Morbidity and mortality due to infections with Toxoplasma gondii is an increasing problem in the HIV infected population. Management of toxoplasmosis in these patients is complicated by a high incidence of side effects with the most commonly used compounds and the inactivity of these compounds against the latent stage of the parasite. Hence, there is a continuing need to define novel therapeutic targets and develop new therapeutic agents for this pathogen. One feature of the basic metabolism of these organisms which has not been fully exploited for chemotherapy is the characteristics of their adenosine kinase and its unique specificity in the activation of """"""""subversive"""""""" substrates"""""""". Results from our laboratory indicate that the parasite, but not the host, adenosine kinase can uniquely phosphorylate certain 6-substituted purine nucleosides as """"""""subversive"""""""" substrates and thereby selectively kill T. gondii in vitro and extend the life span of infected mice. Therefore, T. gondii adenosine kinase was cloned, overexpressed and purified. Structure activity relationships as well as comparative metabolic and molecular studies indicate that T. gondii adenosine kinase is indeed substantially different from that of the host in substrate specificity, structure and other characteristics. The purpose of this application is to extend our studies to further characterize the T. gondii adenosine kinase and to use the information gained to develop potent and selective subversive substrates as potential anti-toxoplasmic agents. This approach may well apply to other opportunistic infections which share with toxoplasma this unique feature of purine analogue metabolism.
The Specific Aims are:
Aim 1 : Detailed studies of the cloned T. gondii adenosine kinase.
Aim 2 : Crystallization and resolution of the three dimensional structures of the enzyme with bound 6-substituted subversive substrates.
Aim 3. Molecular modeling studies for the rational design of 6-substituted subversive substrates.
Aim 4 : Chemical synthesis and enzymatic evaluation of rationally designed 6-substituted subversive substrates.
Aim 5 : Evaluate promising rationally designed 6-substituted subversive substrates as potential anti-toxoplasmosis agents in vitro and in vivo.
Aim : 6. Determine the mechanism of selective toxicity of active compounds. These studies will utilize the complementary strengths of the investigators in a collaborative, integrated effort to achieve these goals. ? ?
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