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

## TL;DR

Scientists modified a plant protein to make it more effective against Candida fungi while reducing harm to human cells.

## Contribution

Modified analogues of NaD1 show antifungal activity with reduced cytotoxicity compared to the original protein.

## Key findings

- Modified analogues inhibited C. albicans growth but were less fungicidal than NaD1.
- NaD1-3 and NaD1-4 showed tolerance to NaCl and CaCl2, respectively.
- Modified analogues had lower hemolytic activity and cytotoxicity to human cells.

## Abstract

Background/Objectives: The growing resistance development among fungi, including those of Candida 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 C. albicans, 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). Methods: 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. Results: We showed that the modified NaD1 analogues effectively inhibited the growth of C. albicans 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. Conclusions: 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.