Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity

Abstract

Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein.The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.

Exploring the molecular structure of lipids in the design of artificial lipidated antifungal proteins

“…4). The LysM-lipids showed no detrimental effects on Hek293T cells and these results were consistent with our previous studies with LysM-C16 12,20 . The treatment of cells with 1 µM LysM-lipid, and ≤1.25 µM LysM-lipid in the presence of AMB, showed little effect on the cell viability.…”

“…Further investigations were carried out using a fusion protein of LysM and green fluorescent protein (muGFP) with a C -terminal MTG-reactive Q-tag (LysM-muGFP-Q) 13 to study how the formulation interacted with T. viride (Figs. 3 and S6).…”

“…Then, the absorbance at 450 nm was measured using a microplate reader. The following formula, where A test , A blank , and A control are the absorbances of the test, blank, and control samples, respectively, was used to determine the cell viability 13 . …”

“…In particular, a lysin motif (LysM) found in the carbohydrate-binding domains of PrChiA exhibited higher antifungal activity than the catalytic domain of PrChiA in the growth inhibition of Trichoderma viride 12 . In addition, all the palmitoylated chitinase domains tested showed minimal cytotoxicity toward mammalian cells 13 . The lipid modification of antifungal proteins and enzymes was thus found to be an effective strategy for the development of new antifungal reagents; however, the mechanism of the antifungal action of the palmitoylated LyM domain (LysM-C16) has not been fully elucidated.…”

Liposomal doxorubicin and free doxorubicin in vivo quantitation method developed on CE‐LIF and its application in pharmacokinetic analysis

“…Since the first liposome (AmBisome, amphotericin B liposome) was introduced in the 1990s [3,4], various liposomal drugs have been developed following each other. Compared with nonliposomal drugs, liposome significantly changed metabolic process of drugs in vivo, improved therapeutic effect and reduced drug toxicity [5][6][7]. The liposome drugs existed three forms in vivo, including liposome-encapsulated drugs (L-drugs), free drugs, and protein-bound drugs [8][9][10].…”