Dissertations / Theses on the topic 'Cytochrome P45Os'
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Stok, Jeanette Elizabeth. "Biosynthetic cytochrome P450s /." [St. Lucia, Qld.], 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16210.pdf.
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Orr, Catherine. "Characterisation of equine cytochrome P450s." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33315/.
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Cytochrome P450s (CYPs) are a superfamily of enzymes involved in the phase I metabolism of endogenous and exogenous substances. They are present in almost all forms of life and have been studied extensively, particularly in relation to human medicine, where knowledge of their activities is essential for predicting drug-drug interactions. In the horse, little is currently known about CYP-specific drug metabolism, which holds importance for animal welfare and for doping control within the horseracing industry where drug-specific metabolites are tested for on race days. Recently the first recombinant equine CYPs have been produced, allowing specific data on equine P450 activity to be gathered for the first time. During the current study,46 full-length P450 sequences were identified from the equine genome. RT-PCR analysis was then carried out on equine liver in order to detect hepatic expression of P450s across various families. After this, cold-induction (pCold) E. coli were used for production of recombinant P450 proteins for subsquent functional testing. Four recombinant equine P450s were successfully expressed (CYP1A1, CYP2A13, CYP2C92 and CYP2D50). Due to being the isoforms most likely to be involved in drug metabolism, rCYP2D50 and rCYP2C92 were selected to be screened against ten of the most commonly used horse drugs to identify potential substrates. rCYP2C92 appeared to metabolise all four NSAIDs tested (flunixin, ketoprofen, phenylbutazone and diclofenac), however presence of the known hydroxylated metabolites of diclofenac and phenylbutazone (4-hydroxydiclofenac and oxyphenbutazone, respectively) could not be confirmed despite being present within equine liver microsome and human recombinant CYP2C9 samples. In spite of the apparant acivity displayed by rCYP2C92 towards all four NSAIDs, no conclussions can be made about this enzyme’s role in NSAID metabolism due to a lack of known hydroxylated metabolite production.
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Rylander, Rudqvist Tove. "Extrahepatic cytochrome P450s : relation to cancer susceptibility /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-601-4/.
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Kusimo, Michael Olugbenga. "Characterisation of cytochrome P450s in Anopheles gambiae." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/9313/.
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Cytochrome P450s provide a natural mechanism by which insects defend themselves against the relatively small number of insecticides approved by WHO in fighting malaria vectors. Three dimensional structures of these enzymes are important to aid our understanding of the mechanism of their action in dissipating the harmful effects of such insecticides. However, to date no coordinates of any insect P450 structure have been deposited in the Protein Data Base. In this study a C-terminally, His-tagged, recombinant CYP6Z2, CYP for cytochrome P450, was constructed genetically and its expression and biochemical properties evaluated in an attempt to produce material suitable for crystallisation trials and ultimately X-ray structural analysis. The recombinant enzyme was purified in a soluble form through the introduction of a range of chemical agents including sodium cholate. A series of truncating mutants were constructed to determine the minimum set of primary structural elements that create a barrier to crystallisation. Genetically engineered truncations (11 amino acids and 22 amino acids) of the N-terminal hydrophobic region did not produce holoprotein. The N-terminal region appears to be essential to ensure the proper folding of insect P450 investigated, which may be the principal reason underpinning the lack of success in producing crystal structures for this group of enzymes. The homology model of CYP6Z2 used in this thesis, compared with those of the confirmed pyrethroid metabolisers; CYP6D1 and CYP6P3, revealed three key amino acid differences at their active sites. Single and double mutations of these three amino acids were constructed to explore their individual and joint roles in the metabolic activities of CYP6Z2. The single mutants, CYP6Z2 (Y102F) and CYP6Z2 (F212L) retained similar affinities as wild type for both benzyloxyresorufin (BR) and methoxyresorufin (MR) fluorescent probes. However, the turnover number of CYP6Z2 (Y102F) was 1.7-fold lower than the wild type for BR but shows improved activity with MR achieving 2-fold increase in turnover. CYP6Z2 (F212L) maintained the same turnover number with the wild type against BR but attained a 3.9-fold increase in activity against the methoxy derivative. Deletion of the positively charged arginine in