Protists cause numerous human diseases, including malaria, African sleeping sickness, Chagas' disease, leishmaniasis, giardiasis, and amebic meningitis. Treatments for each of these is inadequate, as few pharmaceutically exploitable metabolic differences have been found between humans and protists. Sterol biosynthesis is a promising but underexplored area for finding vital protist enzymes that lack human homologs, and the primary goal of this proposal is identifying novel drug targets in protist sterol metabolism. Animals and fungi synthesize the sterol ring system in one step using lanosterol synthase, which cyclizes oxidosqualene to lanosterol. The overwhelming majority of protists in which sterol biosynthesis has been examined synthesize that ring system in two steps: cycloartenol synthase cyclizes oxidosqualene to cycloartenol, which is then converted to lanosterol by cyclopropyl isomerase. These organisms should be susceptible to specific inhibitors of either cycloartenol synthase or cyclopropyl isomerase. Presented in this proposal are experiments to characterize these enzymes from several protists. A series of compounds designed to specifically inhibit cycloartenol synthase is described. Recombinant organisms that express the protist cycloartenol synthases and cyclopropyl isomerases will be constructed to provide enzymes with which to test the drug candidates. Metabolic research in pathogenic protists is hindered by difficulties in obtaining sufficient biomass for classical experiments with radiolabeled tracers. The approach described here uses molecular biological techniques to clone and express protist genes responsible for forming the sterol ring and to determine which pathway various protists use. This laboratory has constructed several metabolically engineered yeast strains in which the normal sterol pathway (which goes through lanosterol) is supplemented with components of the plant sterol biosynthetic pathway (which goes through cycloartenol). These strains can utilize either lanosterol or cycloartenol as sterol precursors, and will serve as hosts to clone cycloartenol synthase, cyclopropyl isomerase, or lanosterol synthase by genetic complementation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041598-04
Application #
6170487
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Gottlieb, Michael
Project Start
1997-07-01
Project End
2002-06-30
Budget Start
2000-07-01
Budget End
2002-06-30
Support Year
4
Fiscal Year
2000
Total Cost
$121,982
Indirect Cost
Name
Rice University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
050299031
City
Houston
State
TX
Country
United States
Zip Code
77005
Xiong, Quanbo; Hassan, Saad A; Wilson, William K et al. (2005) Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors. Antimicrob Agents Chemother 49:518-24
Xu, Ran; Fazio, Gia C; Matsuda, Seiichi P T (2004) On the origins of triterpenoid skeletal diversity. Phytochemistry 65:261-91
Segura, Michael J R; Jackson, Beth E; Matsuda, Seiichi P T (2003) Mutagenesis approaches to deduce structure-function relationships in terpene synthases. Nat Prod Rep 20:304-17
Hinshaw, Jerald C; Suh, Dae-Yeon; Garnier, Philippe et al. (2003) Oxidosqualene cyclase inhibitors as antimicrobial agents. J Med Chem 46:4240-3
Lu, Shan; Xu, Ran; Jia, Jun-Wei et al. (2002) Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression. Plant Physiol 130:477-86
Milla, P; Viola, F; Oliaro Bosso, S et al. (2002) Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast. Lipids 37:1171-6
Ruan, Benfang; Lai, Peggy S; Yeh, Christine W et al. (2002) Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance. Steroids 67:1109-19
Meyer, Michelle M; Xu, Ran; Matsuda, Seiichi P T (2002) Directed evolution to generate cycloartenol synthase mutants that produce lanosterol. Org Lett 4:1395-8
Joubert, B M; Buckner, F S; Matsuda, S P (2001) Trypanosome and animal lanosterol synthases use different catalytic motifs. Org Lett 3:1957-60
Joubert, B M; Hua, L; Matsuda, S P (2000) Steric bulk at position 454 in Saccharomyces cerevisiae lanosterol synthase influences B-ring formation but not deprotonation. Org Lett 2:339-41

Showing the most recent 10 out of 17 publications