GENETIC ANALYSIS OF PURINE METABOLISM IN LEISHMANIA DONO

Summary

Principal Investigator: B Ullman
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.
Funding Period: 1983-06-01 - 2009-03-31
more information: NIH RePORT

Top Publications

  1. pmc Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani
    Jessica L Martin
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, United States of America
    PLoS Pathog 10:e1003938. 2014
  2. pmc Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine
    Cassandra S Arendt
    Pacific University School of Pharmacy, 222 SE 8th Avenue, Suite 451, Hillsboro, OR 97123, USA Electronic address
    Mol Biochem Parasitol 191:75-9. 2013
  3. pmc Substrate inhibition of uracil phosphoribosyltransferase by uracil can account for the uracil growth sensitivity of Leishmania donovani pyrimidine auxotrophs
    Radika Soysa
    From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 3098 and
    J Biol Chem 288:29954-64. 2013
  4. pmc Adenine and adenosine salvage in Leishmania donovani
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 190:51-5. 2013
  5. pmc KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes
    Khoa D Tran
    Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239, USA
    J Biol Chem 288:22721-33. 2013
  6. pmc Adenylosuccinate synthetase and adenylosuccinate lyase deficiencies trigger growth and infectivity deficits in Leishmania donovani
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    J Biol Chem 288:8977-90. 2013
  7. pmc A rapid, efficient and economical method for generating leishmanial gene targeting constructs
    Audrey L Fulwiler
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 175:209-12. 2011
  8. pmc Purine salvage in Leishmania: complex or simple by design?
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    Trends Parasitol 28:345-52. 2012
  9. pmc IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice
    Audrey L Fulwiler
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 180:123-6. 2011
  10. pmc The Leishmania donovani UMP synthase is essential for promastigote viability and has an unusual tetrameric structure that exhibits substrate-controlled oligomerization
    Jarrod B French
    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA
    J Biol Chem 286:20930-41. 2011

Scientific Experts

  • Nicola S Carter
  • Cassandra S Arendt
  • Jan M Boitz
  • Buddy Ullman
  • Phillip A Yates
  • Audrey L Fulwiler
  • Armando Jardim
  • Radika Soysa
  • Jessica L Martin
  • Khoa D Tran
  • Johannes Elferich
  • Jarrod B French
  • D Radika Soysa
  • Joshua F Alfaro
  • Feng Yang
  • Richard D Smith
  • Phillip A Wilmarth
  • Kristin E Burnum-Johnson
  • Karl K Weitz
  • Peter J Myler
  • Vladislav A Petyuk
  • Gowthaman Ramasamy
  • Larry L David
  • David G Camp
  • Zachary N Wilson
  • Wandy Beatty
  • Michael K Riscoe
  • Dayana Rodriguez-Contreras
  • Scott M Landfear
  • Danielle P Vieira
  • Isaac Forquer
  • Ujwal Shinde
  • Rona Strasser
  • Larry David
  • Caslin Gilroy
  • Bailey Chang
  • Steven E Ealick
  • Normand Cyr
  • Kenneth Choi

Detail Information

Publications16

  1. pmc Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani
    Jessica L Martin
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, United States of America
    PLoS Pathog 10:e1003938. 2014
    ....
  2. pmc Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine
    Cassandra S Arendt
    Pacific University School of Pharmacy, 222 SE 8th Avenue, Suite 451, Hillsboro, OR 97123, USA Electronic address
    Mol Biochem Parasitol 191:75-9. 2013
    ..These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity. ..
  3. pmc Substrate inhibition of uracil phosphoribosyltransferase by uracil can account for the uracil growth sensitivity of Leishmania donovani pyrimidine auxotrophs
    Radika Soysa
    From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 3098 and
    J Biol Chem 288:29954-64. 2013
    ....
  4. pmc Adenine and adenosine salvage in Leishmania donovani
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 190:51-5. 2013
    ..donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation. ..
  5. pmc KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes
    Khoa D Tran
    Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239, USA
    J Biol Chem 288:22721-33. 2013
    ..Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle. ..
  6. pmc Adenylosuccinate synthetase and adenylosuccinate lyase deficiencies trigger growth and infectivity deficits in Leishmania donovani
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    J Biol Chem 288:8977-90. 2013
    ..donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania...
  7. pmc A rapid, efficient and economical method for generating leishmanial gene targeting constructs
    Audrey L Fulwiler
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 175:209-12. 2011
    ..These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species...
  8. pmc Purine salvage in Leishmania: complex or simple by design?
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    Trends Parasitol 28:345-52. 2012
    ..Here, we review recent developments that reveal how purines flux in Leishmania and offer a potential 'Achilles' heel' for future validation...
  9. pmc IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice
    Audrey L Fulwiler
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 180:123-6. 2011
    ..The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host...
  10. pmc The Leishmania donovani UMP synthase is essential for promastigote viability and has an unusual tetrameric structure that exhibits substrate-controlled oligomerization
    Jarrod B French
    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA
    J Biol Chem 286:20930-41. 2011
    ..We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS...
  11. pmc Adaptive responses to purine starvation in Leishmania donovani
    Nicola S Carter
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 3098, USA
    Mol Microbiol 78:92-107. 2010
    ..Moreover, qRT-PCR analyses coupled with cycloheximide inhibition studies suggest that the underlying molecular mechanism for this augmentation in purine salvage occurs post-transcriptionally and is reliant on de novo protein synthesis...
  12. ncbi Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms
    Audrey L Fulwiler
    Oregon Health and Science University, Department of Biochemistry and Molecular Biology, Portland, OR 97239 3098, USA
    Mol Biochem Parasitol 175:76-82. 2011
    ....
  13. pmc Amplification of adenine phosphoribosyltransferase suppresses the conditionally lethal growth and virulence phenotype of Leishmania donovani mutants lacking both hypoxanthine-guanine and xanthine phosphoribosyltransferases
    Jan M Boitz
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA
    J Biol Chem 285:18555-64. 2010
    ..Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Deltahgprt/Deltaxprt parasites displayed persistence and increased virulence in mice...
  14. pmc Role of transmembrane domain 4 in ligand permeation by Crithidia fasciculata equilibrative nucleoside transporter 2 (CfNT2)
    Cassandra S Arendt
    School of Pharmacy, Pacific University Oregon, Hillsboro, Oregon 97123, USA
    J Biol Chem 285:6024-35. 2010
    ..Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2...
  15. ncbi Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase
    Buddy Ullman
    Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97239, USA
    Int J Biochem Cell Biol 42:253-62. 2010
    ..These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases...
  16. pmc Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis
    Cassandra S Arendt
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    Anal Biochem 365:185-93. 2007
    ..This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position...