Transition state theory for enzymatic reactions indicates that molecules which resemble the enzyme-bound transition state will capture a fraction of the transition state binding energy and provide powerful and specific noncovalent inhibitors. This theory has gained wide acceptance but applications have been limited by the lack of methods to experimentally determine reliable transition state structures for enzymatic reactions. Recent advances in the application of kinetic isotope effects have permitted unprecedented understanding of the geometry and electronic nature of enzyme-stabilized transition states. The inosine-preferring nucleoside hydrolase from the trypanosome Crithidia fasciculata has provided a test case to establish that transition state information can be determined with sufficient accuracy to permit the design of powerful inhibitors. A family of inhibitors is now available to establish the interactions between the enzyme and the transition state. The inhibitors can also be used to explore a surprising diversity of transition state structures which exist in the nucleoside hydrolases. Nucleoside hydrolases are found in protozoa including Trypanosoma (Chagas' disease and sleeping sickness), Leishmania (leishmaniasis), Entamoeba (amebic dysentery), Plasmodium (malaria), Giardia (giardiasis) and Trichomonas (vaginitis). Protozoan parasites are incapable of de novo purine synthesis and require purine salvage for DNA and RNA synthesis. Mammalian tissues lack nucleoside hydrolases. Transition state inhibitors for inosine-preferring nucleoside hydrolase are highly specific and do not inhibit mammalian purine nucleoside phosphorylase or guanosine-preferring nucleoside hydrolase from C. fasciculata. The hypothesis for this proposal is that transition state inhibitors for nucleoside hydrolases are isozyme- and Protozoan-specific. The basis of this specificity will be established by transition state analysis of the guanosine-preferring nucleoside hydrolase from C. fasciculata. At least one additional nucleoside hydrolase will be obtained from cDNA and genomic libraries of Trypanosoma or Plasmodium. Inhibitors will be designed based on the geometric and electrostatic potential surface of the transition state. The genes for nucleoside hydrolases will be isolated from cDNA and genomic libraries and overexpressed in E. coli. The basis for specificity will be established by X-ray crystallography, pH profiles and transition state specificity studies. The results will provide novel information on transition state structure, catalysis, and transition state inhibitors which may be antimetabolites for protozoan parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041916-09
Application #
2459396
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-08-01
Project End
1998-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
9
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Harijan, Rajesh K; Zoi, Ioanna; Antoniou, Dimitri et al. (2018) Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels. Proc Natl Acad Sci U S A 115:E6209-E6216
Evans, Gary B; Tyler, Peter C; Schramm, Vern L (2018) Immucillins in Infectious Diseases. ACS Infect Dis 4:107-117
Ducati, Rodrigo G; Namanja-Magliano, Hilda A; Harijan, Rajesh K et al. (2018) Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum. Proc Natl Acad Sci U S A 115:2114-2119
Mason, Jennifer M; Yuan, Hongling; Evans, Gary B et al. (2017) Oligonucleotide transition state analogues of saporin L3. Eur J Med Chem 127:793-809
Ducati, Rodrigo G; Firestone, Ross S; Schramm, Vern L (2017) Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum. Biochemistry 56:6368-6376
Namanja-Magliano, Hilda A; Evans, Gary B; Harijan, Rajesh K et al. (2017) Transition State Analogue Inhibitors of 5'-Deoxyadenosine/5'-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. Biochemistry 56:5090-5098
Stratton, Christopher F; Poulin, Myles B; Du, Quan et al. (2017) Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase. ACS Chem Biol 12:342-346
Gebre, Sara T; Cameron, Scott A; Li, Lei et al. (2017) Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase. J Biol Chem 292:15907-15915
Namanja-Magliano, Hilda A; Stratton, Christopher F; Schramm, Vern L (2016) Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. ACS Chem Biol 11:1669-76
Du, Quan; Wang, Zhen; Schramm, Vern L (2016) Human DNMT1 transition state structure. Proc Natl Acad Sci U S A 113:2916-21

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