Development of Novel Drugs and Drug Combinations to Treat Persistent and Drug-Resistant Fungal Infections

The incidence of fungal infections has risen sharply in recent decades due to increasing numbers of susceptible immunosuppressed persons. Prevention and treatment of these infections rely on a very limited number of antifungal drugs in only three major drug classes. Global epidemics are increasingly being caused by drug-resistant (and sometimes multi-drug resistant) fungal pathogens including Aspergillus fumigatus, Candida glabrata, Cryptococcus neoformans, and, most recently, Candida auris, a pathogen with the potential for extensive multi-drug resistance. Notably, infections with drug-resistant fungi are associated with mortality rates in excess of 50%, placing them as high priority targets for new drug development. Our goal is to pursue strategies in parallel for the identification of combination therapies by repurposing existing drugs, and the design of novel drugs, both of which effectively treat drug-tolerant, as well as resistant, fungal infections. Our main strategy is the development of fluorescent derivatives of antifungal drugs and study their effects and subcellular localization in live cells of pathogenic yeast. We focus our studies on two classes of antifungal drug that currently serve as the first line of treatment against invasive fungal infections; azoles and echinocandines. 

We recently demonstrated that targeting an antifungal drug to a particular organelle can significantly improve the therapeutic properties. Azole antifungal drugs are amongst the very few classes of antifungal drugs and are often the first line of antifungal infection therapy. We developed antifungal azoles that were designed to localize to the endoplasmic reticulum (ER), which harbors the target of the azole class of antifungals, the CYP51. The antifungal activity of these azoles against a panel of Candida pathogens was over two orders of magnitude more potent than those of the commonly used antifungal azole drug fluconazole and of antifungal azoles that were found to localize to the mitochondria, which does not harbor CYP51. The results of this study suggest that adoption of the “target-organelle directed drug” approach in general, and the design of the ER-directed antifungal azoles in particular, has the potential to markedly improve the potency of antifungal azoles.

ER localization 

Mitochondrial localization 

Echinocandins are the most recently approved of the three main classes of antifungal drugs currently used clinically for the treatment of invasive fungal infections. These drugs are increasingly used as first-line therapy for invasive fungal infections, largely because of their efficacy and low toxicity. Echinocandins inhibit the biosynthesis of the glucan component of the fungal cell-wall. We are engaged in an investigation of the mechanisms and dynamics with which echinocandins enter yeast cells and the relationship between drug uptake and antifungal activity. Using newly developed fluorescent caspofungin probes, we monitored the subcellular distribution of the drug in live pathogenic yeast cells and the relationship between the degree of intracellular drug internalization and the level of caspofungin resistance in laboratory strains and in clinical isolates.

 

We showed that echinocandins do not accumulate throughout the yeast cell cytoplasm or in its membrane. Based on images of Candida yeast cells treated with the fluorescently labeled echinocandin drug caspofungin, it accumulates in vacuoles shortly after  introduction. The uptake of the fluorescently labeled drug is facilitated by endocytosis: The fluorescent drug formed vesicles similar to fluorescently labeled endocytic vesicles, the vacuolar accumulation of fluorescent caspofungin was energy-dependent, and inhibitors of endocytosis reduced uptake. 

 

Time-dependent subcellular distribution of fluorescent caspofungin revealed, for the first time, that echinocandins cause cell lysis under conditions that promote rapid yeast cell growth, and not under conditions that lead to quiescent cell maintenance: in cells maintained in a nutrient-free buffer, the drug accumulates and remains in the vacuole and does not confer cell-damage.

 

A major and surprising finding was the discovery that, on average, the level of fluorescent caspofungin uptake into yeast cells was significantly higher in echinocandin-resistant strains than echinocandin-susceptible strains. This correlated with the content of the cell-wall polysaccharide chitin, which was high in all strains in which the uptake of the fluorescent echinocandin was high. We trust that the fluorescent echinocandin probes we developed will prove to be useful in clinical laboratories for rapid prediction of echinocandin-resistance in pathogenic yeast.

