Rationally designed indole-Thiosemicarbazides as potent Ergosterol-targeting antifungal agents against Candida spp

The increasing antifungal resistance in Candida species highlights the critical need for new medications with unique mechanisms and antibiofilm properties. In this study, indole-thiosemicarbazide compounds were designed, synthesized, and evaluated against both sensitive and resistant strains of Candida. Compounds 3b, 3d, and 3i were most potent and among them, 3d and 3i showed strong activity against C. albicans, with MICs of 2.44 μg/mL, similar to fluconazole's 2.0 μg/mL. Compound 3b had broad-spectrum activity with MICs of 4.88 μg/mL against C. albicans, C. tropicalis, and C. auris. These compounds also exhibited activity against resistant strains. Biofilm assays showed 3d inhibited ∼90% of C. albicans and 83% of C. tropicalis biofilms, and reduced resistant C. tropicalis biofilms by 60%. Compound 3b exhibited broad antibiofilm activity, achieving 77-84% inhibition across species; 3i was most effective (∼92%) against sensitive C. albicans but less effective against resistant strains. Mechanistic experiments using qRT-PCR demonstrated significant downregulation of ERG11, a key gene in the ergosterol biosynthesis pathway in cell wall formation. ERG11 transcripts decreased by roughly 70%, 84%, and 90% compared with controls in 3b, 3d, and 3i, with 3i's effect comparable to fluconazole. X-ray analysis confirmed the molecular structure. Docking and molecular dynamics demonstrated stable binding for 3b, 3d, and 3i, with the greatest stable free energy for 3i at ΔG = -51.20 kcal/mol. The combined antifungal, antibiofilm, mechanistic, structural, and computational findings identify compound 3d as the most promising, highlighting indole-thiosemicarbazides as a valuable scaffold for ergosterol-targeted antifungals effective against drug-sensitive and resistant Candida.