This research proposal focuses on structural properties that govern the functional behavior of the nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in their respective environments. Three genes encode NOS enzymes, and other products of these genes are expressed in various tissues as a result of alternative RNA splicing. L-Arginine is the natural substrate for NOS isoforms, producing L-citrulline and NO, which serves as a gaseous messenger in the processes of neurotransmission, cytotoxicity or vasodilatation, among others, depending upon the isoform and tissue localization. The hypotheses to be addressed in this proposal are that the molecular design of the NOS isoforms, while requiring the same cofactors and prosthetic groups (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin), is adapted in each isoform to satisfy its specific cellular function. For example, sequence inserts in the constitutive NOS enzymes (nNOS and eNOS) confer regulatory properties that do not exist in the inducible isoform (iNOS). Therefore, molecular studies will be focused on further examination of structural properties, using new techniques to examine intrinsic regulatory elements and relationships and the determination of mechanisms that bear on O2 metabolism.
Specific Aim 1 : To determine the structural properties of the NOS holoenzymes and derivative domains, using crystallography and cryo-electron microscopy;
Specific Aim 2 : To address intrinsic regulation of the nNOS and eNOS by nuclear magnetic resonance spectroscopy, laser flash photolysis and protein film voltammetry to determine the mechanistic properties of these proteins;
and Specific Aim 3 : To address the mechanisms involved in O2 metabolism and the process of oxygenation, using rapid-freeze-quench ENDOR, and conventional O2 metabolism measurements. These experiments will test the following hypotheses: 1) that the overall architecture of each NOS isoform determines the intrinsic control of electron transfer through these enzymes;2) that these isoform-specific architectural features influence their catalytic regulation;and 3) that differential O2 metabolism is also based on structural features of the three isoforms.
The nitric oxide produced by the nitric oxide synthases plays vital roles in human neurotransmission, hemodynamic control, and the immune response. Aberrant production of nitric oxide has been implicated in endothelial dysfunction, septic shock, and other disease processes. Studying the structure and regulation of NOS in vitro will lead to greater the understanding of, and the ability to positively affect, these processes in humans.
|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|
|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|
|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; 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 24:4504-10|
|Volkmann, Niels; MartÃ¡sek, 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|
|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|
|Huang, He; Li, Huiying; Martasek, Pavel et al. (2013) Structure-guided design of selective inhibitors of neuronal nitric oxide synthase. J Med Chem 56:3024-32|
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