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

Abstract

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.IMPORTANCEThe rise of fungal infections in recent years is alarming due to an increase in the vulnerable immunocompromised population, global temperature increase, and limited antifungal treatment options. One of the major hurdles in developing new drugs is the identification of fungal-specific antifungal drug targets due to highly conserved cellular machinery between fungi and humans. Here, we describe a small molecule inhibitor, 4456dh, of the trehalose biosynthesis pathway. This pathway is present in fungi but not in humans. Trehalose plays a critical role in stress responses such as thermotolerance in fungal pathogens and is essential for their virulence. We show that treatment with 4456dh blocks the production of trehalose and renders fungal cells inviable. Thus far, 4456dh is active against two fungal pathogens of critical importance, suggesting broad-spectrum activity.

Keywords: Candida; Cryptococcus; Tps1; antifungal; trehalose.

Fig 1

Seven Tps1 inhibitors were identified using a high-throughput FP screen. (A) Diagram of the Transcreener assay for Tps1 activity. (B) Coomassie-stained SDS-PAGE of purified recombinant 6xHis-CaTps1. Molecular weight markers are shown with the 50 kDa protein standard highlighted with an arrow. (C) Pipeline and results from the Tps1 UDP Transcreener assay. (D) Dot plot of % inhibition and compounds that were tested for inhibition of 6xHis-CaTps1 activity. The 32 compounds above 10% inhibition at 500 µM are highlighted. Highlighted in green are six of the compounds that resulted in a dose-dependent inhibition of 10% or greater of 6xHis-CaTps1 activity. In blue and highlighted with an arrow is SJ6675. (E) The chemical structures and identities of validated 6xHis-CaTps1-inhibiting hit compounds. The compound SJ6675 used in additional studies is boxed.

Fig 2

4456dh binds Tps1 and inhibits Tps1 activity. (A) Structure of 4456dh. (B and C) Glo-melt thermal shift assays reveal 4456dh destabilizes 6xHis-CaTps1 (B) and 6xHis-CnTps1 (C). The first derivative of the raw data was used to determine the melting temperature of 1 µM 6xHis-CaTps1 (B) and 1 µM 6xHis-CnTps1 (C) treated without (black) or with 2 mM 4456dh (red). (D and E) Microscale thermophoresis (MST) assays demonstrate 4456dh binds to 6xHis-CaTps1 (D) and 6xHis-CnTps1 (E) with the indicated K_d. The top panel shows a representative of the MST trace with the cold region labeled highlighted in blue and the hot region highlighted in red. The bottom panel shows the dose-response curve. The resulting dose-response curves were fitted to a one-site binding model to extract K_d values; the SD was calculated using the K_d values from each independent experiment (n = 3). (F) Tps1 enzyme activity assay with 5 µM 6xHis-CaTps1 (F) and 6xHis-CnTps1 (G) ±1 mM 4456dh. Error bars represent SE. Comparison to control, untreated 6xHis-CaTps1, was performed using Student’s t-test; **P < 0.01 and ***P < 0.001.

Fig 3

Structure of C. albicans Tps1 in complex with 4456. (A) One subunit of the CaTps1-4456 complex shown as a ribbon diagram. (B) Surface-rendered structure revealing 4456, shown as atomic-colored sticks, binds in the deep cavity of the substrate-binding pocket proximal to known substrate-binding residues. (C) Tps1 residues within 4 Å of 4456 are labeled, shown as stick representations and indigo colored. (D) Residues within 4 Å of 4456 are proximal to residues that also interact with product and substrate, UDP and G6P (atom-colored sticks), in the CaTps1-UDP-G6P complex (PDB: 5HUU) (29).

Fig 4

4456dh inhibits the growth of Candida and Cryptococcus species. Two-fold dose response assays were performed against (A) C. albicans at 42°C and C. auris at 39°C, (B) C. glabrata at 42°C, and (C) C. neoformans, C. deneoformans, and C. gattii at 37°C. 4456dh was administered at the concentrations indicated. Cultures were grown in YPD at the listed temperatures for 48 hours for Candida species and 72 hours for Cryptococcus species. Experimental temperatures reflect the temperatures at which the fungi experience heat stress. Left: relative growth of treated samples was calculated by averaging technical triplicates and normalizing the OD₆₀₀ of each treated well to the average OD₆₀₀ of the untreated WT controls, after the absorbance due to the compound was subtracted. The MIC₈₀ values, calculated based on the OD₆₀₀ values, are indicated in the white text on each heat map. Right: representative spot assays show the ability of cells to grow after exposure to 4456dh and indicate fungicidal activity of 4456dh.

Fig 5

4456dh treatment results in reduced trehalose accumulation and a decrease in Tps1-dependent virulence factors. Trehalose accumulation measurements for C. albicans (A) and C. neoformans (B) cultured at indicated temperatures in the absence or presence of 0.5 mM 4456dh. Trehalose was measured using a colorimetric glucose assay after treatment of cultures with trehalase. Statistical comparisons were done with a one-way analysis of variance (ANOVA) analysis followed by a post hoc Tukey’s test. Error bars represent SE. (C) C. albicans WT strain SC5314 cells were treated with 0.5 mM, 1.0 mM, and 5 mM 4456dh. Untreated C. albicans WT cells and the C. albicans tps1Δ/tps1Δ deletion strain were included in this experiment as positive and negative controls, respectively. Increasing concentrations of 4456dh negatively affect filamentation in C. albicans in YPD plus 10% fetal bovine serum incubated at 37°C for 2 hours. Representative images from two independent experiments are shown. The scale bars represent 10 microns. (D) Analysis of capsule formation in C. neoformans WT H99 using India Ink counter-staining in cells treated with 0.1 mM, 0.5 mM, and 1 mM 4456dh. Untreated C. neoformans WT cells and the C. neoformans tps1Δ deletion strain were included in this experiment as positive and negative controls, respectively. The cells were grown for 72 hours at 30°C in capsule-inducing conditions. Cells were harvested, stained in India Ink, and analyzed using differential interference contrast imaging. Representative images from three independent experiments are shown. Scale bar represents 10 microns.

Fig 6

Model for 4456dh activity in fungal cells. Trehalose biosynthesis in Cryptococcus and Candida is initiated by Tps1, which converts G6P and UDPG into T6P. In the second step of the trehalose biosynthesis pathway, T6P is dephosphorylated by Tps2, and trehalose is generated. As shown, Tps1 from C. neoformans forms a homotetramer (31). 4456dh binds the exposed substrate-binding pockets of Tps1. Additionally, 4456dh may have an additional target that is likely another glycosyltransferase in some fungal organisms.