A First-in-Class Pyrazole-isoxazole Enhanced Antifungal Activity of Voriconazole: Synergy Studies in an Azole-Resistant Candida albicansStrain, Computational Investigation and in Vivo Validation in a Galleria mellonellaFungal Infection Model

The widespread and irrational use of azole antifungal agents has led to an increase of azole-resistant Candida albicansstrains with an urgent need for combination drug therapy, enhancing the treatment efficacy. Here, we report the discovery of a first-in-class pyrazole-isoxazole, namely, 5b, that showed remarkable growth inhibition against the C. albicansATCC 10231 strain in combination with voriconazole, acting as a downregulator of ERG 11 (Cyp51) gene expression with a significant reduction of the yeast-to-hypha morphological transition. Furthermore, C. albicansCYP51 enzyme assay and in-depth molecular docking studies unveiled the unique ability of the combination of 5band voriconazole to completely fill the CYP51 binding sites. In vivostudies using a Galleria mellonellamodel confirmed the previously in vitroobserved synergistic effect of 5bwith voriconazole. Also considering its biocompatibility in a cellular model of human keratinocytes, these results indicate that 5brepresents a promising compound for a further optimization campaign.