The overall purpose of this research project is to employ biochemical, molecular biological and biophysical means to discover an effective and nontoxic antischistosomal agent. The specific approach is to try to design a specific, potent inhibitor of the hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) in Schistosoma mansoni through a thorough understanding of the structures and properties of S. mansoni and human HGPRTases as well as the discrepancies between the two enzymes. The reasons justifying this particular plan of study are based on the knowledge that schistosomes are incapable of de novo synthesis of purine nucleotides, and have to rely on the function of HGPRTase as the primary means of fumishing guanine nucleotides. Full-length cDNAs encoding the S. mansoni and human enzymes have been cloned and expressed in transformed Escherichia coli to produce native enzymes in large quantities (15 to 25 mg of purified enzyme per liter of bacterial pulture). Both enzymes have been crystallized. Preliminary X-ray diffraction patterns of a 2.9 Angstrom resolution have been recorded from the crystals of S. mansoni HGPRTase. For the next granting period, we plan to focus on resolution of detailed 3-dimensional structures of both S. mansoni and human HGPRTase by X-ray crystallography. By all the initial indications we have collected thus far, we have every confidence to believe that this objective will be fully accomplished. We shall then use computer graphic programs to search for the appropriate chemical structure of a specific inhibitor of S. mansoni HGPRTase, and design site-directed mfitagenesis to generate mutant enzymes for additional structural and kinetic analysis aimed at further in-depth understandings of the distinctive properties between the host and parasite enzymes. Meanwhile, chemical rnodifications of the two enzymes by a photoaffinity label 8-azidohypoxanthine and 2', 3'-dialdehyde derivatives of IMP, GMP and PRPP will be performed to identify specific amino acid residues in the active pockets involved with substrate-bindings. Iodoacetate labelings of the enzymes in the presence of PRPP may also identify the potential cysteine residue(s) responsible for PRPP binding to HGPRTase. Circular dichroism spectral analysis will provide information on conformational changes of the two proteins with changing environments, and may explain the remarkable stabilides of the two enzymes at elevated tempemtures (80 degrees C). Finally, the fine collection of hypoxanthine and guanine analogs at the Wellcome Research Laboratories will be tested on the transformed E. coli whose survival depends on a functioning S. mansoni HGPRTase in order to discover a specific inhibitor of this enzyme. Thus, we are approaching the final stage of a long struggle toward establishing a model for biochemical approaches to antiparasitic chemotherapy (or any chemotherapy). There is every reason for us to feel optimistic about the eventual outcome.
