Summary: Researchers used information generated by artificial intelligence to develop a promising new inhibitor that targets the cyst form of Toxoplasma gondii, the parasite that causes toxoplasmosis.
Source: University of Kentucky
New findings from a University of Kentucky College of Medicine study, published in the Journal of Biological Chemistry on May 28, describe the development of a novel small molecule that inhibits a parasite enzyme and could form the basis of a future treatment for toxoplasmosis.
Toxoplasma gondii is a common intracellular parasite. The Centers for Disease Control and Prevention estimates that roughly 40 million people in the United States carry the parasite, though most never experience symptoms because a healthy immune system keeps the infection in check. Nevertheless, toxoplasmosis can cause serious illness in people with weakened immune systems and can lead to severe consequences if a person becomes newly infected during pregnancy.
A key clinical challenge is the parasite’s ability to form tissue cysts—dormant bradyzoite stages—that persist in brain and muscle tissue and can reactivate later, producing life-threatening disease in immunocompromised individuals. Current FDA-approved therapies treat active symptoms but do not specifically target these cyst forms.
To address that gap, teams led by Matthew Gentry, Ph.D., and Craig Vander Kooi, Ph.D., in the Department of Molecular and Cellular Biochemistry, Anthony Sinai, Ph.D., in the Department of Microbiology, Immunology and Molecular Genetics at the University of Kentucky, and Zhong-Yin Zhang, Ph.D., at the Purdue Institute for Drug Discovery, collaborated to develop a compound that inhibits a parasite enzyme thought to be essential for bradyzoite energy metabolism.
Earlier work from the Gentry lab identified a parasite enzyme called TgLaforin, a glucan phosphatase the team believes is important for breaking down stored carbohydrates that bradyzoites use for energy. Inhibiting TgLaforin could therefore deprive the cyst form of an essential energy source and make the parasite more vulnerable.
The multidisciplinary effort combined biochemical characterization, structural modeling, and medicinal chemistry. Robert Murphy, Ph.D., a member of the Gentry and Sinai laboratories, performed initial experiments that defined TgLaforin’s biochemical behavior and established a functional baseline for the enzyme. In parallel, Tiantian Chen, a graduate student in Vander Kooi’s lab, used AlphaFold2—an artificial intelligence–driven protein structure prediction tool—to model the enzyme’s structure. These models revealed distinctive features of TgLaforin that suggested it could be selectively targeted by small molecules.
Using the biochemical data and structural insights generated by Murphy and Chen, Jianping Lin, Ph.D., a postdoctoral researcher in Zhang’s lab, applied medicinal chemistry techniques to synthesize and optimize a first-generation inhibitor of TgLaforin. Initial in vitro tests showed that the compound blocks TgLaforin’s activity against multiple carbohydrate substrates, preventing the enzyme from performing its normal function in test-tube assays.
“I was excited to find that the drug was effective against TgLaforin in test tubes and that it prevented TgLaforin from performing its normal activity against a variety of substrates, including carbohydrates,” said Murphy. The discovery demonstrates how integrating AI-based structural models with classical biochemical and chemical methods can accelerate target validation and inhibitor design.
The study also employed complementary biophysical and structural approaches to define TgLaforin’s distinctive properties. AlphaFold2 modeling, together with hydrogen–deuterium exchange mass spectrometry and differential scanning fluorimetry, helped reveal an unusual split carbohydrate-binding module, unique structural dynamics related to glucan binding, and a dimeric arrangement mediated by a dual specificity phosphatase domain. These attributes informed the design of molecules that exploit the enzyme’s distinctive catalytic features.
Next steps will focus on testing the compound against live parasites, improving its potency and selectivity for TgLaforin, and optimizing chemical properties required for animal studies. The research team plans to refine the inhibitor series with the goal of producing a candidate suitable for preclinical evaluation.
“This study is a great example of what Provost DiPaola consistently promotes regarding transdisciplinary research,” Gentry said. “This work was a true team effort and it is very exciting to see where we take it next.”
About this neuropharmacology and toxoplasmosis research news
Author: Press Office
Source: University of Kentucky
Contact: Press Office – University of Kentucky
Image: The image is in the public domain
Original Research: Closed access. “The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors” by Robert D. Murphy et al., Journal of Biological Chemistry
Abstract
The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors
Toxoplasma gondii generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that characterize chronic infection. AGs are believed to serve as an essential energy reserve supporting bradyzoite persistence, transmission, and reactivation—events that can lead to life-threatening toxoplasmosis upon reactivation.
T. gondii encodes glucan dikinase and glucan phosphatase enzymes homologous to plant and animal proteins involved in reversible glucan phosphorylation, processes required for efficient polysaccharide degradation and utilization. The structural determinants regulating this reversible phosphorylation in T. gondii were previously unclear.
This study defines key functional features of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). The enzyme contains an atypical split carbohydrate-binding-module domain and exhibits unique structural dynamics related to glucan binding. Using AlphaFold2 modeling together with hydrogen–deuterium exchange mass spectrometry and differential scanning fluorimetry, the authors characterize TgLaforin’s interaction with glucans and demonstrate that TgLaforin forms a dimer mediated by its dual specificity phosphatase domain.
Finally, the investigators exploited the distinct properties of TgLaforin’s catalytic domain to identify a small molecule inhibitor of its phosphatase activity. By revealing a unique mechanism of TgLaforin function, this work opens a new avenue for targeting bradyzoite biology and advancing therapeutic strategies against T. gondii cysts.