Enzymatic transition state structure can be experimentally approached by a combination of intrinsic kinetic isotope effects (KIEs) and computational quantum chemistry. Experimental KIEs provide boundaries for computational transition states. Transition state knowledge provides chemical insights and practical application of molecular electrostatic maps of transition states are blueprints for transition state analog design. Transition state analysis has provided femtomolar to picomolar inhibitors for N-ribosyltransferases, some of the most powerful enzyme inhibitors. Several transition state analogs are in human clinical trials with more in preclinical studies. These analogs follow the predictions of Pauling and Wolfenden that transition state mimics bind tightly by engaging the forces permitting enzymes to accelerate reactions up to 20 orders of magnitude. Yet, the application of transition state structure to inhibitor design is in its infancy, with development fr only a few chemical reaction classes. The fundamental processes leading to transition state formation remain in contention. This project will expand the utility of transition state analysis ad inhibitor design to two new reaction classes. A fundamental property of enzymatic transition state formation will be tested by individual heavy amino acid substitution into catalytic sites.
Th first aim will target human phenylethanolamine N- methyltransferase (PNMT), the SAM-based formation of epinephrine. N-Methylation reactions are critical for hormones and neurotransmitter metabolism. PNMT is a target for blood pressure regulation and has potential links to Alzheimer's disorder. Hydrolysis of beta-lactamases is our second target class. New Delhi zinc metallo-beta-lactamase (NDM-1) and serine-beta- lactamase are clinically important targets for antibiotic resistance and provide new transition state insight for beta-lactam hydrolysis. The thir aim develops a new experimental approach to resolve fast (fs-ps) protein motions linked to transition state barrier-crossing. Pioneering enzyme chemistry experiments have revealed that increased protein mass slows on-enzyme chemistry. The ultimate extension of heavy-enzyme technology will replace selected amino acids specifically in the catalytic site with their heavy (2, 13C, 15N) counterparts by cell-free protein synthesis with a single amino acid substitution. This project advances transition state chemistry, transition state analog design and the fundamental properties of enzymatic catalysis.

Public Health Relevance

The National Institutes of Health, the World Health Organization and the Center for Disease Control are concerned that the development of new drugs is lagging behind the rise of drug resistance and the evolution of new pathogens. Drug design by transition state analysis provides an efficient approach to drug design. However, the field is limited to only a few classes of enzymatic transition states. This research expands the fundamental knowledge of enzymatic transition states to two new classes of enzyme chemistry. They are pertinent to central nervous system disorders and antibiotic resistance. Drug design from transition state structure is still developing. However, several candidates have reached clinical trials, providing proof of concept for this method.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041916-31
Application #
9531366
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
1989-08-01
Project End
2020-07-31
Budget Start
2018-08-01
Budget End
2019-07-31
Support Year
31
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine, Inc
Department
Type
DUNS #
079783367
City
Bronx
State
NY
Country
United States
Zip Code
10461
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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
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
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; 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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