GENETIC ANALYSIS OF PURINE METABOLISM IN LEISHMANIA DONO

Oregon Health & Science University, Portland OR

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Abstract

This grant application pertains to a critical issue in the treatment and[unreadable] control of parasitic diseases, the need for better chemotherapies.[unreadable] Amalgamating techniques of molecular biology, biochemistry, genetics,[unreadable] structural biology, and computational chemistry this proposal offers a[unreadable] multidisciplinary dissection of the hypoxanthine-guanine[unreadable] phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani, an[unreadable] enzyme that renders an important, if not essential, nutritional function[unreadable] for the parasite, and that initiates the metabolism of allopurinol, a drug[unreadable] that exhibits therapeutic efficacy in both leishmaniasis and Chagas[unreadable] disease. These studies constitute a logical step in the implementation of[unreadable] a rational, structure-based strategy of drug discovery, and ultimately[unreadable] drug design, for the treatment and prevention of leishmaniasis and other[unreadable] diseases of parasitic origin. Reagents available for these studies[unreadable] include: i. the L. donovani, T. brucei, T. cruzi and C. fasciculata hgprt[unreadable] genes; ii., the L. donovani aprt gene; iii., hgprt- populations of L.[unreadable] donovani that were generated by targeted gene replacement;' iv., E. coli[unreadable] that overproduce each of the trypanosomatid HGPRTs and v., effectively[unreadable] unlimited amounts of L. donovani, T. brucei, T. cruzi, and C. fasciculata[unreadable] HGPRT proteins that appear homogeneous by SDS-PAGE. In addition, an[unreadable] homology-based 3-D molecular model of the L. donovani HGPRT has been[unreadable] computationally constructed and serves as a cornerstone for our structural[unreadable] studies. The first objective of this application is to evaluate the[unreadable] contributions of HGPRT and APRT to purine metabolism in L. donovani[unreadable] promastigotes by phenotypic characterization of hgprt- and aprt- null[unreadable] mutants that will be created by homologous gene replacement. Whether hgprt[unreadable] and/or aprt gene function is essential for infectivity and virulence will[unreadable] also be tested by generating null mutants in infective Leishmania strains.[unreadable] The second specific aim entails the structural characterization of the L.[unreadable] donovani HGPRT. The first component of this specific aim will be to[unreadable] evaluate the 3-D model of the protein by site-directed mutagenesis of key[unreadable] amino acid residues that are postulated to participate in catalytic[unreadable] activity or govern substrate specificity and biochemical characterization[unreadable] of the genetically altered proteins. The second aspect of Specific Aim II[unreadable] will be to introduce crystallographic methods to the structural studies[unreadable] for the ultimate purpose of either refining the 3-D molecular model or[unreadable] determining the structure of the L. donovani HGPRT protein itself. The[unreadable] penultimate specific aim involves the identification of key active site[unreadable] residues of the L. donovani HGPRT that participate in catalysis using[unreadable] affinity and photoaffinity labeling techniques and further evaluation of[unreadable] the functional role of these amino acids in catalysis by site-directed[unreadable] mutagenesis. Lastly, we will perform computational screens of 3-D small[unreadable] molecule structural databases with our molecular models, and ultimately[unreadable] with resolved structures, to discover novel 'lead' compounds that target[unreadable] the active site pocket of the L. donovani HGPRT. Computationally[unreadable] identified compounds from the database screens, as well as 40 procured[unreadable] purine base analogs, will be evaluated as potential antileishmanial[unreadable] compounds using a simple, yet multifaceted, screen comprising of purified[unreadable] recombinant HGPRT enzymes, E. coli that overexpress hgprt genes, and[unreadable] intact parasites.