In recent years, drug-resistant and multidrug-resistant fungal strains have been more frequently isolated in clinical practice. This phenomenon is responsible for difficulties in the treatment of infections. Therefore, the development of new antifungal drugs is an extremely important challenge. Combinations of selected 1,3,4-thiadiazole derivatives with amphotericin B showing strong synergic antifungal interactions are promising candidates for such formulas. In the study, microbiological, cytochemical, and molecular spectroscopy methods were used to investigate the antifungal synergy mechanisms associated with the aforementioned combinations. The present results indicate that two derivatives, i.e., C1 and NTBD, demonstrate strong synergistic interactions with AmB against some Candida species. The ATR-FTIR analysis showed that yeasts treated with the C1 + AmB and NTBD + AmB compositions, compared with those treated with single compounds, exhibited more pronounced abnormalities in the biomolecular content, suggesting that the main mechanism of the synergistic antifungal activity of the compounds is related to a disturbance in cell wall integrity. The analysis of the electron absorption and fluorescence spectra revealed that the biophysical mechanism underlying the observed synergy is associated with disaggregation of AmB molecules induced by the 1,3,4-thiadiazole derivatives. Such observations suggest the possibility of the successful application of thiadiazole derivatives combined with AmB in the therapy of fungal infections.

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.

Amphotericin B (AmB) is a potent antifungal agent with minimal resistance among clinical isolates, but its use is limited by severe side effects. Reducing its toxicity through combination therapy with synergistic compounds is a promising strategy. This study investigates the antifungal potential of 1,3,4-thiadiazole derivatives, focusing on AT2 and AT10, against Candida species. AT2 demonstrated the highest activity, achieving complete inhibition at 128 µg/mL and notable suppression at lower concentrations. The combination of AT2 and AT10 with amphotericin B exhibited synergistic effects, leading to significant structural alterations in the fungal cell wall, including reduced β-glucan levels and increased synthesis of mannan and phosphomannan. These modifications correlated with enhanced antifungal efficacy without exacerbating cytotoxicity toward human fibroblasts and renal epithelial cells. The spectroscopic analysis suggested that the synergy arose from both cell wall disruptions and amphotericin B disaggregation. These findings highlight the potential of thiadiazole-based combination therapies for combating resistant fungal infections.