S. Bruce King, PhD

Associate Professor, Wake Forest University Department of Chemistry

Email: kingsb@wfu.edu

Education:

Cornell University, PhD

Postdoctoral, The Scripps Research Institute, K. Barry Sharpless

Research Interests: Design and synthesis of new compounds capable of interaction with nitric oxide synthase, design, synthesis and evaluation of new nitric oxide and nitroxyl delivery agents, reactions of hydroxyurea with heme proteins.

Current Research: Organic Synthesis / Bioorganic Chemistry / Biochemistry and Biophysics

Our research program is based on a combination of organic chemistry and biochemistry directed towards understanding the various roles nitric oxide (NO) performs in biological systems. Nitric oxide directly participates in the control of blood flow and pressure, neurotransmission, and the immune response and the regulation of NO levels represents a therapeutic strategy for disease states characterized by abnormal NO production. Our laboratory examines three major areas of NO chemistry: 1) the reaction of nitric oxide and hydroxyurea with hemoglobin to understand the chemistry responsible for the use of these agents as sickle cell disease treatments, 2) the interaction of small molecules with nitric oxide synthase (NOS), the enzyme responsible for biochemical NO production from L-arginine and 3) the synthesis and evaluation of new organic NO delivery agents. In addition to these projects, our laboratory works to develop new synthetic organic methodology for the preparation of biologically active compounds.

Chemistry of N-Hydroxyureas and Reactions of Nitric Oxide and Hydroxyurea with Heme Proteins

Much of our basic research involves understanding the oxidative chemistry of N-hydroxyureas and the ability of these compounds to liberate nitric oxide or the one electron reduced form of nitric oxide, nitroxyl (HNO). Nitroxyl displays many of the same pharmacological properties as nitric oxide and can be oxidized to NO by many biological oxidants. Our results indicate that N-hydroxyureas can be chemically oxidized to the corresponding nitroxide radicals and nitroso compounds, which react with water to produce the corresponding amine, carbon dioxide, and nitroxyl. The presence of nitroxyl is implied by the gas chromatographic identification of nitrous oxide, the dimerization and dehydration product of HNO. Current investigations are aimed at more clearly understanding the chemical mechanisms of NOand HNO formation from N-hydroxyureas.

Identification of proteins and enzymes capable of oxidizing hydroxyureas with nitric oxide release represents a major research project in the group. Our recent efforts have focused on nitric oxide release during the reactions of hydroxyureas with heme-containing proteins including hemoglobin, horseradish peroxidase and catalase. Hydroxyurea, the simplest hydroxyurea, is an approved treatment of sickle cell anemia and understanding the chemical reactions of this drug with hemoglobins could be important in explaining the action of this drug. Much of this work is done in collaboration with Dr. Dany Kim-Shapiro, Wake Forest University, Department of Physics. Each of the above proteins react with hydroxyurea to produce either NO or HNO as determined using a combination of various spectroscopic methods and analytical techniques including electron paramagnetic resonance and ultraviolet spectroscopy as well as chemiluminescence NO detection. Current efforts are focused on determining the site and mechanism of in vivo metabolism of hydroxyurea to NO or HNO. In addition to the direct oxidation of hydroxyurea to NO, we are also investigating an alternative mechanism of NO production from hydroxyurea which entails the initial hydrolysis of hydroxyurea to hydroxylamine with the subsequent oxidation of hydroxylamine to NO or HNO. In addition to these studies, our group also participates in collaborative work with laboratories at the National Institutes of Health and the UCLA medical school to determine how hydroxyurea-derived NO benefits sickle cell patients. These studies examine the ability of hydroxyurea to stimulate soluble guanylate cyclase, which ultimately controls fetal hemoglobin biosynthesis.

In collaboration with Dr. Kim-Shaprio, a series of biophysical experiments designed to evaluate the effects of hydroxyurea on the physical properties of sickle cell hemoglobin and sickle red blood cells has been initiated. Recently, we have shown that iron nitrosylation has little effect on the solubility of sickle cell deoxyhemoglobin, an important result when one considers the recent suggestion to use inhaled NO gas as a treatment for sickle cell disease. Other collaborative work with Dr. Kim-Shapiro examines the chemistry and biochemistry of the reactions of nitric oxide with hemoglobin. An important result from this work is that binding of nitric oxide to partially oxygenated hemoglobin does not follow cooperative binding behavior.

Hydroxyurea also inhibits the enzyme ribonucleotide reductase, the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. Inhibition of ribonucleotide reductase blocks DNA synthesis and stops cell division and hydroxyurea has been used as a treatment for a number of cancers. Hydroxyurea inhibits ribonucleotide reductase by quenching the catalytically essential tyrosyl radical of the enzyme. This reaction also generates the same hydroxyurea radical formed in the heme protein catalyzed oxidation of hydroxyurea that ultimately forms nitric oxide. Currently, we are evaluating the reaction of hydroxyurea with ribonucleotide reductase for NO formation. In addition, we have prepared carbohydrate and peptide-derived hydroxyureas as ribonucleotide reductase inhibitors and are testing these compounds both as inhibitors of ribonucleotide reductase and cytotoxic agents.

Recent Publications:

Sha X, Isbell TS, Patel RP, Day CS, King SB. Hydrolysis of acyloxy nitroso compounds yields nitroxyl (HNO). J Am Chem Soc. 2006 Aug 2;128(30):9687-92.

Alexander RL, Bates DJ, Wright MW, King SB, Morrow CS. Modulation of nitrated lipid signaling by multidrug resistance protein 1 (MRP1): glutathione conjugation and MRP1-mediated efflux inhibit nitrolinoleic acid-induced, PPARgamma-dependent transcription activation. Biochemistry. 2006 Jun 27;45(25):7889-96.

Gorczynski MJ, Huang J, King SB. Regio- and stereospecific syntheses and nitric oxide donor properties of (E)-9- and (E)-10-nitrooctadec-9-enoic acids. Org Lett. 2006 May 25;8(11):2305-8.

Huang J, Yakubu M, Kim-Shapiro DB, King SB. Rat liver-mediated metabolism of hydroxyurea to nitric oxide. Free Radic Biol Med. 2006 May 1;40(9):1675-81.

Poole LB, Zeng BB, Knaggs SA, Yakubu M, King SB. Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug Chem. 2005 Nov-Dec;16(6):1624-8.

Publications:
For a listing of additional publications, refer to PubMed, a service provided by the National Library of Medicine