Developing Kinase Inhibitors to Counteract Parasite Virulence

Toxoplasma gondii is a widespread protozoan parasite of wild, domestic, and companion animals that also commonly infects humans. Severe infections are normally only found in immunocompromised patients, including HIV infection, cancer chemotherapy, organ transplant, or infants infected in utero. However, emerging studies indicate that severe ocular toxoplasmosis can also occur in healthy adults, and that chronic infection is an underlying risk factor for some forms of psychiatric disease. <P> Toxoplasma isolates differ dramatically in their virulence in animal models and also in human infections. Recent findings reveal that the major virulence determinants of this parasite are secretory proteins derived from the rhoptries (ROPs), which are injected to the host cell at the time of invasion. Many ROPs contain a conserved serine / threonine kinase domain and while most appear not to be active, several contain a conserved catalytic triad and have been shown to function as kinases. In preliminary studies, we have developed biochemical assays to examine the ability of ROP kinases to phosphorylate a variety of substrates in vitro. <P> In the proposed studies, proteomic, biochemical, mass spectrometry, and bioinformatic analyses will be used to identify substrates of ROP kinases in infected host cells. These studies will provide insight into the mechanism of enhanced virulence conferred by ROP kinases. In vitro kinase assays will be further optimized to provide high-throughput screening for specific inhibitors of ROP kinases. We will also will solve the crystal structure of key members of the ROP kinase family and use molecular modeling to examine specific interactions with inhibitors. Compounds discovered though this process will be analyzed for their potential to prevent infection in vivo using the mouse model. <P> These studies will help define the substrate specificity and inhibitor profile of an important class of parasite virulence factors and may lead to improved therapeutic intervention against toxoplasmosis. <P> PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is an important food borne pathogen of humans that causes disease in those with weakened immune systems. Our studies are designed to identify new chemical inhibitors that counteract the parasite's major virulence factors. Such inhibitors may lead to improved therapeutic intervention against severe infections caused by this parasite.