CYP6Z2 (R210A) significantly improved the activity of the enzyme. The turnover number increased by 3-fold against BR and the enzyme effectiveness also increased by 2-fold. To further investigate the roles of these residues, the same mutations were introduced into CYP6P3. The addition of a hydroxyl group in CYP6P3 (F110Y) changed the kinetic behaviour of the enzyme drastically: the mutant lost activity with the resorufin probe and the affinity for the DEF probe was lowered by 7-fold. Conversely, introduction of benzyl ring in CYP6P3 (L216F) did not affect the affinity, but the enzyme’s activity increased by 15-fold with DEF. CYP6P3 (L216F) also improved the pyrethroid metabolising ability of the enzyme. These experiments clearly point to the important contribution of these amino acids in modulating the metabolic characteristics of these P450s. Similar studies were carried out to investigate the function of F115 in CYP6Z2 and CYP6Z3. This residue is frequently found close to the heam in P450s, where it is believed to play a key stabilising role in substrate recognition. A range of mutants were prepared and all produced holoproteins except F115H. The results presented in this thesis, point to a significant role in metabolic function for this residue, F115A, in CYP6Z3, and a rationalisation of the data is presented. Finally, the metabolic activities of CYP6Z2 and CYP6Z3 were compared in this study. Results presented here, suggest that both act independently in the presence of cytochrome b5 in their activities against the resorufin fluorescent probes but their activities with diethyl fluorescein (DEF) probe increased significantly when cytochrome b5 is present. CYP6Z3 has 2-fold turnover rate and 5-fold greater efficiency more than CYP6Z2 in dealkylation of BR and 8-fold and 10-fold in turnover and efficiency respectively for MR. These metabolic comparisons suggest strongly that CYP6Z3 is an improved “version” of CYP6Z2.
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Hodgkinson, Conrad Phillip. "Expression of cytochrome P450s in rat hepatocyte culture." Thesis, Queen Mary, University of London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244085.
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Gillett, Lorna. "Function of cytochrome P450s in the CYP4 family." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408051.
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Pineau, Emmanuelle. "Formation des acides gras poly-hydroxylés et incorporation dans la cutine chez Arabidopsis thaliana." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ052/document.
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Les plantes sont des organismes sessiles qui ne peuvent fuir des conditions souvent défavorables et doivent par conséquent s’adapter à un environnement hostile pour survivre. La cutine partie intégrante de la cuticule qui joue un rôle de barrière pour la plante est un polymère lipidique constitué principalement d’acides gras en C16 et C18 hydroxylés et époxydés reliés entre eux par des liaisons ester mettant en jeu les fonctions carboxyl et ω-hydroxyl des acides gras. La cutine ne joue pas seulement un rôle de barrière physique mais joue un rôle de réservoir de molécules possédant des propriétés physiologiques fondamentales. Grâce à des approches biochimiques et génétiques, nos travaux ont permis de mettre en évidence AtEH1, une époxyde hydrolase responsable de la formation des diols incorporés dans la cutine d’Arabidopsis thaliana. Ces diols sont décrits dans la littérature comme intervenant dans les interactions plante-pathogène. Nous avons également montré que ces composés ainsi que d’autres dérivés d’acides gras sont perçus par la plante. Nous avons identifié et caractérisé CYP77B1, une époxygénase d’acide gras qui a un rôle potentiel à jouer dans la formation d’acides gras polyhydroxylés incorporés dans la cutinePlants are sessile organisms that are not able to escape from difficult environmental conditions and therefore have to adapt to multiple abiotic and biotic stress to survive. Cutin is a part of the cuticle which plays a major role as a barrier for the plant. It’s a lipid polymer composed mainly by hydroxylated and epoxidized C16 and C18 fatty acids linked together by ester links involving the carboxyl and ω-hydroxyl functions of those fatty acids. Cutin plays also a role as a reservoir of molecules with fundamental physiological properties. With biochemical and genetic approaches, we characterized AtEH1, an epoxide hydrolase responsible for the formation of diols incorporated in Arabidopsis thaliana cutin. These diols are described as being involved in plant-pathogen interactions. We also showed that these compounds as well as others fatty acids derivatives are perceived by plants. We have also identified and characterized CYP77B1, an epoxidase that has a potential role in the formation of polyhydroxylated fatty acids incorporated in cutin
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Hunter, Arwen Leigh. "The role of peri-transplant ischemia and reperfusion injury in cardiac allograft vasculopathy." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/2503.
