Polyamine and glutathione metabolism in trypanosomes.

Summary

Principal Investigator: Margaret Phillips
Abstract: DESCRIPTION (provided by applicant): The parasitic protozoa of the genus Trypanosoma are the causative agents of African sleeping sickness and of Chagas' disease in South America. These parasites cause significant mortality and drug therapy to combat these diseases is inadequate. Polyamines are essential growth factors, and inhibition of polyamine biosynthesis provides a mechanism to inhibit cell growth. The first committed step in the biosynthesis of polyamines is catalyzed by ornithine decarboxylase (ODC). This enzyme is one of the few validated targets for the treatment of parasitic diseases. A number of enzymes in the pathway are essential for cell growth, and inhibitors of several polyamine biosynthetic enzymes have anti-trypanosomal activity. In addition the parasites synthesize a unique cofactor that is a conjugate of spermidine and glutathione, termed trypanothione that functions to maintain the reduced thiol pool in the cell. In this proposal our planned studies focus on the three enzymes in the pathway (ODC, S-adenosylmethionine decarboxylase and gamma-glutamylcysteine synthetase) that catalyze the first committed steps in the biosynthesis of the polyamines and trypanothione. In the first two aims we will explore the structural basis for inhibition of these enzymes. These studies will lay the foundation for future inhibitor design. In Aim one we plan to explore the role of active site interactions in the energetics of ligand binding and catalysis, using site-directed mutagenesis and X-ray crystallography. In Aim two mechanisms of allosteric inhibition will be studied to explore the potential to develop novel mechanisms of inhibition that target regions outside of the active site. While ODC is a fully validated target, other enzymes in the pathway are not. The nature of the rate-determining steps in polyamine and trypanothione biosynthesis in T. brucei has not been fully elucidated. Understanding the metabolic flux through the pathway will provide insight into which additional enzymes are best targeted for drug design. Polyamine metabolism is highly regulated in most cells, yet no evidence for regulation has been described in trypanosomatids. If the parasite alters polyamine levels in response to pathway inhibitors this regulation may impact on the effectiveness of drugs targeted at these enzymes. In Aim 3 we propose to study the effects of both genetic and metabolic perturbations in the pathway on the levels of key enzymes and metabolites.
Funding Period: 1994-12-01 - 2009-09-29
more information: NIH RePORT

Top Publications

  1. ncbi Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase
    Tracy Clyne Beswick
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Boulevard, Dallas, Texas 75390 9041, USA
    Biochemistry 45:7797-807. 2006
  2. pmc Trypanosoma brucei S-adenosylmethionine decarboxylase N terminus is essential for allosteric activation by the regulatory subunit prozyme
    NAHIR VELEZ
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 9041, USA
    J Biol Chem 288:5232-40. 2013
  3. pmc State of the art in African trypanosome drug discovery
    Robert T Jacobs
    SCYNEXIS, Inc, Research Triangle Park, North Carolina 27709 2878, USA
    Curr Top Med Chem 11:1255-74. 2011
  4. pmc A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei
    Savitha Kalidas
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 9041, United States
    Mol Biochem Parasitol 178:51-5. 2011
  5. pmc Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota
    Colin C Hanfrey
    Institute of Food Research, Norwich, NR4 7UA, United Kingdom
    J Biol Chem 286:43301-12. 2011
  6. pmc Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine
    Frances L Shaw
    Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom
    J Biol Chem 285:14711-23. 2010
  7. pmc Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes
    David C Smithson
    Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA
    Assay Drug Dev Technol 8:175-85. 2010
  8. pmc Discovery of potent and selective inhibitors of Trypanosoma brucei ornithine decarboxylase
    David C Smithson
    Department of Chemical Biology and Therapeutics, St Jude Children s Research Hospital, Memphis, Tennessee 38105, USA
    J Biol Chem 285:16771-81. 2010
  9. pmc Evolution of substrate specificity within a diverse family of beta/alpha-barrel-fold basic amino acid decarboxylases: X-ray structure determination of enzymes with specificity for L-arginine and carboxynorspermidine
    Xiaoyi Deng
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 9041, USA
    J Biol Chem 285:25708-19. 2010
  10. pmc Cross-species activation of trypanosome S-adenosylmethionine decarboxylase by the regulatory subunit prozyme
    Erin K Willert
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, TX 75390 9041, USA
    Mol Biochem Parasitol 168:1-6. 2009

