Cholesterol-terminated cationic lipidated oligomers (CLOs) as a new class of antifungals†

Infections caused by fungal pathogens are a global health problem, and have created an urgent need for new antimicrobial strategies. This report details the synthesis of lipidated 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers via an optimized Cu(0)-mediated reversible-deactivation radical polymerization (RDRP) approach. Cholesterol-Br was used as an initiator to synthesize a library of oligo-VDM (degree of polymerisation = 5, 10, 15, 20, and 25), with an α-terminal cholesterol group. Subsequent ring-opening of the pendant oxazolone group with various functional amines [i.e., 2-(2-aminoethyl)-1,3-di-Boc-guanidine (BG), 1-(3-aminopropyl)imidazole (IMID), N-Boc-ethylenediamine (BEDA), or N,N-dimethylethylenediamine (DMEN)] yielded an 11 functional cationic lipidated oligomer (CLOs) library, which comprised different cationic elements with the same terminal lipid cholesterol element. These CLOs exhibited greater activity against all tested fungal pathogens (Candida albicans, Cryptococcus neoformans, Candida tropicalis, Candida glabrata, Cryptococcus deuterogattii, and Candida auris), compared to the bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus [MRSA]). Specifically, the DMEN and BEDA (after deprotection) series exhibited superior antifungal activities 4–16 times greater [determined by the minimum inhibitory concentration (MIC) in μg mL⁻¹] than the clinically relevant antifungal fluconazole. Two ‘hit’ CLOs (Chol-DMEN-25 and Chol-BEDA-10) were identified, which inhibited both single sp. (C. albicans, C. tropicalis, C. neoformans, or C. gattii) and dual sp. (C. albicans and C. tropicalis) biofilm formation, and were able to attenuate mature biofilms, with a >50% mature biofilm biomass reduction at 128 μg mL⁻¹. Co-delivery of fluconazole with two ‘hit’ CLOs demonstrated additive and synergistic effects on the aforementioned single-species and dual-species fungi biofilms, with a synergy score (SS) ranging from ∼3 to 15 and most synergistic area score (MSAS) ∼13–29 (by a Bliss independence model). The mechanistic studies (PI assay and nucleic acid release assay) revealed that these CLOs disrupted the integrity of fungal cell membranes. These results demonstrate that cholesterol terminated CLOs are potential antifungal candidates.