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Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of I/R during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV.
ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats being treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs.
In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction.
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Rosic, Nedeljka. "Molecular breeding of cytochrome P450s for indigoid pigment production /." [St. Lucia, Qld.], 2005. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18978.pdf.
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Dodhia, Vikash Rajnikant. "Engineering human cytochrome P450s for structural and biosensing studies." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429524.
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Davies, Lindsay. "Molecular cloning and characterization of cytochrome P450s metabolising ecdysteroids." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269568.
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Nisbar, Nur Dayana Binti. "Characterisation of orphan cytochrome P450s from Mycobacterium tuberculosis H37Rv." Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-orphan-cytochrome-p450s-from-mycobacterium-tuberculosis-h37rv(5f953958-9722-4531-b8b2-b034adaaabb0).html.
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Tuberculosis is a disease that kills more people every year than any other infectious disease and is caused by the human pathogen, Mycobacterium tuberculosis (Mtb). This disease can be treated by a standard six month course of four antimicrobial drugs that have been in use since the 1960s. However, the rise of multi-drug resistant and extensively drug-resistant strains of TB has complicated the efforts to eradicate the disease. Therefore, there is a critical need for the development of new anti-TB drugs with a novel mechanism of action that can speed up treatment duration and help avoid resistance. The discovery of twenty genes encoding cytochrome P450 enzymes in the Mtb H37Rv genome sequence has pointed to the significance of these enzymes in the physiology and pathogenicity of this bacterium. Consequently, the characterisation of these Mtb P450 enzymes may define their physiological roles of which can be a novel anti-tubercular drug target. To date, the characterisations of selected Mtb P450 enzymes have highlighted their diverse and unexpected roles in the metabolism of cholesterol and lipids and the production of secondary metabolites. Biochemical and biophysical studies of these enzymes provided knowledge of their active site properties that may be exploited for drug discovery. Therefore, with the prospect of defining novel functions and identifying novel drug targets, characterisations of the remaining orphan Mtb P450s is of interest. M. tuberculosis CYP141A1 and CYP143A1 are orphan enzymes with unknown physiological function in Mtb which is characterised in this study through use of various spectroscopic and biophysical techniques. Interestingly, CYP141A1 can be expressed in form of which 54 amino acids (Del54CYP141A1) are deleted from the N-terminus. Although Del54CYP141A1 still retain spectroscopic characteristics, this form of P450 cannot be crystallized. Optimisation of full-length CYP141A1 buffer composition resulted to the formation of reproducible crystals and determination of CYP141A1 structure. Spectroscopic and structural characterisations presented in this thesis revealed many characteristics of CYP141A1 and CYP143A1 are comparable to previous Mtb P450s reported to date. CYP141A1 and CYP143A1 active site consist of b-type heme iron ligated by cysteine residue and a water molecule at its proximal and distal face, respectively. Both enzymes bind tightly to azole antifungal drugs highlighting their potential as a drug target. In addition, fragment-based screening applied to CYP141A1 and CYP143A1 provided the starting point for the development of potent, isoform-specific inhibitors for both orphan Mtb P450 enzymes. The first crystal structure of CYP141A1 and identification of new fragment binders of CYP141A1 and CYP143A1 are presented in this thesis. Overall, this research remains significant in providing new knowledge on the spectroscopic and structural properties of the M. tuberculosis P450s CYP141A1 and CYP143A1.
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Basom, Edward J. "Dynamics and Conformational Heterogeneity in Cytochrome P450s via Infrared Spectroscopy." Thesis, Indiana University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10604874.
