This proposal centers on structure-function relationships in heme proteins with a special emphasis on the heme thiolate enzymes cytochrome P450 and nitric oxide synthase (NOS). P450s play critical roles in drug detoxification, steroid biosynthesis, and in the oxidative assimilation of various natural organic compounds by microorganisms. With P450s our efforts currently are focused on the interaction between P450s and their redox partners. There are only 3 crystal structures of a P450-partner complex and one of these, the complex formed between P450cam and its redox partner (a ferredoxin called Pdx), shows that Pdx induces a large structural change in P450cam that we hypothesize is required for proton coupled electron transfer. P450cam is quite specific for Pdx and no other ferredoxin or related protein can support P450cam catalysis. The goal now is to ask whether or not this property is a more general feature of P450s and if not, why not. What is the biological advantage for such a high level of control? To probe these questions we plan to study in depth other P450 redox partners to better understand how redox partner binding effects the proton coupled electron transfer reaction. Our studies on P450s also extend to mammalian P450s and especially P4503A4, the most abundant and important human P450 for drug metabolism. Our goal here is to develop a pharmacophore using novel inhibitors that probe the dynamics and adaptability of the P4503A4 active. This will provide important information on drug-drug interactions and allosteric mechanisms in P4503A4. With NOS, our efforts center on structure-based inhibitor/drug design. The overproduction of NO is well known to be associated with a number of pathological conditions and we currently are focusing on neurodegenerative diseases, melanoma, and pathogenic bacteria. In each of these 3 cases we know that inhibiting NO production by blocking NOS has potential therapeutic benefits. We now are using a broad range of approaches toward developing selective NOS inhibitors for each of the 3 potential targets. One final area is the structural biology of heme transport in bacterial pathogens. Certain bacterial pathogens must acquire host iron by taking up free heme followed by heme degradation and release of iron. This requires a complex transport system involving several proteins many of which are membrane bound. The goal here is to work out the structural biology of heme transport and especially to better understand the many protein-protein interactions required for successful delivery of heme from hemoglobin to the bacterial cytoplasm.

Public Health Relevance

Cytochromes P450, nitric oxide synthase (NOS), and pathogenic bacterial heme transport proteins all play critical roles in human health. P450s are essential for drug metabolism while nitric oxide (NO) produced by human NOS enzymes plays essential roles in the neuronal, cardiovascular, and immune systems. Critical to bacterial pathogenesis is the ability of these organisms to acquire host iron from heme and understanding the structural biology of the proteins involved opens the way for structure-based drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM057353-17A1S1
Application #
8929464
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Smith, Ward
Project Start
1998-05-01
Project End
2019-08-31
Budget Start
2014-09-15
Budget End
2015-08-31
Support Year
17
Fiscal Year
2014
Total Cost
$34,298
Indirect Cost
$5,123
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Poulos, Thomas L (2014) Heme enzyme structure and function. Chem Rev 114:3919-62
Chreifi, Georges; Li, Huiying; McInnes, Craig R et al. (2014) Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase. Biochemistry 53:4216-23
Astashkin, Andrei V; Chen, Li; Zhou, Xixi et al. (2014) Pulsed electron paramagnetic resonance study of domain docking in neuronal nitric oxide synthase: the calmodulin and output state perspective. J Phys Chem A 118:6864-72
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
Li, Huiying; Jamal, Joumana; Plaza, Carla et al. (2014) Structures of human constitutive nitric oxide synthases. Acta Crystallogr D Biol Crystallogr 70:2667-74
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
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
Li, Huiying; Jamal, Joumana; Delker, Silvia et al. (2014) The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases. Biochemistry 53:5272-9
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
Holden, Jeffrey K; Lim, Nathan; Poulos, Thomas L (2014) Identification of redox partners and development of a novel chimeric bacterial nitric oxide synthase for structure activity analyses. J Biol Chem 289:29437-45

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