Evelyne Deplazes

[This corrects the article DOI: 10.1021/acsmedchemlett.6c00070.].

Amphotericin B (AmB) is a polyene antifungal that, despite its severe dose-limiting toxicity, is the most effective drug to treating invasive fungal infections. The drug’s toxicity arises from its poor selectivity for ergosterol over cholesterol and its tendency to aggregate. Understanding the relationship between toxicity and aggregation is challenging due to the drug’s complex mechanism of action, yet imperative to improve the safety profiles of existing and new polyene-based antifungal drugs. In this review, we critically examine and summarise studies that investigate the aggregation of AmB and its relationship to toxicity. As the aggregation behaviour of AmB is highly sensitive to environmental conditions, we provide an in-depth analysis of sample preparation, characterisation of aggregates and control experiments. While data from haemolysis, animal, and cell-based studies consistently show that oligomeric AmB is more toxic than larger aggregates, findings on the toxicity of monomeric AmB inconsistent. Our collated data underscores the need for careful consideration of sample preparation and proper use of vehicle controls when studying the complex relationship between the supramolecular organisation of AmB and its biological effects.

The fungal cell envelope, consisting of the cell wall and plasma membrane, is a dynamic structure crucial for cell shape, viability, pathogenicity, and the cell’s ability to interact with and respond to its environment. Most antifungal drug development target components of the fungal plasma membrane and cell wall, thus understanding its composition and interactions with small molecules is vital for biomedical research and drug development. However, studying cell walls and membranes is challenging due to their high degree of complexity, their heterogeneous and dynamic structure and their sensitivity to environmental conditions. Our review provides a unique exploration of how biophysical techniques have advanced our understanding of the cell envelope’s structure, its role in fungal pathogenicity, and drug resistance, which are critical issues for global health and food security. We highlight recent advances in microscopy and spectroscopy approaches, combined with analytical techniques and lipidomics, that have enabled detailed study of fungal cell walls and plasma membranes at unprecedented spatial and temporal resolutions. These studies have helped provide structural models of fungal cell walls and plasma membranes, including important differences between clinically relevant fungal species that are critical for antifungal drug development. Our review also summarises commonly used model membranes systems and discusses challenges and considerations in bridging gaps between simplified models and cellular systems, and why they are lacking compared to bacterial and mammalian systems and what is required to improve these systems.

Amphotericin B (AmB) is a potent, life-saving antifungal, but its clinical use is limited by toxicity due to poor selectivity for ergosterol over cholesterol. The AmB derivative Am-2-19 (turletricin) exhibits improved ergosterol selectivity and reduced toxicity, which is attributed to enhanced ergosterol extraction kinetics via C16-amidation rather than altered sterol binding affinity. Using all-atom molecular dynamics simulations, we reveal key differences in the aggregate morphologies of AmB and Am-2-19 that provide mechanistic insights into the increased ergosterol extraction kinetics. In both the absence and presence of sterols, Am-2-19 aggregates slower than AmB, forming less dense and more solvated aggregates, driven by the hydrogen-bonding capacity of the C16-amidation. In vivo, these looser aggregates likely enable a greater exchange of sterols, increasing the likelihood of cholesterol-ergosterol exchange. Our findings provide the first atomistic-level insight into Am-2-19's mechanism, emphasizing the critical role of aggregation in structure-activity relationships in a physiologically relevant solution environment.

ABC toxins are toxin-translocating, pore-forming proteins found in a wide range of insecticidal bacteria and some mammalian pathogens. The Yersinia entomopahaga toxin complex (YenTc) belongs to a distinct subclass of ABC toxins, defined by a divergent molecular architecture. Structural details that define their mechanism of action remain to be elucidated. Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM in conjunction with Alphafold2-assisted structural modelling of flexible domains. We define the structural mechanism via which enzymatically-active chitinase subunits are incorporated, and show using phylogenetic analyses that this subclass-defining feature has evolved relatively recently. Our structures point to the existence of distinct conformational states in YenTc, which may distinguish it from other structurally-characterised ABC toxins, or represent states on a shared mechanistic trajectory. Thus, our findings enhance our understanding of the structural diversity that defines distinct ABC toxin subclasses.

