The principal objective of this research program is to determine the structural parameters that define the functions of the isoforms of nitric oxide synthase (NOS) in their various milieus. L-Arginine is the single natural substrate for the NOS enzymes, producing both L-citrulline and NO., which serves as a gaseous messenger in effecting neurotransmission, cytotoxicity, or vasodilatation, among other biological effects. Three genes encode the NOS proteins: neuronal NOS (NOS-1; nNOS), inducible NOS (NOS-2; iNOS), and endothelial NOS (NOS 3; eNOS) and a number of other gene products found in various tissues resulting from alternative RNA splicing. All NOS isoforms require NADPH as a source of reducing equivalents for oxygenation of L-arginine to form NO.. The basic chemical mechanisms of NOS isoforms are similar to those demonstrated for cytochrome P450- mediated reactions but the extent of coupling of electron equivalents to the production of metabolites, overall reaction rates, and regulation of catalytic activity vary significantly among the isoforms. By understanding their individual structural properties, specific chemical interventions can be designed to regulate the activities of each of the isoforms. The overall hypothesis is that, despite requiring the identical complement of prosthetic groups and cofactors (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin) to catalyze the same enzymatic reaction, NOS isoforms have evolved different sequences and structural properties to accommodate their distinct functions.
The Specific Aims are: 1) to continue examining structural properties at the atomic level, using crystallographic methods, and to determine other biophysical properties of the NOS holoenzymes and derivative domains, using analytical ultracentrifugation, electron microscopy, and high pressure spectroscopy that determine their unique characteristics; 2) to characterize the protein-protein interactions that regulate NOS activities, using various biophysical methods including co-crystallization and surface plasmon resonance techniques, to measure interactions with bradykinin receptors, caveolins, dynamin, and nostrin or their respective interactive domains; and 3) to identify and quantify the various reduced oxygen species (O2-, H202, OONO-) produced by the NOS isoforms under a variety of conditions, including interactions with protein regulators, such as those in the cellular environment. A combination of site-directed mutants, chimeras, and modular constructs to dissect and characterize these processes has been used successfully in performing such studies in this laboratory.
|Kang, Soosung; Li, Huiying; Tang, Wei et al. (2015) 2-Aminopyridines with a Truncated Side Chain To Improve Human Neuronal Nitric Oxide Synthase Inhibitory Potency and Selectivity. J Med Chem 58:5548-60|
|Astashkin, Andrei V; Chen, Li; Elmore, Bradley O et al. (2015) Probing the Hydrogen Bonding of the Ferrous-NO Heme Center of nNOS by Pulsed Electron Paramagnetic Resonance. J Phys Chem A 119:6641-9|
|Jing, Qing; Li, Huiying; Roman, Linda J et al. (2014) Combination of chiral linkers with thiophenecarboximidamide heads to improve the selectivity of inhibitors of neuronal nitric oxide synthase. Bioorg Med Chem Lett 24:4504-4510|
|Cinelli, Maris A; Li, Huiying; Chreifi, Georges et al. (2014) Simplified 2-aminoquinoline-based scaffold for potent and selective neuronal nitric oxide synthase inhibition. J Med Chem 57:1513-30|
|Kang, Soosung; Tang, Wei; Li, Huiying et al. (2014) Nitric oxide synthase inhibitors that interact with both heme propionate and tetrahydrobiopterin show high isoform selectivity. J Med Chem 57:4382-96|
|Volkmann, Niels; Martásek, Pavel; Roman, Linda J et al. (2014) Holoenzyme structures of endothelial nitric oxide synthase - an allosteric role for calmodulin in pivoting the FMN domain for electron transfer. J Struct Biol 188:46-54|
|Huang, He; Li, Huiying; Yang, Sun et al. (2014) Potent and selective double-headed thiophene-2-carboximidamide inhibitors of neuronal nitric oxide synthase for the treatment of melanoma. J Med Chem 57:686-700|
|Trane, Andy E; Pavlov, Dmitri; Sharma, Arpeeta et al. (2014) Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance. J Biol Chem 289:13273-83|
|Jing, Qing; Li, Huiying; Chreifi, Georges et al. (2013) Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors. Bioorg Med Chem Lett 23:5674-9|
|Panda, Satya Prakash; Polusani, Srikanth R; Kellogg 3rd, Dean L et al. (2013) Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding. Arch Biochem Biophys 533:88-94|
Showing the most recent 10 out of 100 publications