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Cytochrome P450s (P450s) are a superfamily of enzymes that catalyze oxidation of unactivated hydrocarbons. However, the means by which P450s control (1) regioselectivity of their activity and (2) specificity in their molecular recognition remain largely elusive. Toward investigation of the role of dynamics in the regioselectivity of the archetypal cytochrome P450cam (P450cam), two-dimensional infrared spectroscopy has been applied with heme-bound carbon monoxide (CO) as an infrared probe of the active site. The data support a model for P450cam regioselectivity in which binding of different substrates to P450cam variably stabilizes the active site into two distinct states, each associated with different dynamics linked to different levels of regioselectivity. To investigate the role of conformational heterogeneity in P450cam substrate specificity, infrared spectoscopy was combined with the site-specific incorporation of nitrile probes at distinct P450cam microenvironments. This approach enabled differentiation of changes experienced at each of those environments when d-camphor and/or CO binds to the active site. Finally, the impact of conformational heterogeneity on the affinity of substrate molecular recognition by wild-type and mutant P450cam was evaluated using both CO and nitrile probes. This study suggests that the nature of the conformations populated in the unbound states influences the affinity for different substrates. Collectively, these studies provide new insight into the roles of conformational heterogeneity and dynamics in P450cam activity. Furthermore, these studies help to lay the foundation for efforts toward understanding the roles of conformational heterogeneity and dynamics in the function of human P450s, for which unraveling the mechanisms involved in Phase I metabolism is a topic of great pharmacological concern.
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Hahn, Christopher Norman. "Regulation of chicken cytochrome P450s and 5-Aminolevulinic acid synthase." Title page, contents and summary only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phh148.pdf.
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Brundage, Meghan Elizabeth. "CLONING OF KNOWN AND NOVEL CYTOCHROME P450S IN SCUTELLARIA BAICALENIS." University of Cincinnati / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin998483270.
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Trnka, Michael J. "Photoaffinity labeling of cytochrome P450s with imidazole-tethered benzophenone compounds /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/8516.
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Richmond, Alison. "Expression and characterisation of biomedically and biotechnologically important bacterial cytochrome P450s." Thesis, University of Strathclyde, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275059.
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Ing, Rachael Helen. "Enzymology and induction of hepatic cytochrome P450s in the guinea pig." Thesis, De Montfort University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393360.
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Changenet, Valentin. "Towards new roles for cytochrome P450s and strigolactones in Fusarium Head Blight of Brachypodium distachyon." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS354/document.
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La fusariose des épis est l’une de maladies les plus dommageables des céréales tempérées et est principalement causée par le champignon toxinogène Fusarium graminearum (Fg). Ces dix dernières années, de nombreuses études ont rapporté l’induction transcriptionnelle de gènes de la plante codant pour des cytochromes P450 (P450) en réponse l’infection par Fg. Les P450s constituent une famille enzymatique impliquée dans de nombreuses voies métaboliques, certaines avec des intérêts potentiels dans la résistance face aux maladies. Nous avons utilisé la petite graminée modèle Brachypodium distachyon (Bd) pour caractériser fonctionnellement le premier gène codant pour un P450 induit chez la plante au cours de la fusariose des épis par l’utilisation de lignées altérées dans la séquence ou l’expression du gène Bradi1g75310 codant le P450 BdCYP711A29. Nous avons montré qu’en plus d’être un facteur de sensibilité à la maladie, le gène Bradi1g75310 est impliqué dans une voie de biosynthèse hormonale chez Bd, celle des strigolactones (SLs). En effet, en plus de complémenter génétiquement