Polyenes are among the most commonly used broad-spectrum antifungals but have significant associated renal toxicity. Attempts to develop newer drugs of the same class without these adverse effects led to nothing, mostly because of the perception that their antifungal activity was based on membrane permeabilization.
Amphotericin B is a natural small-molecule antifungal that is essential in clinical practice but has severe renal toxicity. Today, it is known that amphotericin B kills fungal cells mostly by forming extramembrane sponges that pull out the vital ergosterol molecules from lipid bilayer membranes, cellular or subcellular.
This was the starting point of an investigation that led to the production of powerful antifungals of the polyene class without nephrotoxicity. This was achieved using compounds that rapidly remove ergosterol but do not extract cholesterol, the latter action being responsible for the kidney cell damage.
What did the study show?
In a study published in XXX, scientists examined the structure of amphotericin B in its sponge-like state. They observed that it forms a clathrate-like lattice, trapping either ergosterol or cholesterol at a highly conserved region of the polyene macrolide molecule. However, its affinity for ergosterol is higher than for cholesterol.
By using controlled destabilization, the complexes formed by the antifungal molecule with either of these “guest” molecules could be manipulated so as to avert cholesterol binding while retaining ergosterol binding capacity. The challenge was to identify the specific conformational changes in protein structure that drive this biological function so that the small molecule amphotericin can be made to interact with antifungal proteins for therapeutic purposes selectively.
This led to the synthesis of a sterol-related compound via C2’ epimerization. This modification inhibits cholesterol binding and is thus non-toxic to the renal cells even at the highest concentrations tested in human cells as well as in mice, unlike the marked injury caused by amphotericin B, even at 2 mg/kg.
This new compound is less potent than the parent because of the lower rate of ergosterol extraction, however. The extent of decreased potency varied with the fungal strain, being least for Candida krusei but largest for Aspergillus fumigatus. This indicated that other factors contributed to its potency rather than only ergosterol binding strength.
In fact, when used along with ketoconazole, an antifungal that inhibits the biosynthesis of ergosterol, this compound became as potent as amphotericin B, even against those that had been highly resistant, like A. aspergillus. This indicated the need to increase ergosterol extraction rates above the rate of biosynthesis, thus promoting fungicidal activity, but without increasing ergosterol binding affinity which drives nephrotoxicity.
To achieve this, another structural modification was introduced whereby the C16 position was converted into a non-ionizable functional group. This resulted in a new polyene compound that extracts ergosterol at rapid rates but does not damage renal cells in a mouse animal model, as well as in human cell lines. This compound, termed AM-2-19, has activity against hundreds of pathogenic fungi.
“It is notable that both of these chemical modifications are positioned on the portion of [amphotericin B] that is conserved in all of the glycosylated polyene macrolide natural products that operate through the same sterol sponge mechanism.”
Serial passaging of fungi promotes the development of spontaneous resistance-inducing mutations. However, the compound remains active in this situation.
In animal models, invasive fungal infections were treated effectively using this compound. There was no adverse effect observed on the genome, no drug-drug interactions, and no renal toxicity at up to 45 mg/kg. Candida infection was controlled proportionate to the dose of this compound and completely eradicated at dosage levels of 10 mg/kg or more in neutrophil-deficient mice vs. 0.3 mg/kg dosage in mice with normal immunologic function.
At higher doses, disseminated or pulmonary infection with A. fumigatus, and mouse mucormycosis was also reduced in a dose-dependent fashion, though eradication occurred in only a few cases. Survival rates increased in the latter infection.
What are the implications?
Advanced understanding of the mechanism by which polyene macrolide natural products kill fungal and human cells enabled the selective tuning of sterol extraction kinetics to yield a new polyene antifungal that is both renal sparing and highly potent in clinically relevant animal models of invasive fungal infections.”
This lays the foundation for a reason-based tuning of why and how small molecules interact with other potential therapeutic targets, resulting in better antifungal treatment. This could also promote the development of other antimicrobial drugs capable of overcoming resistance, including those that act via lipid targets.