Toxoplasma gondii chronically infects approximately 30% of the United States population. Chemotherapy, largely based on anti-folates, is available for acute infection with tachzyzoites, but life-long treatment is required to protect against infections in AIDS. Unfortunately, a high frequency of adverse reactions are associated with chronic treatment, frequently complicating clinical treatment of T. gondii. Thus it is a high priority to develop new and improved treatment strategies for T. gondii infection in AIDS. This grant focuses on new approaches to develop improved treatments for T. gondii infection based on targeting the parasite carbamoyl phosphate synthetase II (CPSII) activity required for de novo pyrimidine biosynthesis. Preliminary biochemical and genetic studies indicate that T. gondii expresses a novel CPSII that can be disrupted to induce starvation for uridine monophosphate (UMP), and other pyrimidine nucleotides required for parasite RNA and DNA synthesis. Disruption of CPSII activity in T. gondii blocks parasite replication in vitro, rapidly resulting in loss of parasite viability. Disruption of CPSII abolished parasite growth and virulence in mice that lack interferon-y. The present proposal will expand our studies of CPSII to focus on the development of specific inhibitors of this essential parasite enzyme. We will characterize the essential activities and regulation to gain information on CPSII functions that can be effectively targeted for chemotherapy. We will determine the crystal structure of active T. gondii CPSII enzyme(s) to establish a structural basis for drug development. We will evaluate lead inhibitor compounds of T. gondii CPSII, and will identify or synthesize new inhibitors. It is our hypothesis that targeting CPSII and de novo pyrimidine biosynthesis can lead to improved treatments. The proposed research formally brings together expertise from three research groups at Dartmouth. Dr. David Bzik is a molecular parasitologist with training in genetics and biochemistry. Dr. Amy Anderson is a structural biochemist with training in biochemistry and crystallography. Dr. Gordon Gribble is a medicinal/synthetic chemist with training in chemical synthesis of compounds for the treatment of disease. Together, the expertise of these three research groups will provide a unique opportunity to develop and evaluate new drugs for treatment of T. gondii infection based on inhibition of parasite CPSII.
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