Transition state (TS) analysis from isotope effects and computational chemistry provides frontier technology for understanding the chemistry of bond change at the instant of enzymatic TS formation. Transition state analogues can be designed from the molecular electrostatic potential surfaces of TSs and have provided unique design parameters for some of the most powerful enzymatic inhibitors known. First and second generation TS analogues for human purine nucleoside phosphorylase (PNP) have advanced from first principles of TS design into human clinical trials for cancer and autoimmune diseases. Third generation PNP inhibitors will be compared to 1st and 2nd generation analogues for binding, structure, thermodynamics and biological lifetimes on PNP in cells. Binding isotope effects are an emerging technology for understanding the geometric and electronic constraints experienced by molecules as they become immobilized at their binding sites on macromolecules, including enzymes and receptors. Binding isotope effects will explore the atomic constraints of substrates and tight- binding TS analogues at the binding sites of human PNP. A surprising diversity of TS structure exists in the same enzyme isolated from different species, establishing the possibility of species-specific TS analogue design. Transition state structures of bacterial 5'- methylthioadenosine nucleosidases (MTANs) will be solved and matched to specific analogues for affinity and structures of reactant and TS-complexes. Biological efficacy of MTAN inhibitors will be analyzed in bacterial quorum sensing pathways. In theory, all enzymatic TSs should be accessible to isotope effect analysis but some provide technical challenges because the chemical step is obscured by non-chemical steps. Human thymidine phosphorylase is a prototype for kinetically difficult TS analyses. TS analysis methods will be established to expose the chemical step by rapid reaction kinetics and altered reaction conditions. Atomic understanding of enzymatic TS chemistry has been developed primarily in enzymes involved in N-ribosyltransferases and deaminases. Expanding the frontier of TS analysis to hydrolysis at carbonyl carbons will be accomplished in the well-known system of HIV-protease and in the important but poorly understood target of human 2'-O-acetyl-ADP-ribosyl esterase. Goals of this research are to push the frontier of enzymatic TS theory to enhance understanding of catalysis and drug design for human targets.

Public Health Relevance

Transition state theory provides an approach to design better drugs for human disease. Expanded methods of drug design will be applied to targets of human disease. Purine nucleoside phosphorylase is a target for leukemia and for autoimmune diseases including psoriasis and tissue transplant rejection;methylthioadenosine phosphorylase is a target for antibiotic-resistant bacteria;thymidine phosphorylase is a target for solid tumors;HIV protease is a target for AIDS infections;and acetyl-ADP-ribosyl hydrolase is a target for diseases of ageing. New methods will be established for the broader application of this theory and the results may lead to new drugs to treat cancer, autoimmunity, bacterial infections and diseases of ageing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041916-23
Application #
8133136
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Gerratana, Barbara
Project Start
1989-08-01
Project End
2012-07-31
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
23
Fiscal Year
2011
Total Cost
$653,028
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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
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
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
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
Yuan, Hongling; Stratton, Christopher F; Schramm, Vern L (2016) Transition State Structure of RNA Depurination by Saporin L3. ACS Chem Biol 11:1383-90
Schramm, Vern L (2015) Transition States and transition state analogue interactions with enzymes. Acc Chem Res 48:1032-9
Wang, Shanzhi; Cameron, Scott A; Clinch, Keith et al. (2015) New Antibiotic Candidates against Helicobacter pylori. J Am Chem Soc 137:14275-80
Suarez, Javier; Schramm, Vern L (2015) Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase. Proc Natl Acad Sci U S A 112:11247-51

Showing the most recent 10 out of 129 publications