This paper introduces a test system for the investigation of biotin transport following inactivation of the SLC5A6 gene, which encodes the sodium-dependent multivitamin transporter SLC5A6. The aim was to develop a method for assessing the efficiency of biotin penetration across the cell membrane following inactivation of the SLC5A6 gene and to explore the feasibility of delivering biotin derivatives into cells independent of SLC5A6. The test system is built upon modified HEK293 cell lines with overexpression of the BirA* biotin ligase, with the first line comprising a functional SLC5A6 gene and the second one involving an inactivated version of this gene mimicking impaired biotin transport. This test system was used to investigate the transport of biotin and its two derivatives, namely the biotin conjugate with p-aminophenylalanine (Bio-1) and biotin methyl ester (Bio-2), through the cell membrane. It has been determined that biotin and its methyl ester (Bio-2) can enter cells independently of the SLC5A6 transporter, which points to the presence of alternative transport pathways. The biotin derivative Bio-1, which contains p-aminophenylalanine, is internalized into cells solely through the hSMVT transporter. The novel test system will serve as a tool for investigating the pathways involved in vitamin entry into cells and for developing therapeutic strategies for individuals with mutations in the SLC5A6 gene, as well as other transport-related genes.

Background: The emergence of antifungal resistance and the limited number of clinically available antifungal drug classes necessitate the development of new agents with improved efficacy and safety. We investigated how linker architecture influences the antifungal activity and lead properties of hybrid triazole-thiazolidine derivatives. Methods: A focused library of triazole-thiazolidine hybrids incorporating alkylamine, amide, cyclic amine, 2-hydroxypropyl, and thiazepane linkers was synthesized and characterized. Antifungal activity was evaluated against reference strains and clinical isolates of Candida spp., Aspergillus fumigatus, dermatophytes, and Cryptococcus neoformans. Structure–activity relationships were analyzed by molecular docking. Selected compounds were further assessed by SCRAPPY profiling, fluorescence microscopy, mammalian-cell cytotoxicity assays, acute oral toxicity studies, and evaluation of microsomal stability and interactions with human CYP450 isoforms. Results: Linker architecture strongly influenced antifungal potency. Amide- and cyclic amine-containing hybrids were generally the most active, whereas simple alkylamide derivatives showed narrower activity profiles. Compound 28 emerged as the most promising lead, exhibiting sub-microgram MIC values against several Candida isolates, particularly C. parapsilosis, and retaining measurable activity against an azole-resistant C. albicans strain. Docking generated putative CYP51-binding models, while SCRAPPY profiling and fluorescence microscopy revealed an azole-like cellular response consistent with perturbation of sterol-associated homeostasis. Compound 28 was tolerated at 300 mg/kg in an acute oral study but showed concentration- and time-dependent cytotoxicity and rapid CYP3A4-mediated microsomal metabolism. Conclusions: Systematic variation of linker architecture identified compound 28 as a promising exploratory antifungal lead. Further optimization should focus on improving metabolic stability and cytotoxicity, together with direct target validation, pharmacokinetic characterization, and in vivo efficacy studies.