Detail Information

Publications19

  1. ncbi Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase
    Tracy Clyne Beswick
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Boulevard, Dallas, Texas 75390 9041, USA
    Biochemistry 45:7797-807. 2006
    ..These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation...
  2. pmc Trypanosoma brucei S-adenosylmethionine decarboxylase N terminus is essential for allosteric activation by the regulatory subunit prozyme
    NAHIR VELEZ
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 9041, USA
    J Biol Chem 288:5232-40. 2013
    ..Our findings provide the first insight into the mechanisms that influence catalytic regulation of AdoMetDC and may have potential implications for the development of new inhibitors against this enzyme...
  3. pmc State of the art in African trypanosome drug discovery
    Robert T Jacobs
    SCYNEXIS, Inc, Research Triangle Park, North Carolina 27709 2878, USA
    Curr Top Med Chem 11:1255-74. 2011
    ..This dire situation underscores the need for continued effort to identify new chemical agents for the treatment of HAT...
  4. pmc A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei
    Savitha Kalidas
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 9041, United States
    Mol Biochem Parasitol 178:51-5. 2011
    ..The new vector facilitates high-throughput applications for gene silencing and provides a tool for functional genomics in T. brucei...
  5. pmc Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota
    Colin C Hanfrey
    Institute of Food Research, Norwich, NR4 7UA, United Kingdom
    J Biol Chem 286:43301-12. 2011
    ..The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds...
  6. pmc Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine
    Frances L Shaw
    Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom
    J Biol Chem 285:14711-23. 2010
    ..Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine...
  7. pmc Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes
    David C Smithson
    Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA
    Assay Drug Dev Technol 8:175-85. 2010
    ..90 with 95% confidence interval between 0.82 and 0.97)...
  8. pmc Discovery of potent and selective inhibitors of Trypanosoma brucei ornithine decarboxylase
    David C Smithson
    Department of Chemical Biology and Therapeutics, St Jude Children s Research Hospital, Memphis, Tennessee 38105, USA
    J Biol Chem 285:16771-81. 2010
    ..These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites...
  9. pmc Evolution of substrate specificity within a diverse family of beta/alpha-barrel-fold basic amino acid decarboxylases: X-ray structure determination of enzymes with specificity for L-arginine and carboxynorspermidine
    Xiaoyi Deng
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 9041, USA
    J Biol Chem 285:25708-19. 2010
    ..These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold...
  10. pmc Cross-species activation of trypanosome S-adenosylmethionine decarboxylase by the regulatory subunit prozyme
    Erin K Willert
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, TX 75390 9041, USA
    Mol Biochem Parasitol 168:1-6. 2009
    ..These data demonstrate that prozyme mediated activation of AdoMetDC is a common mechanism required to regulate AdoMetDC activity in the trypanosomatids...
  11. pmc An alternative polyamine biosynthetic pathway is widespread in bacteria and essential for biofilm formation in Vibrio cholerae
    Jeongmi Lee
    Departments of Pharmacology, Microbiology, and Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA
    J Biol Chem 284:9899-907. 2009
    ..cholerae infection. Notably, the alternative polyamine biosynthetic pathway is widespread in bacteria and is likely to play a previously unrecognized role in the biology of these organisms...
  12. pmc RNA interference-mediated silencing of ornithine decarboxylase and spermidine synthase genes in Trypanosoma brucei provides insight into regulation of polyamine biosynthesis
    Yanjing Xiao
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390 9041, USA
    Eukaryot Cell 8:747-55. 2009
    ..This finding may contribute to the effectiveness of ODC as a target for the treatment of African sleeping sickness, thus providing important insight into the mechanism of action of a key antitrypanosomal agent...
  13. pmc Novel S-adenosylmethionine decarboxylase inhibitors for the treatment of human African trypanosomiasis
    Robert H Barker
    Genzyme Corporation, 153 Second Avenue, Waltham, MA 02451, USA
    Antimicrob Agents Chemother 53:2052-8. 2009
    ..Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration...
  14. pmc Regulated expression of an essential allosteric activator of polyamine biosynthesis in African trypanosomes
    Erin K Willert
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
    PLoS Pathog 4:e1000183. 2008
    ..The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis...
  15. pmc X-ray structure of Paramecium bursaria Chlorella virus arginine decarboxylase: insight into the structural basis for substrate specificity
    Rahul Shah
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 9041, USA
    Biochemistry 46:2831-41. 2007
    ..In conjunction with prior structural studies these data predict that this loop adopts different conformations throughout the catalytic cycle, and that loop movement may be kinetically linked to the rate-limiting step of product release...
  16. ncbi Phylogenetic diversity and the structural basis of substrate specificity in the beta/alpha-barrel fold basic amino acid decarboxylases
    Jeongmi Lee
    Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 9041, USA
    J Biol Chem 282:27115-25. 2007
    ..Our data demonstrate that there is greater structural and functional diversity in bacterial polyamine biosynthetic decarboxylases than previously suspected...
  17. pmc Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog
    Erin K Willert
    Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390 9041, USA
    Proc Natl Acad Sci U S A 104:8275-80. 2007
    ..These data identify a distinct mechanism for regulating AdoMetDC in the parasite that suggests new strategies for the development of parasite-specific inhibitors of the polyamine biosynthetic pathway...
  18. pmc Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei
    Melissa J Rodgers
    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390 9041, USA
    Eukaryot Cell 6:1108-18. 2007
    ..In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis...
  19. pmc Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme
    Yanjing Xiao
    Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 9041, USA
    Mol Microbiol 88:846-61. 2013
    ..These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role...