Anticandidal Activity and Low Cytotoxicity of Modified Analogues of the Tobacco Defensin NaD1

<b>Background/Objectives:</b> The growing resistance development among fungi, including those of <i>Candida</i> species, poses significant challenges to public health, emphasizing the need for the implementation of innovative therapeutic approaches. The tobacco defensin NaD1 exhibits a pronounced activity against <i>C. albicans</i>, but its relatively high cytotoxicity toward mammalian cells limits its potential application. Here, we investigated anticandidal activity and cytotoxicity of four modified analogues of NaD1 (NaD1-1 T44R/K45R, NaD1-2 L38R, NaD1-3 K36R/L38R, NaD1-4 L38R/T44R/K45R). <b>Methods:</b> These peptides contained substitutions with arginine of some amino acid residues in the C-terminal region of NaD1 and in its L5 loop (S₃₅KILRR₄₀), responsible for the "cationic grip" and binding to phosphatidylinositol 4,5-bisphosphate (PIP4,5), one of the primary targets of tobacco defensin action. <b>Results:</b> We showed that the modified NaD1 analogues effectively inhibited the growth of <i>C. albicans</i> cells but had a less fungicidal action than NaD1. As compared to NaD1, its modified analogues differed in their sensitivity to the presence of various salts; antifungal activities of NaD1-3 and NaD1-4 were more tolerant to the presence of NaCl and CaCl₂, respectively. All modified analogues except NaD1-1 did not exhibit hemolytic activity and showed significantly less cytotoxicity towards human immune and epithelial cells compared to NaD1. All modified analogues enhanced the permeability of PIP4,5-containing liposomes, although less effectively than NaD1. Differences in their properties were also demonstrated through experiments on oligomerization and zymosan binding. <b>Conclusions:</b> Thus, we proposed that the modified NaD1 analogues NaD1-2, NaD1-3, and NaD1-4 appear to be promising candidate antifungals. However, further in vitro and in vivo studies are required to evaluate their therapeutic potential against critical fungal pathogens.