les phénotypes aériens de la lignée mutante max1-1 d’Arabidopsis thaliana altérée dans le gène homologue MAX1 (AtCYP711A1), une lignée de Bd surexprimant Bradi1g75310 (lignée OE) exsude davantage d’orobanchol, une SL spécifique, que la lignée sauvage ou mutante. Une analyse préliminaire de l’impact direct de l’orobanchol sur la croissance de Fg semble indiquer une activation des étapes précoces du développement du champignon (germination) qui pourrait être à l’origine de l’induction plus rapide de gènes de défenses observée chez une lignée OE de Bradi1g75310. Nous avons également montré que les 4 paralogues de Bradi1g75310 chez Bd, qui codent également pour des CYP711A, sont tous capable de complémenter la lignée max1-1 et avons généré du matériel végétal fondamental pour la poursuite de l’étude de la diversification des SLs chez la plante monocotylédone modèle Bd. Au global, ce projet constitue une première étape dans la caractérisation de l’implication des P450 dans la réponse de la plante face à l’infection par Fg en plus de donner de nouveaux indices concernant le rôle des SLs dans les interactions plante-pathogène. Les résultats obtenus au cours de ce travail de thèse pourront permettre l’amélioration de caractères tant développementaux que de résistance à la fusariose chez les céréales cultivéesFusarium Head Blight (FHB) is one of the most important diseases of temperate cereals and is mostly caused by the toxin producing-fungus Fusarium graminearum (Fg). This last decade, several studies reported the transcriptional activation of cereal cytochrome P450-encoding genes (P450s) in response to Fg infection. P450s constitute an enzymatic family participating in very diverse metabolic pathways with potential interest for disease resistance. We used the model temperate cereal Brachypodium distachyon (Bd) to functionally characterize the first FHB-induced P450- encoding gene using Bd lines altered in the locus or gene expression of the Bradi1g75310 gene encoding the BdCYP711A29 P450. We showed that in addition to be a plant susceptibility factor towards the disease, the Bradi1g75310 gene is involved in the hormonal biosynthetic pathway of strigolactones (SLs) in Bd. Indeed, in addition to genetically complement the shoot phenotypes of the Arabidopsis thaliana mutant line for the homologous gene MAX1 (AtCYP711A1, max1-1 line), a Bd linewhich overexpresses the Bradi1g75310 gene (OE) exudes more orobanchol, a specific SL, compared to wild-type or mutant lines. Preliminary analysis of the direct impact of orobanchol on Fg growth suggests an activation of early fungal development (germination) likely to induce faster induction of defense-related genes during FHB, observed in Bradi1g75310 OE line. We showed that the four paralogs of Bradi1g75310 encoding BdCYP711A P450s are all able to genetically complement max1-1 line and provide important plant material for studying SLs diversification in the model monocot B. distachyon. Overall, this project constitutes a first step in the characterization of P450s involvement in plant response towards Fg infection in addition to give new evidences about the role of SLs in plant-pathogen interactions. Results obtained during this Ph.D. project will allow the improvement of both developmental and FHB-related traits in cereal crops
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Nikou, Dimitra. "Molecular analysis of multiple cytochrome P450s from the maleria vector Anopheles gambiae." Thesis, University of Liverpool, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275044.
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Withers, John C. "CHARACTERIZATION OF TWO NOVEL CYTOCHROME P450S INVOLVED IN GRAVITROPISM IN ARABIDOPSIS THALIANA." Ohio University / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1187184960.
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Zhu, Shiqian. "The roles of cytochrome P450s in the toxicity of polycyclic aromatic hydrocarbons (PAHs) /." Full text available from ProQuest UM Digital Dissertations, 2007. http://0-proquest.umi.com.umiss.lib.olemiss.edu/pqdweb?index=0&did=1800288781&SrchMode=1&sid=12&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1258491890&clientId=22256.
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Yadav, Jaydeep. "EVALUATING PHARMACOKINETIC DRUG-DRUG INTERACTIONS DUE TO TIME DEPENDENT INHIBITION OF CYTOCHROME P450s." Diss., Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/524248.
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Pharmaceutical SciencesPh.D.
