Targeting fungal drug resistance: Mogen scientists advance antifungal therapy with structure-guided drug design

The Cowen Lab advances antifungal therapy with structure-guided drug design

Fungal infections caused by Candida albicans remain a major clinical challenge, especially as drug resistance continues to rise and treatment options remain limited.

A recent publication by researchers at the Cowen Lab in the Department of Molecular Genetics at the University of Toronto, including first author Dr. Emily Puumala, PhD graduate from the Cowen lab, charts a promising path forward through structure-guided drug optimization.

Published in Nature Communications early this year, the study demonstrates a multidisciplinary effort to develop and screen new antifungal compounds that selectively target a fungal protein kinase called Yck2, which is necessary for fungal growth, virulence, and drug resistance.

Why Yck2?

The Cowen Lab has long focused on antifungal resistance, a growing concern according to the World Health Organization’s Fungal Priority Pathogens list, a global effort to drive research, development and policy interventions to combat fungal infections and antifungal resistance.

In this study, the team focuses on Yck2, a fungal protein kinase that enables C. albicans to survive under stress and resist frontline treatments, such as Caspofungin, a widely used echinocandin antifungal.

Yck2 belongs to the casein kinase 1 family, a class of enzymes conserved across species but the main differences between the fungal and human versions of this kinase offered an opportunity.

“We were building on foundational work from a previous graduate student in the lab, who had identified a molecule that inhibited Yck2 in Candida albicans and showed promising antifungal activity,” explained Dr. Puumala. “That initial molecule had low toxicity to human cells in vitro, which made Yck2 a compelling target. Our goal was to optimize that scaffold and generate new compounds with improved properties that could be tested in a model of disease.”

From Hits to Lead

The study builds on earlier work by former Cowen Lab graduate student Tavia Kaplan, who identified GW461484A (GW), a small molecule that inhibits Yck2 but lacked the metabolic stability for use in animal models.

“This was a really exciting starting point,” said Dr. Nicole Robbins, Senior Research Associate at the Cowen lab and a co-author on the publication. She noted that while the compound showed strong biochemical activity, small structural changes often led to a complete loss of antifungal function, indicating how difficult it is to get molecules into fungal cells and retain their potency.

Working closely with chemists at the University of North Carolina Chapel Hill, pharmacologists at the University of Texas, Southwestern, and microbiologists and crystallographers across several institutions, Dr. Puumala took the lead on the efforts to optimize GW.

The team synthesized and screened various chemical analogues, modifying key parts of the molecule to their improve drug-like properties, taking various factors into account such as target selectivity, cell entry, and metabolic stability.

Among the most promising small molecules were two new compounds, 1e and 2a. Forming new stabilizing contacts that helped explain their improved activity and stability, structural studies using crystallography revealed how 1e and 2a interacted with Yck2 at the atomic level similarly to GW.  However, 1e and 2a featured subtle but important chemical changes; replacing a methyl group with a cyano group, known for increasing metabolic stability, at a specific site significantly improved their metabolic stability while preserving, and in some cases enhancing, their antifungal potency.

Selectivity and Potency

Importantly, these modified molecules, 1e and 2a were selective for the fungal Yck2 over the human kinase, which reduces the risk of side effects. These molecules also preserved the ability to work synergistically with Caspofungin, enhancing its efficacy in mice models, making them promising leads for potential therapeutic use against echinocandin-resistant C. albicans strains.

The compounds showed strong antifungal activity without harming host cells in co-culture assays (experiments where human cells and the C. albicans are grown together to mimic infection conditions) with human liver and immune cells. Another exciting outcome came from mouse models of invasive candidiasis. After oral administration, compounds 1e and 2a achieved therapeutically relevant concentrations in the bloodstream and tissues, including the brain, a notoriously difficult site for antifungal delivery due to the blood-brain barrier. Remarkably, in mice infected with drug-resistant C. albicans, these compounds significantly reduced fungal burden, especially in combination with Caspofungin.

A Team Effort

Behind these results lies an impressive feat of cross-disciplinary coordination. “Collaboration is how the best and most exciting science gets done,” said Robbins. “This was not a study we could have done in isolation, and we were just so thrilled to bring on board these fantastic collaborators to help us with all of the aspects of the project.” Led by the Cowen lab, this study required coordination across chemistry, structural biology, pharmacology, microbiology, and animal modeling, all different languages of science.

Puumala took the lead not only on at the bench for the lab experiments but also on the complex project management required to keep collaborations at this scale running smoothly across all groups.

“It was a huge learning experience,” Puumala reflected. “From setting agendas to coordinating reagent shipments and aligning data across teams, I had to grow as a communicator and leader.”

This coordination has more than paid off.

A Lasting Legacy

After completing her PhD in the Cowen Lab, Dr. Emily Puumala has moved on to a clinical microbiology fellowship at the Mayo Clinic, where she continues to work at the intersection of diagnostics and infectious disease. Puumala’s impact on the Yck2 project is still felt deeply in Toronto, where she led the work from both the bench and behind the scenes, running experiments, coordinating collaborators, and keeping the team moving forward.

The Cowen lab misses Puumala and wishes her the best.

What Comes Next

Looking ahead, the Cowen lab is now working to refine the current chemical scaffold, explore new compound classes, and investigate how Yck2 mechanistically governs drug resistance in C. albicans at the molecular level. With co-author and current graduate student Bonnie Yiu leading efforts to identify downstream targets of Yck2, Robbins shared that the team is also pursuing a deeper understanding of Yck2’s interactors, that may be involved in resistance and stress responses. With promising proof-of-concept results from this study, the Cowen Lab’s work continues to push forward our understanding of fungal biology and drug discovery, at a time when fungal infections pose a growing threat worldwide.

This work was supported by the National Institutes of Health (NIH), the Canada Research Chairs Program, CIFAR, the Structural Genomics Consortium, UT Southwestern Medical Center, the Canadian Light Source, and the University of Toronto.