Fluorescently labeled caspofungin 

Synthetic Approaches for Reducing Auditory Cell Damage Induced by Aminoglycoside Antibiotics

 

For almost eight decades, aminoglycosides have been clinically useful antibiotics. These antibiotics are key drugs for the treatment of neonatal sepsis, a potentially fatal infection, and for treatment of cystic fibrosis patients suffering from reoccurring lung infections. Aminoglycosides act by interfering with bacterial protein synthesis by binding to the A-site decoding rRNA region of the bacterial ribosome. Unfortunately, the efficacy of aminoglycosides is overshadowed by severe and irreversible ototoxicity caused by these antibiotics. 

Ototoxicity is the tendency of a drug or chemical agent to cause inner ear dysfunction with symptoms of hearing loss and/or dizziness. Some aminoglycosides are more toxic to the cochlea, the inner ear part responsible for hearing, whereas others are more toxic to the vestibular apparatus, responsible for balance. Ototoxicity is dose-dependent, and certain patients are genetically more susceptible than others. Due to life-threatening infections and lack of suitable alternative antibiotics, it is often necessary to continue treatment with aminoglycoside antibiotics, despite their toxicity.

 

Although there has been some success in the development of novel aminoglycosides that are less prone to modifications by enzymes that confer resistance to these antibiotics, fewer efforts have been made to reduce the toxicity of these antibiotics through chemical modifications.

In search of structural modifications that significantly affect the ototoxicity of aminoglycosides, we integrate synthetic strategies and biological and in-silico evaluation strategies. The vast structural diversity among different aminoglycosides results in major differences in both their desirable and undesirable biological effects. By focusing on chemical modifications i.e., glycosylation of aminoglycosides with a ribofuranose ring or N-demethylation of natural aminoglycosides, we showed that aminoglycosides inhibit the activities of mammalian and bacterial ribosomes when tested in cell-free assays albeit with different efficacies. We established that, depending on the specific aminoglycoside, toxicity to auditory cells can stem from acute and rapid plasma membrane permeabilization. Moreover, these antibiotics are potent inhibitors of translation in cell-free extracts, in intact cells these drugs can cause an elevation in the levels of proteins, presumably, due to stress responses to these antibiotics. Importantly, we identified modifications that improve the selectivity of aminoglycosides for inhibition of bacterial translation and that can significantly reduce undesired mammalian cell permeabilization and damage.   

 

Metabolic Labeling  Strategies to Unreveal the Organization of Lipopolysacchairdes in the Outer Membrane of Gram-Negative Bacteria   

To survive, Gram-negative bacteria depend on proper assembly of a complex cell envelope structure, which comprises two lipid bilayers sandwiching a thin cell wall layer. The inner membrane (IM) encloses the cytoplasm while the outer membrane (OM) faces the extracellular environment. The OM functions as an effective permeability barrier at the cell surface, contributing to high intrinsic resistance of Gram-negative bacteria to antibiotics and detergents. A defining component of the OM lipid bilayer is lipopolysaccharides (LPS) in the outer leaflet; phospholipids (PLs) largely reside in the inner leaflet of the OM. This lipid asymmetry is required for OM function; aberrant localization of PLs on the cell surface compromises the permeability barrier.

 

Given its importance, extensive research efforts have been directed at understanding OM structure, assembly, and maintenance. How the PS and PLs, are organized in the complex OM structure is still largely unknown. This knowledge gap stemmed largely from the lack of molecular tools for direct visualization of the various components, particularly lipid molecules, at the OM.

 

In this project, we, therefore, propose to develop strategies to incorporate novel chemical functionalities into LPS and PLs, eventually allowing fluorescence imaging of lipid organization at the outer leaflet of the OM. We will exploit metabolic pathways involved in the biosynthesis of LPS and PLs to label these molecules with chemical analogs, and covalently attach fluorophores via bio-orthogonal chemistry. Our metabolic labeling approaches will enable spatial analysis of lipid organization at the bacterial cell surface using fluorescence microscopy and will provide new insights into how structure directly influences function at the OM. Given the role of the OM in intrinsic Gram-negative antibiotic resistance, our findings here will also provide a good foundation for future studies into OM permeability and will influence future antibiotics development efforts.

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Research

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