Discovery and Characterization of a Potent Antifungal Peptide through One-Bead, One-Compound Combinatorial Library Screening

Abstract

This work describes the discovery of a bead-bound membrane-active peptide (MAP), LBF127, that selectively binds fungal giant unilamellar vesicles (GUVs) over mammalian GUVs. LBF127 was re-synthesized in solution form and demonstrated to have antifungal activity with limited hemolytic activity and cytotoxicity against mammalian cells. Through systematic structure–activity relationship studies, including N- and C-terminal truncation, alanine-walk, and d-amino acid substitution, an optimized peptide, K-oLBF127, with higher potency, less hemolytic activity, and cytotoxicity emerged. Compared to the parent peptide, K-oLBF127 is shorter by three amino acids and has a lysine at the N-terminus to confer an additional positive charge. K-oLBF127 was found to have improved selectivity toward the fungal membrane over mammalian membranes by 2-fold compared to LBF127. Further characterizations revealed that, while K-oLBF127 exhibits a spectrum of antifungal activity similar to that of the original peptide, it has lower hemolytic activity and cytotoxicity against mammalian cells. Mice infected with Cryptococcus neoformans and treated with K-oLBF127 (16 mg/kg) for 48 h had significantly lower lung fungal burden compared to untreated animals, consistent with K-oLBF127 being active in vivo. Our study demonstrates the success of the one-bead, one-compound high-throughput strategy and sequential screening at identifying MAPs with strong antifungal activities.

Osmotic Pressure Enables High-Yield Assembly of Giant Vesicles in Solutions of Physiological Ionic Strengths

“…Giant unilamellar vesicles, GUVs, vesicles with a single bimolecular wall of phospholipids with diameters ≥1 μm, mimic the minimal configuration of biological cells. , GUVs are useful for applications in bottom-up synthetic biology and biomedicine. − Assembling GUVs in solutions with ionic concentrations ∼100–150 mM Na/KCl (salty solutions) using thin-film hydration, however, is difficult. ,, This limitation presents a longstanding challenge for the use of GUVs in applications since biomolecules such as proteins, nucleic acids, and polysaccharides require salty solutions to function. − …”

Osmotic pressure enables high yield assembly of giant vesicles in solutions of physiological ionic strengths

“…Giant unilamellar vesicles, GUVs, vesicles with a single bimolecular wall of phospholipids with diameters ≥ 1 µm, mimic the minimal configuration of biological cells 1,2 . GUVs are useful for bottom-up synthetic biology and biomedical applications [3][4][5][6][7][8][9][10][11][12] . Conventionally it has been challenging to obtain GUVs in solutions with ionic concentrations ~100 -150 mM Na/KCl (salty solutions) using thin-film hydration 1,13,14 .…”

His-Ala-Phe-Lys peptide from Burkholderia arboris possesses antifungal activity

“…Several mechanisms through which antifungal peptides inhibit fungal growth have been elucidated, which include the blocking of the biosynthesis of chitin and b-glucan, two major fungal cell wall components ( Donzelli et al, 2001 ; Pushpanathan et al, 2012 ; Moghaddam et al, 2017 ; Tong et al, 2020 ). Additionally, some antifungal peptides can disrupt the fungal membrane, while others can pass through cell membranes to target intracellular DNA and RNA ( Han et al, 2019 ; Khani et al, 2020 ; Bansal et al, 2022 ; Ding et al, 2022 ). Moreover, accumulating evidence indicates that short peptides containing 2–10 amino acids also exhibit antifungal activity ( Velivelli et al, 2018 ; Gong et al, 2022 ).…”