Antifungal drugs developed around this target would represent a new antifungal class. Currently, there are no known antifungal agents that target the trehalose pathway enzymes. However, our previous work has shown that growth at the human body temperature (37 °C) of Cryptococcus neoformans and Cryptococcus gattii requires the activity of the trehalose pathway. Furthermore, of more than 50 attenuated cryptococcal mutants that we have studied in animal models, the most fragile fungal mutants are the trehalose pathway mutants (tps1Δ and tps2Δ). These mutants are rapidly and completely cleared from the host. At least two other major human invasive fungal pathogens (candida and aspergillus) are also negatively impacted in their ability to cause significant disease in the absence of the trehalose pathway. These observations, plus the lack of the trehalose pathway in humans, suggest that targeting this pathway is ideal and may lead to broad-spectrum antifungal agents that could also extend to many other fungi causing disease, including Mucor circinelloides, in animals and plants.
Our hypothesis is that structural characterization coupled with fungal biology, drug screening, and medicinal chemistry will enable rational antifungal drug design targeting this pathway and identify lead compounds for further antifungal development. Thus, our long-range goal is to provide a comprehensive understanding of the trehalose pathway that will lead ultimately to improved treatment of fungal diseases through drug targeting of this pathway.
Specific Aims
- Determine the structures of the proteins of the fungal trehalose biosynthetic pathway and define the specific interactors with the trehalose pathway. The trehalose biosynthetic pathway primarily consists of just two key enzymes, Tps1 (the trehalose-6-phosphate synthase) and Tps2 (the trehalose-6-phosphate phosphatase). High-resolution structures of the Tps1 and Tps2 from cryptococcus, candida, aspergillus and mucor species bound to substrates, substrate analogues, and inhibitors are needed to understand fully the enzymes’ catalytic mechanisms, to map their protein-protein interaction sites, and to initiate rational drug design. Another key aspect of drug design is understanding the protein(s) that may interact with the drug target. Our hypothesis is that identifying the enzymes’ partners will also characterize functions and may identify other additional synergistic drug targets. Lastly, by defining how resistance might develop around this target, we will deepen our knowledge on how the trehalose pathway functions in the cell as well as how the pathway interacts with networks that contribute to the temperature-sensitive phenotype of tps1Δ and tps2Δ.
Aim 1A. Conduct structural and functional analyses of Tps1, Tps2, and Tps3 from fungal pathogens.
Aim 1B. Identify and characterize proteins interacting with Tps1 and Tps2.
Aim 1C. Define gene(s)/pathway(s) that interact with the trehalose pathway to affect growth at 37 °C.
2. Identify and develop lead compounds targeting the trehalose pathway. We hypothesize that trehalose pathway inhibitors will lead to death of cryptococcus and candida in the host, have synergistic effects with known and novel antifungal drugs, protect the host from infection, and/or have broad-spectrum activity against multiple fungal pathogens.
Aim 2A. Screen to identify compounds that block the activity of either Tps1 or Tps2.
Aim 2B. Select and develop lead compounds.
Aim 2C. Assess lead compounds for in vitro and in vivo toxicity, efficacy, and pharmacokinetics.
Impact
The impact of Project 3 will be that it positions the trehalose pathway for structure-guided drug development, which has significant potential to generate well-tolerated, broad-spectrum antifungals that could substantially improve treatment of human disease. The interactions of Project 3 with the other Projects and Cores will be the following: it will receive critical assistance with our structural studies (Core A) and with validation of the in vitro and in vivo activity of inhibitors and Cryptococcus mutants (Core B); in connection with Projects 1 and 2, we will pursue connections between the trehalose pathway, calcineurin (Project 1), and Ras (Project 2) to assess the synergistic potential of combination treatment by free exchange of expertise, inhibitors, structures, and models among all three projects.
Team
Richard Brennan, PhD
Charles Giamberardino
Richard Lee
John Perfect, MD
William Shadrick, PhD
Jennifer Tenor, PhD
Dena Toffaletti