Fungal infections are becoming an increasingly difficult clinical and global threat, driven in part by antimicrobial resistance and challenges in developing new antifungal treatments. Polyenes, a powerful class of antifungal drugs that includes amphotericin B and nystatin A1, remain among the most effective options for some life-threatening infections. But their usefulness is limited by significant toxicity and poor solubility, because fungal and human cells share features that make it difficult to kill pathogens without harming healthy tissue.
Now, researchers at Imperial College London and The University of Manchester say they have used genome mining and an enzyme-based approach to reshape polyene bioactivity, generating derivatives that showed improved antifungal activity and reduced toxicity in preclinical testing.
The work, described in “Enzymatic glycosylation and amidation reshapes polyene bioactivity,” began with a search for pathways capable of producing previously undescribed polyenes. The researchers then characterized enzymes involved in making and modifying the compounds, including glycosyltransferases that add sugar groups and an amidotransferase that can alter a carboxylate substituent associated with unfavorable properties to reduce toxicity while increasing potency.
“The most effective antifungal agent currently available is a polyene molecule called amphotericin produced by soil bacteria,” says first author Saadia Nasr Mirza, PhD, a postdoctoral research associate at The University of Manchester. “Although amphotericin is very potent, it is highly toxic, so we set out to discover if bacteria can produce different types of polyenes that are safer than amphotericin. We developed a bioinformatics pipeline, which surprisingly showed that many bacterial species have the capability to produce novel polyenes.”
The team used nuclear magnetic resonance (NMR) to determine the structures of recently discovered polyenes and built a library of derivatives for testing. “The addition of a second sugar, combined with carboxylate modification, leads to new polyene derivatives with increased antifungal activity, lower toxicity and higher solubility than the parent polyenes used at present in the clinic,” write the authors.
The researchers identified Nys34 as a leading candidate after it demonstrated three-to-eightfold lower toxicity than amphotericin B in multiple human cell lines and reduced fungal burden in a mouse model of invasive aspergillosis. While mice tolerated three repeat doses of the compound, toxicity was observed after a fourth dose, underscoring the need for further optimization and testing.
Senior author Jason Micklefield, PhD, professor in the department of chemistry at Imperial College London, says the team was encouraged by the activity of the derivatives. “We were pleased to find that several of the new polyene derivatives were more potent and less toxic than amphotericin and nystatin, which is another important polyene that is also used in the clinic,” he said. “The key advance was the use of these methods to find biosynthetic gene clusters that produce polyenes with multiple sugars, which can have a profound effect on bioactivity. Most of the existing polyenes have just one sugar moiety.”
The researchers also reported that Nys34 appeared to act differently from amphotericin, a finding that could be important if confirmed in further studies. “We were particularly surprised that one of polyene compounds we developed, Nys34, has a different mechanism of action to the existing polyene drugs,” Micklefield told GEN. “Polyenes are thought to target components of the cell membrane rather than a specific enzyme like most antimicrobial drugs. This means that a simple mutation in one enzyme cannot confer resistance, but rather the makeup of the cell membrane must be altered (or the drug degraded by an enzyme). The fact that Nys34 acts via a different mechanism to amphotericin is a promising sign that it can evade resistance.”
Beyond the individual compounds, the study highlights an alternative approach to improving polyene drugs. Because polyenes are structurally complex, modifying them through conventional chemistry can require “many steps, extensive use of protecting groups, and deleterious reagents,” write the authors. The authors point to a recently reported amphotericin B derivative with reduced toxicity that required 12 chemical steps with a 0.7% overall product yield, illustrating the challenges of optimizing these molecules through traditional synthesis.
“In future work, we aim to explore Nys34’s mechanism of action. Also, the methods we have developed here are broadly applicable across a wide variety of polyene scaffolds and substrates (e.g. different sugars). We are currently exploring more combinations of these modifications to identify additional promising drug candidates,” added Micklefield.
By contrast, this new approach could allow researchers to generate modified polyenes through fermentation or enzymatic routes. The team suggests that such an approach may be more scalable and cost-effective than conventional multistep synthesis, though additional work will be needed to determine whether the platform can support development and manufacturing beyond the laboratory.
