Novel carbazate derivatives impair Candidozyma auris immune evasion by β-1,3-glucan unmasking and disruption in metabolic reprogramming.

Identification of novel drug candidates against Candidozyma auris (C. auris), an emerging fungal pathogen associated with global healthcare outbreaks, is of considerable importance. A major clinical challenge in C. auris infection is its ability to evade host immune responses through distinct processes, including reduced exposure of the immunostimulatory cell-wall component β-1,3-glucan and metabolic reprogramming through the glyoxylate cycle (GC), which supports survival under low-carbon conditions. In the present study, we evaluated the antifungal efficacy of two novel carbazate derivatives, C4 [butyl 2-(4-chlorophenyl)hydrazine-1-carboxylate] and C13 [phenyl 2-(4-chlorophenyl)hydrazine-1-carboxylate], against immune-evasion-associated phenotypes in C. auris. The efficacy of C4 and C13 was investigated using a THP-1-derived macrophage infection model, which showed enhanced macrophage-mediated fungal killing, increased phagocytosis-associated fluorescence, and increased ROS production together with apoptosis-associated staining following treatment. Dectin-1-based fluorescence microscopy further indicated increased β-1,3-glucan exposure, suggesting enhanced immune visibility of treated fungal cells. The effects of C4 and C13 on isocitrate lyase (ICL) and malate synthase (MS), two key enzymes of the glyoxylate cycle, were also examined. Phenotypic susceptibility assays under low-carbon-source conditions showed impaired fungal growth in the presence of both derivatives. Crude extract-based enzymatic assays showed that C4 and C13 reduced ICL activity, with no considerable effect on MS activity under the tested conditions. Lineweaver-Burk analysis further indicated that C4 and C13 inhibited ICL through apparent competitive and non-competitive modes, respectively. Homology-based molecular docking provided supportive structural evidence for these findings, where C4 and C13 showed stronger binding affinity toward ICL, with binding energies of -9.4 and -8.6 kcal/mol and two hydrogen bonds with key active-site residues, whereas the derivatives showed comparatively lower binding affinity toward MS, with binding energies of -6.8 and -6.4 kcal/mol with no appreciable interactions. Together, these findings suggest that C4 and C13 interfere with two immune-evasion-associated phenotypes in C. auris, namely β-1,3-glucan masking and ICL-dependent glyoxylate-cycle function, highlighting these carbazate derivatives as promising candidates for further antifungal optimization.