Developing the trehalose biosynthesis pathway as an antifungal drug target

Abstract

Invasive fungal infections are responsible for millions of deaths worldwide each year. Therefore, focusing on innovative approaches to developing therapeutics that target fungal pathogens is critical. Here, we discuss targeting the fungal trehalose biosynthesis pathway with antifungal therapeutics, which may lead to the improvement of human health globally, especially as fungal pathogens continue to emerge due to fluctuations in the climate.

Fig. 1. The canonical trehalose biosynthesis pathway in Cryptococcus and Candida species and virulence-associated phenotypes.

a The canonical, two-step trehalose biosynthesis pathway in fungal pathogens. Tps1 (shown as the C. neoformans homo-tetramer) converts UDPG and G6P into T6P, releasing the byproduct UDP. Tps2 is a phosphatase that removes the phosphate group from T6P to generate trehalose. Trehalases can breakdown trehalose into two glucose molecules. Shown here are representations of trehalases from C. neoformans (Nth1 and Nth2). b There are multiple fungal virulence phenotypes attributed to Tps1 in several fungi including cryptococcal species and C. albicans. c Known fungal virulence phenotypes attributed to Tps2 amongst fungal pathogens are fewer than Tps1. This figure was created in Biorender.

Fig. 2. Key structural features of Tps1 amongst fungal pathogens are conserved.

a Tps1 enzymes are GT-B family glycosyltransferases that consist of a N-terminal lobe and a C-terminal lobe. The two lobes are connected by a kinked C-terminal α-helix. Shown in light brown is E. coli OtsA-UDP-2-fluoroglucose (PDB: 1UQU). Shown in blue is M. oryzae MoTps1-UDP structure (PDB: 6JBW). In purple is the C. albicans Tps1 bound to UDP and G6P (PDB: 5HUU). Shown in pink is M. thermoresistible MtrOtsA in complex with GDPG (PDB: 5K42). In green is the A. fumigatus AfTps1A-UDP-validoxylamine A structure (PDB: 5HVM). In dark beige is a protomer from the C. neoformans cryo-EM homo-tetramer structure (PDB: 8FO1). For clarity, substrates are not shown in the binding pocket of these structures. b The cryo-EM structure of the C. neoformans Tps1 homo-tetramer (PDB: 8FO1). c The substrate-binding pocket of M. thermoresistible MtrOtsA reveals the nearly buried binding site of GDP-glucose. d Conservation of selected substrate-binding residues with the numbers listed from the CaTps1-UDP-G6P (PDB: 5HUU) structure. Residues highlighted with the red stars have been implicated in substrate-binding^,. This figure was created in Biorender with structures generated in ChimeraX.

Fig. 3. Key structural features of Tps2 are conserved.

a Tps2 enzymes are HAD superfamily phosphatases that consist of a cap and core domain. The two domains are connected by a flexible linker to enable movement during catalysis. Shown in magenta is C. neoformans Tps2PD (D24N)-T6P (PDB: 5DX9) and in teal is the A. fumigatus Tps2PD (PDB: 5DXL). Shown in yellow is the S. typhimurium StT6PP-T6-sulfate (PDB: 6UPB). In light gray, is the Brugia malayi Tps2 structure (PDB: 4OFZ). b Conservation of the residues in C. neoformans Tps2PD (D24N)-T6P (PDB: 5DX9) demonstrates that the most conserved residues are near the substrate-binding pocket, while the variable regions are surface-exposed. The heat map is shown in the legend. c The details of the substrate-binding pocket of C. neoformans Tps2PD (D24N)-T6P (PDB: 5DX9), demonstrating the interaction between the Mg²⁺ molecule, T6P and the conserved DXD motif of HADSF enzymes. This figure created in Biorender with structures generated in ChimeraX. Conservation analysis was completed with the ConSurf server.

Fig. 4. Multiple approaches to developing inhibitors of the trehalose biosynthesis pathway.

There are multiple methods to utilize to develop an inhibitor of trehalose biosynthesis, some of which are outlined in this figure. Structure-based approaches combined with high-throughput inhibitor screening can lead to the development of compounds that inhibit Tps1 and Tps2. This approach is enhanced with the use of in silico screening. There are currently multiple compounds that can act as scaffolds to begin the process of designing Tps1 inhibitors^,,. Additionally, if trehalose biosynthesis enzymes form complexes that contribute to their function, inhibiting complex formation if another method to inhibit trehalose biosynthesis. Lastly, there may be synergy between current antifungal drugs, particularly those that target the plasma membrane, and an inhibitor of trehalose biosynthesis. This figure was generated in Biorender.