Mechanisms of action and resistance of FDA-approved antifungals targeting fungal beta-(1,3)-glucan synthase

The National Institute of Allergy and Infectious Diseases, part of the Department of Health and Human Services, awarded Duke University $824,451 on June 15, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to investigate the mechanisms of action and resistance of FDA-approved antifungal drugs targeting fungal beta-(1,3)-glucan synthase.

The research focuses on echinocandins and ibrexafungerp, first-line therapies for invasive Candida infections that have begun showing emerging resistance despite their clinical importance. The project addresses a key gap: while both drug classes are known to target the glucan synthase enzyme complex—composed of the catalytic subunit FKS and regulatory subunit RHO1—their precise binding sites and inhibition mechanisms remain unclear. The recipient has overcome technical barriers in preparing functional glucan synthase components and has already resolved a high-resolution cryo-EM structure of yeast FKS1 in complex with caspofungin, nascent glucan, and RHO1. The four research aims extend this work to elucidate mechanisms of inhibition and resistance for caspofungin, other FDA-approved echinocandins, and ibrexafungerp, and to clarify the molecular mechanisms of glucan synthase catalysis and regulation.

Place of performance is Durham, North Carolina. The period of performance runs through May 31, 2031.

Abstract

Fungal diseases affect over a billion people globally and cause 2.5 million deaths each year. In the united states, candida species are the second leading cause of hospital-acquired bloodstream infections, posing a serious risk to immunocompromised patients. Echinocandins and ibrexafungerp (IBX), the latest FDA- approved antifungal classes, serve as first-line therapies for invasive candida infections due to their potency and low toxicity. However, resistance has begun to emerge because of the widespread use of echinocandins. Despite the clinical significance, their molecular mechanisms of action and resistance remain poorly understood. Echinocandins and IBX target the Β-1,3-D-glucan synthase (GS), an integral membrane enzyme complex essential for fungal cell wall synthesis. GS is composed of the catalytic subunit FKS and the regulatory subunit RHO1. While both drug classes have been proposed to bind FKS, there is currently no structure of FKS in complex with any of these antifungals. Therefore, their precise binding sites and mechanisms of inhibition remain unclear. We have overcome the key technical barrier in preparing functional and homogeneous FKS and RHO1 and successfully solved a high-resolution cryo-EM structure of saccharomyces cerevisiae FKS1 (SCFKS1) in complex with caspofungin (CFN), nascent glucan, and RHO1, revealing many unexpected insights into the mechanisms of GS function and inhibition. We also developed a series of functional assays and computational methods. Based on these developments, we propose four research aims to elucidate the mechanisms of action and resistance of echinocandins and IBX, as well as the mechanisms of catalysis and regulation of GS at the molecular level. Aim 1 focuses on the molecular mechanism of GS inhibition by and resistance against CFN. Aim 2 extends the studies to other FDA- approved echinocandins. Aim 3 studies the mechanism of action of IBX and its activity against echinocandin-resistant FKS1. Aim 4 explores the mechanism of FKS1 catalysis and its regulation by RHO1. To achieve these aims, we will integrate structural biology, enzymology, chemical biology, microbiology, and computational biology. These studies will provide the first atomic-level insights into how echinocandins and IBX inhibit GS and uncover their molecular mechanisms of resistance. These findings will establish a critical foundation for the future rational development of antifungals targeting GS while significantly enhancing our understanding of fungal cell wall biosynthesis and processive membrane-bound glycosyltransferases in general.