Emily E Scott

University of Kansas, Lawrence, Kansas, United States

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Publications (43)188.37 Total impact

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    Full-text · Dataset · Feb 2016
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    ABSTRACT: This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29th in Boston MA. The symposium focused on (1) the interactions of P450s with their redox partners, and (2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-cytochrome P450 reductase (CPR) and cytochrome b5. First, solution NMR was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6 - the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methodologies, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of CPR with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely-related CYPs, CYP1A1 and CYP1A2 resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.
    Full-text · Article · Feb 2016 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: To accomplish key physiological processes from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase (CPR). Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the CPR FMN domain delivers both electrons and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1·FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide including 87-90 (loop 1) and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the three-component system. These results and comparison with previous NMR studies of the CYP17A1·b5 complex suggest a model of CYP17A1 enzyme regulation.
    Preview · Article · Dec 2015 · Journal of Biological Chemistry
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    ABSTRACT: The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multi-functional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically-relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ~60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn202 in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggest a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. While 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.
    No preview · Article · Oct 2014 · Journal of Biological Chemistry
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    ABSTRACT: Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bi-functional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, as well as binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1/pregnenolone induced a set of conformational states closely resembling those of CYP17A1/17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.
    Preview · Article · Mar 2014 · Journal of Biological Chemistry
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    ABSTRACT: This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology in April 2013. Presentations discussed the status of cytochrome P450 knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b 5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein NMR is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of HIV drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.
    No preview · Article · Oct 2013 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Purpose: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. Methods: Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. Results: A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. Conclusions: Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.
    No preview · Article · Jun 2013 · Pharmaceutical Research
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    ABSTRACT: The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Herein the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bi-functional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction which is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicate that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (E48 or E49) or the corresponding cationic CYP17A1 residues (R347, R358, or R449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase (CPR). To probe the differential effects of b5 on the two CYP17A1-mediated reactions and thus communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.
    No preview · Article · Apr 2013 · Journal of Biological Chemistry
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    Agnes A Walsh · Grazyna D Szklarz · Emily E Scott
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    ABSTRACT: Cytochrome P450 (CYP) 1A1 is an extrahepatic monooxygenase involved in the metabolism of endogenous substrates and drugs, as well as the activation of certain toxins and environmental pollutants. CYP1A1 is particularly well known for its ability to biotransform polycyclic aromatic hydrocarbons, such as benzo[a]pyrene in tobacco smoke, into carcinogens. CYP1A1 possesses functional similarities and differences with human CYP1A2 and CYP1B1 enzymes, but the structural basis for this has been unclear. We determined a 2.6 Å structure of human CYP1A1 with the inhibitor α-naphthoflavone. α-Naphthoflavone binds within an enclosed active site, with the planar benzochromen-4-one core packed flat against the I helix that composes one wall of the active site, and the 2-phenyl substituent oriented toward the catalytic heme iron. Comparisons with previously determined structures of the related cytochrome P450 1A2 and 1B1 enzymes reveal distinct features among the active sites that may underlie the functional variability of these enzymes. Finally, docking studies probed the ability of CYP1A structures to assist in understanding their known in vitro interactions with several typical substrates and inhibitors.
    Preview · Article · Mar 2013 · Journal of Biological Chemistry
  • Natasha M DeVore · Emily E Scott
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    ABSTRACT: Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.
    No preview · Article · Jun 2012 · Journal of Biological Chemistry
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    Eva S Stephens · Agnes A Walsh · Emily E Scott
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    ABSTRACT: Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 μM⁻¹ · min⁻¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 μM⁻¹ · min⁻¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, β-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes.
    Preview · Article · Jun 2012 · Drug metabolism and disposition: the biological fate of chemicals
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    Natasha M DeVore · Emily E Scott
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    ABSTRACT: Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.
    Preview · Article · Feb 2012 · Nature
  • Agnes Walsh · Grazyna D. Szklarz · Emily E. Scott

    No preview · Conference Paper · Jan 2012

  • No preview · Conference Paper · Jan 2012
  • Emily Scott

    No preview · Conference Paper · Jan 2012
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    Natasha M. DeVore · Emily E. Scott

    Preview · Dataset · Dec 2011
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    Natasha M. DeVore · Emily E. Scott

    Preview · Dataset · Dec 2011
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    ABSTRACT: Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine. Database Structural data are available in the Protein Data Bank database under the accession numbers 3T3Q (CYP2A6 I208S/I300F/G301A/S369G with pilocarpine), 3T3R (CYP2A6 with pilocarpine), 3T3S (CYP2A13 with pilocarpine), and 3T3Z (CYP2E1 with pilocarpine)
    Preview · Article · Nov 2011 · FEBS Journal
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    ABSTRACT: Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-Å resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S]2+) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr60, Trp264, and Trp355 provide the framework for the “aromatic bowl” that serves as a trimethylamine-binding site in TMADH is comprised of Gln65, Trp267, and Asp358, respectively, in HADH. The surface Tyr442 that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.
    Full-text · Article · Aug 2010 · Journal of Biological Chemistry
  • Patrick R Porubsky · Kevin P Battaile · Emily E Scott
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    ABSTRACT: Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9-C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with omega-imidazolyl-octanoic fatty acid, omega-imidazolyl-decanoic fatty acid, and omega-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the omega-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe(298) side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B' helices at successive distances from the active site.
    No preview · Article · May 2010 · Journal of Biological Chemistry