Time-dependent inactivation (TDI) of CYPs is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to over-predict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human liver microsomes. Inhibitors evaluated include troleandomycin (TAO), erythromycin (ERY), verapamil (VER), Paroxetine (PAR), itraconazole (ITZ) and diltiazem (DTZ) along with primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (MA). Complexities incorporated in the models included multiple binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The different factors affecting TDI kinetics were evaluated such as lipid partitioning, inhibitor depletion, presence of transporters. The inactivation parameters obtained from numerical method were incorporated into static in-vitro – in-vivo correlation (IVIVC) models to predict clinical DDIs. For 123 clinically observed DDIs, using a hepatic CYP3A synthesis rate constant of 0.000146 min-1, the average fold difference between observed and predicted DDIs was 2.97 for the standard replot method and 1.66 for the numerical method. Similar results were obtained using a synthesis rate constant of 0.00032 min-1. These results suggest that numerical methods can successfully model complex in-vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach. Chapter one presents the detailed introduction along with the hypothesis and significance of the project. Chapter 2 includes the development of the bioanalytical method for quantitation of various compounds which includes inactivators and their primary metabolites. Chapter 3 entails the discussion on in-vivo studies in rats involving TDI mediated DDI studies. Chapter 4 discusses the in-vitro studies and use of the numerical method for evaluation of TDI kinetics. Chapter 5 and chapter 6 provides discussion on the impact of inhibitor depletion and partitioning of TDI kinetics and how these two could lead to misinterpretation of TDI results. Chapter 6 also provides a discussion on how transporters could affect TDI results mainly from hepatocyte studies. Chapter 7 involves prediction of TDI mediated DDI using static modeling. Chapter 8 is a case study on bosentan involving induction mediated DDI.
Temple University--Theses
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Gravot, Antoine. "Étude de P450s impliqués dans la biosynthèse des furocoumarines chez Ruta graveolens." Vandoeuvre-les-Nancy, INPL, 2002. http://www.theses.fr/2002INPLA63N.
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Ruta graveolens est une plante présentant des fortes teneurs en 5-méthoxypsoralène (5-MOP), une furocoumarine particulièrement intéressante d'un point de vue médical. Nous avons choisi d'étudier les étapes enzymatiques clés de la biosynthèse de cette molécule. Ces étapes sont catalysées par des cytochromes P450. Les travaux effectués au cours de cette thèse ont porté sur le clonage et la caractérisation de plusieurs de ces P450s (la Cinnamate 4-hydroxylase (C4H) et deux psoralène monooxygénases). Un ADNc complet codant pour la C4H a été isolé, et exprimé dans la levure. Après avoir caractérisé l'enzyme recombinante, nous avons mis en évidence un phénomène d'inactivation auto-catalytique de la C4H par le psoralène et le 8-MOP. Nous avons par ailleurs mis en évidence que les C4Hs de R. Graveolens et de P. Crispum - deux plantes productrices de furocoumarines - sont moins sensibles à l'inactivation par les furocoumarines que la C4H de H. Tuberosus.
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Al-Nuaemi, Inas. "Investigating the industrial application potentials of cytochrome P450 BM-3 and four novel putative cytochrome P450s isolated from Cupriavidus necator H16." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/22435/.
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Cytochrome P450 is a superfamily of heme-dependent monooxygenases. These enzymes play a very important role in drug metabolism and detoxification since they catalyse a broad range of reactions including hydroxylation, dehalogenation, deamination, epoxidation, peroxidation, desulphurisation and dealkylation involving various substrates, such as alkanes, phenols, steroids and fatty acids. This thesis presents results from two P450 hemoprotein investigations: I) targeting study of the most common cytochrome P450 BM-3 from Bacillus megaterium, and II) discovery study of four novel putative cytochrome P450s from Cupriavidus necator H16. In Part II of this project, P450 BM-3 WT and W5F5 variants were expressed and purified, and their activities in three green solvents, 1-butanol, 2-butanol and dimethyl carbonate, were examined to check the tolerance of the wild type and the mutant W5F5 variants towards green solvents used for the first time with this enzyme. The results revealed a new green solvent with lower denaturation effect on P450 BM-3, the wild type and the mutant, with potential application in industrial biosynthesis. Part III focuses on the characterisation of four novel cytochrome P450 proteins of unknown structure and function. The proteins were analysed structurally and the first predicted models were discussed. Optimisations of the expression and purification of these proteins as well as optical properties were analysed. Scanning the changes in light absorbance of the substrate-protein complex with eleven substrates resulting in Type I changes when lauric acid and 3-phenoxytoluene were added to the purified P450-H16-B1743, as well as the evaluation of the catalytic activity of P450-H16-B1279 showed possible catalytic activity towards indol, malonic acid, adipic acid, 3-phenoxytoluene and S-Ethyl-N,N-dipropylthiocarbama (EPTC).
