. Toxoplasma gondii, a pervasive parasitic protozoan, causes significant morbidity and mortality among patients with Acquired Immune Deficiency Syndrome (AIDS), as well as among organ transplant patients. Current treatment regimens for T. gondii infections often produce severe side effects, leading to the cessation of therapy. Thus, it is widely recognized that new drugs having novel mechanisms of action are urgently needed for the treatment of toxoplasmosis. Since T. gondii are incapable of de novo synthesis of purine nucleotides, but instead salvage host purines, the applicant's long term research goal is to use this crucial aspect of parasitic metabolism to advantage: They want to design purine salvage inhibitors and salvageable toxic purine substrates (subversive substrates) that will inactivate or kill toxoplasma. Two enzymes control purine salvage in toxoplasma: hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and adenosine kinase. In this grant application, the applicants are targeting T. gondii HGPRT. they have successfully overexpressed and crystallized T. gondii HGPRT, and thus are now in a position to determine its crystal structure, for the purpose of carrying out structure-based drug design. In this grant application, they propose to solve the crystal structure of Toxoplasma gondii HGPRT, and to use this structure to design potent and selective inhibitors and subversive substrates of this enzyme. The centerpiece of their approach is the determination of the three-dimensional structure of T. gondii HGPRT using X-ray crystallography. They have crystallized T. gondii HGPRT in two different crystal forms that diffract X-rays to 2.75 ( resolution. In support of this central aim, they propose to: complete the characterization of both T. gondii HGPRT isozymes; prepare and characterize several point mutations of T. gondii HGPRT active site residues that they hypothesize control the selective utilization of xanthine by the enzyme; synthesize several HGPRT transition state analogues likely to be potent inhibitors; and test these and other inhibitors against the enzyme, and against the parasite in in vitro cell culture.
| White, E L; Ross, L J; Davis, R L et al. (2000) The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers. J Biol Chem 275:19218-23 |
| Heroux, A; White, E L; Ross, L J et al. (2000) Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis. Structure 8:1309-18 |
| Heroux, A; White, E L; Ross, L J et al. (1999) Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2+) ions bound: insights into the catalytic mechanism. Biochemistry 38:14495-506 |
| Heroux, A; White, E L; Ross, L J et al. (1999) Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex. Biochemistry 38:14485-94 |
