Inhibitors of trehalose-6-phosphate synthase activity in fungal pathogens compromise thermal tolerance pathways

Infections caused by fungal pathogens such as Candida and Cryptococcus are associated with high mortality rates, partly due to limitations in the current antifungal arsenal. This highlights the need for antifungal drug targets with novel mechanisms of action. The trehalose biosynthesis pathway is a promising antifungal drug target because trehalose biosynthesis is essential for virulence in Cryptococcus neoformans and Candida albicans and is also a mediator of fungal stress responses, such as thermotolerance. To exploit its untapped antifungal potentials, we screened the St. Jude 3-point pharmacophore library to identify small molecule inhibitors of the first enzyme in the trehalose biosynthesis pathway, trehalose-6-phosphate synthase (Tps1). Structure-guided optimization of a potent hit, SJ6675, yielded a water-soluble inhibitor named 4456dh. Employing biochemical, structural and cell-based assays, we demonstrate that 4456dh inhibits Tps1 enzymatic activity, suppresses trehalose synthesis and exerts a fungicidal effect. Notably, the structure of Tps1 in complex with 4456 reveals that 4456 occupies the substrate binding pocket. Importantly, 4456dh renders normally thermotolerant fungal pathogens unable to survive at elevated temperatures, which is critical as we investigate the emergence of fungi from the environment due to a warming climate. Overall, this work develops the water-soluble 4456dh as an early-stage antifungal drug that has a distinct mechanism of action compared to existing clinical antifungals.