Metabolic changes during cancer progression produce key differences in phospholipid distributions among normal (RWPE-1), benign hyperplasia (BPH-1), and metastatic prostate (lymph: LNCaP, bone: PC-3, and brain: DU145) cell lines that are markers of disease progression and cancer cell adaptation (Young et al., 2021). The effect of cancer-associated lipidome shifts on membrane structural dynamics has not been investigated in a cell-specific context. Here, we spatially map the specific membrane lipidome from each cell line data set to assess membrane organization and dynamics in normal, benign hyperplasia, and metastatic prostate cell lines. Long time scale molecular dynamics simulations show leaflet-specific alterations in membrane dynamics, with increased fluidity in the intracellular leaflet of metastatic membranes. All membranes exhibited phase-separated domains enriched in polyunsaturated lipids and depleted in cholesterol; however, the extent of phase separation varied with disease state. Specifically, the loss of phosphatidylserine lipid leaflet asymmetry in metastatic cell membranes decreased the level of phospholipid phase separation. In particular, the metastatic LNCaP lymph node cell line showed exaggerated phase separation for distinct phospholipid species compared to all other metastatic and noncancerous lines. These alterations in lipid colocalization and phase separation patterns across prostate cancer cell membrane lines suggest environment-specific adaptations that may play a role in cancer progression.

Steroids are organic compounds found in all forms of biological life. Besides their structural roles in cell membranes, steroids act as signalling molecules in various physiological processes and are used to treat inflammatory conditions. It has been hypothesised that in addition to their well-characterised genomic and non-genomic pathways, steroids exert their biological or pharmacological activities via an indirect, nonreceptor-mediated membrane mechanism caused by steroid-induced changes to the physicochemical properties of cell membranes. While the effect of cholesterol on phospholipid bilayer properties has been extensively studied, much less is known about the effect of other steroids and steroid-like molecules. Here, we combine electrical impedance spectroscopy (EIS) experiments with molecular dynamics (MD) simulations to study the effect of the steroids cortisone, prednisolone and progesterone and the steroid-like compounds enoxolone and carbenoxolone on the ion permeability and structure of phospholipid bilayers composed of the zwitterionic lipid POPC. The EIS data shows that all five compounds increase permeability, while the simulations suggest that this is accompanied by a thinning of the bilayer and reduced lipid order. We show that for steroids, a previously proposed structure–activity relationship that classifies steroids into order-promoting or order-disrupting compounds based on domain formations translates to ion permeability. We confirmed this by additional experiments with cholesterol and 7-ketocholesterol. In contrast, the previously reported relationship between log P and molecular area and a steroid being a promoter does not translate to the steroid-like compounds enoxolone and carbenoxolone. We propose that their membrane-disruption activity can be explained by their hydrogen-bonding capacity that dictates the compound's orientation at the water–lipid interface. Specifically, their membrane-disrupting ability is a result of the steroids to intercalate between lipids and form stable interactions with lipid headgroups and interfacial water, thereby pushing lipids apart and lowering the energy required for ion-induced pores, an effect previously reported for other membrane-altering small molecules.

In biological systems, enzymes and transport proteins can bind anions in aqueous media solely by forming hydrogen bonds with charge-neutral motifs. Reproducing this functionality in synthetic systems presents challenges and incurs high costs, particularly when targeting strongly hydrated anions such as sulfate. Here we report a [2.2.2]urea cryptand (cage), synthesized in one pot, that selectively binds sulfate in a mixture of dimethyl sulfoxide and water and in water with affinities in the micromolar to millimolar range. The neutral cage bearing six urea groups donates 12 strong hydrogen bonds to encapsulate a sulfate anion, showing favourable enthalpy even in pure water. Sulfate binding can be further enhanced by using micelles to provide a low-polarity microenvironment. The cage finds utility in analysing divalent anions in water and beverage samples or in removing sulfate. The work demonstrates the achievability of robust and selective anion binding in water with minimal synthetic efforts, by using neutral NH hydrogen bonds akin to those found in biology.

Haemophilus influenzae is a commensal of the human upper respiratory tract that can infect diverse host niches due, at least in part, to its ability to withstand both endogenous and host-mediated oxidative stresses. Here, we show that hfeA, a gene previously linked to iron import, is essential for H. influenzae manganese recruitment via the HfeBCD transporter. Structural analyses show that metal binding in HfeA uses a unique mechanism that involves substantial rotation of the C-terminal lobe of the protein. Disruption of hfeA reduced H. influenzae manganese acquisition and was associated with decreased growth under aerobic conditions, impaired manganese-superoxide dismutase activity, reduced survival in macrophages, and changes in biofilm production in the presence of superoxide. Collectively, this work shows that HfeA contributes to H. influenzae manganese acquisition and virulence attributes. High conservation of the hfeABCD permease in Haemophilus species suggests that it may serve similar roles in other pathogenic Pasteurellaceae.