type strain and the parent <i>ku80</i>-knockout strain but not against individual Cyp51 knockout mutants. Our data revealed that the two Cyp51 isozymes can be selectively inhibited by different azole antifungals, resulting in a synergistic effect when combined. This synergistic effect was also observed on another fungal species, <i>Aspergillus welwitschiae</i>, which also has two Cyp51 isozymes. These data demonstrate that combining azole antifungals with different Cyp51 isozyme selectivities exerts synergistic effects against fungi possessing multiple Cyp51 isozymes. These findings advance antifungal therapeutic strategies by demonstrating that the combination of antifungals with different Cyp51 isozyme selectivities offers a promising approach for treating fungal infections, opening new avenues for isozyme-specific drug development.