The overall goal of our present research proposal is to establish a model to verify the feasibility of a new approach to discover novel agents against infectious diseases.
This model requires a consortium of researchers in biochemistry, pharmacology, molecular biology, structural biology, computer graphics and organic synthesis to work together. The results from our previous metabolic studies chose Tririchomonas foetus and Giardia lamblia as the two parasitic organisms for further investigations because of their deficiency in de novo synthesis of both purine and pyrimidine nucleotides. Further investigations focused our attention on T. foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), T. foetus IMP dehydrogenase (IMPDH) and G. lamblia guanine phosphoribosyltransferase (GPRTase), because each protein was found to be a pivotal enzyme in purine salvage; their inhibition in each case brought on arrested in vitro growth of the parasite. These enzymes were purified, and their distinctive substrate specificities and catalytic properties were demonstrated in preliminary studies. Genes encoding these enzymes were then identified, cloned, sequenced and expressed to yield large quantities of purified recombinant enzyme in their native forms. They share relatively low sequence identities with their mammalian counterparts; T. foetus HGXPRTase, 27.3 percent, G. lamblia GPRTase, lesser 20 percent; T.foetus IMPDH, 34 percent. The crystal structure of T. foetus HGXPRTase was identified at 1.91 resolution. It is an unusually compact asymmetric dimer with many distinctive features in the active pocket when compared with that of human HGPRTase. The crystal structure of T. foetus IMPDH was also determined up to 2.3A resolution. It is primarily an alpha beta barrel packed into two flat C4-symmetric tetramers. Though the structure of mammalian IMPDH is not yet available for comparison, our biochemical data on the parasite enzyme have already indicated an unusually high K1 for mycophenolic acid and identified Cys319 in the enzyme as the catalytic nucleophile. For the next granting period, we intend to: 1) generate a variety of mutants of the three enzyme proteins by site-directed mutagenesis; 2) examine each mutant enzyme in steady-state kinetic analysis for altered substrate specificities and kinetic constants; 3) analyze the structures of the mutant proteins in X-ray crystallography and correlate structural modifications with functional changes; 4) use the computer graphic DOCK program to screen and identify potential inhibitors of the three enzymes; 5) test the chosen chemical compounds in enzyme assays and in vitro parasite cell cultures for leads, and modify the structures of lead compounds through organic synthesis. Combinatorial libraries of purine analogs will be also screened in the enzyme assays as an alternative route to identify lead compounds. We believe that a solid basis has already been cast upon which significant results may be within reach in the near future.