The central theme of this program involves studies of the mechanisms of action of the drug-metabolizing enzymes cytochrome P450 and other heme proteins such as myoglobin and cytochrome c. We also investigate metalloporphyrin model compounds to elucidate how these enzymatic processes occur. The principal approaches involve kinetic and mechanistic studies of enzyme-substrate interactions, the synthesis and characterization of reactive iron porphyrin species as models of putative enzymic intermediates and to relate the interconversions of these species toward a molecular understanding of these proteins. Two additional applications of the model compounds have evolved in the course of this project. We have discovered that some iron porphyrins are highly bioactive in suppressing protein nitration by peroxynitrite. Other metalloporphyrins have emerged as effective biomimics of P450 action, alowing the facile production of useful quantities of drug metabolites, often a bottleneck in drug development. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while the related nitric oxide synthase is the source of the signal molecules nitric oxide and peroxynitrite. Synthetic metalloporphyrins can be employed as probes to intervene in these processes in diagnostic ways. Thus, these agents may prove to be significant tools for elaborating the biology of superoxide, peroxynitrite and NO. These same metalloporphyrins have shown impressive activity in animals suggesting their application as pharmaceutical agents in degenerative diseases such as diabetes, cardiomyopathy ang age-related disorders. Our effort seeks to provide a foundation of mechanistic and kinetic information that can be applied to in vitro models, cell culture studies and whole animal models of specific disease states such as ischemia-reperfusion, sepsis and autoimmune diseases. Experiments are aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite and these other species, while studies of protein tyrosine nitration will elucidate how proteins are damaged under conditions of oxidative and nitrosative stress. Novel types of rapid kinetic analysis have been developed to study the reactivity observed in these processes. Binding of cytochrome c to synthetic and semi-synthetic phospholipid assemblies, which afford a system of intermediate complexity, are used to model and understand the larger scale events in the role of cytochrome c in triggering lipid oxidation and programmed cell death (apoptosis).

Public Health Relevance

Cytochrome P450 enzymes mediate phase 1 drug metabolism. Other heme proteins such as myoglobin and cytochrome c are involved in nitric oxide catabolism and the initiation of programed cell death (apoptosis). Our program aims to understand these processes. The outcome could lead to more efficatious drugs and new ways of controlling tissue damage that arise from oxidative and nitrosative stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37GM036298-27
Application #
8195682
Study Section
Special Emphasis Panel (NSS)
Program Officer
Okita, Richard T
Project Start
1985-12-01
Project End
2017-03-31
Budget Start
2012-06-01
Budget End
2013-03-31
Support Year
27
Fiscal Year
2012
Total Cost
$354,249
Indirect Cost
$104,249
Name
Princeton University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
Liu, Wei; Groves, John T (2015) Manganese Catalyzed C-H Halogenation. Acc Chem Res 48:1727-35
Wang, Xiaoshi; Ullrich, René; Hofrichter, Martin et al. (2015) Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive. Proc Natl Acad Sci U S A 112:3686-91
Groves, John T; Boaz, Nicholas C (2014) Biochemistry. Fishing for peroxidase protons. Science 345:142-3
Stavniichuk, Roman; Shevalye, Hanna; Lupachyk, Sergey et al. (2014) Peroxynitrite and protein nitration in the pathogenesis of diabetic peripheral neuropathy. Diabetes Metab Res Rev 30:669-78
Groves, John T (2014) Enzymatic C-H bond activation: Using push to get pull. Nat Chem 6:89-91
Liu, Wei; Huang, Xiongyi; Groves, John T (2013) Oxidative aliphatic C-H fluorination with manganese catalysts and fluoride ion. Nat Protoc 8:2348-54
Wang, Xiaoshi; Peter, Sebastian; Ullrich, René et al. (2013) Driving force for oxygen-atom transfer by heme-thiolate enzymes. Angew Chem Int Ed Engl 52:9238-41
Bergstrom, Chris L; Beales, Paul A; Lv, Yang et al. (2013) Cytochrome c causes pore formation in cardiolipin-containing membranes. Proc Natl Acad Sci U S A 110:6269-74
Wang, Xiaoshi; Peter, Sebastian; Kinne, Matthias et al. (2012) Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I. J Am Chem Soc 134:12897-900
Radovits, Tamás; Beller, Carsten J; Groves, John T et al. (2012) Effects of FP15, a peroxynitrite decomposition catalyst on cardiac and pulmonary function after cardiopulmonary bypass. Eur J Cardiothorac Surg 41:391-6

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