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Chenge, Jude. "Investigating orphan cytochrome P450s in Mycobacterium tuberculosis : insights into enzyme structure, function and inhibitor design." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/investigating-orphan-cytochrome-p450s-in-mycobacterium-tuberculosis-insights-into-enzyme-structure-function-and-inhibitor-design(2965ac43-d907-41d8-8222-1f327dc745e4).html.
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The World Health Organization regards tuberculosis as a world pandemic disease. There is increased demand for new drugs to tackle this threat. This threat has been further elevated with the emergence of drug resistant strains of the causative pathogen, Mycobacterium tuberculosis (Mtb), thereby increasing the urgency for development of novel anti-tubercular drugs. Success in whole genome sequence determination of Mtb revealed a large cohort of cytochrome P450 (CYP) enzymes. Research on these Mtb P450s has shown that several of them are critical to the survival of the pathogen. CYP121A1 and CYP128A1 have been demonstrated to be essential using knockout experiments. CYP125A1 and CYP142A1 have been shown to play crucial roles in bacterial catabolism of host steroids, with CYP125A1 also shown to be located within a gene cluster highly important for bacterial virulence and infectivity. CYP144A1 was shown to be one of the genes whose expression is elevated when Mtb was exposed to macrophage-like conditions, and gene knockout studies using the H37Rv virulent strain of Mtb indicated the ΔCYP144A1 mutant to be more sensitive to the clotrimazole antifungal. CYP126A1 was shown to be located within a cluster of genes highly important for the de novo synthesis of purines in Mtb. These and other data suggested these enzymes to be important to the growth process of Mtb and thus potential drug targets for developing novel therapeutics. Findings in this PhD have revealed that many characteristics of CYP144A1 and CYP126A1 are comparable to previous Mtb P450s reported to date. CYP144A1 is highly conserved within the Mycobacterium genus and specifically within pathogenic species. Transcriptomic analysis has revealed an alternative truncated transcript leading to the production of two physiologically relevant versions of CYP144A1. Our comparative biophysical characterization of both versions (CYP144A1-FLV and -TRV) show both enzymes to be similar in their binding tightly to azole antifungals. EPR and DSC studies show that the 30 amino acid truncation (to form CYP144A1-TRV) does not affect the heme electronic environment and the overall thermal stability of the enzymes. X-ray crystallography was utilized to determine the first crystal structure of a Mtb CYP144 family enzyme. The structure reveals that CYP144A1 possesses a large hydrophobic active site primed for accommodating large hydrophobic substrates. Further chemoproteomic profiling identified novel compounds, which bind in both inhibitor-like and substrate-like modes to CYP144A1, resulting in the development of novel CYP144A1 compounds for use as chemical probes for this P450. Fragment and compound screening identified several ligands with varying binding affinities for CYP126A1, suggesting that this P450 is capable of binding and catalyzing reactions with a wide range of substrates. Turnover experiments proved catalytic activity of CYP126A1 on one of these compounds (Compound 4). Crystallization of CYP126A1 with various compound “hits” (compounds 1 and 7, the azole drug ketoconazole) revealed involvement of several important residues within the active site of CYP126A1 in interactions with these molecules, thus providing important information for designing inhibitors for this enzyme. Both CYP144A1 and CYP126A1 display important characteristics that contribute to our general understanding of cytochromes P450 as a whole, and of Mtb P450s in particular. This PhD project has established the first instance of leaderless transcripts in Mtb P450s and has presented the first crystal structures of both CYP144A1 and CYP126A1, as well as identifying novel, useful chemicals that can be used as mechanistic probes for these enzymes as well as providing the basis for Mtb P450 isoform-specific inhibitors.
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Xu, Haibo. "Effects of human milk and formula on the expression of cytochrome P450s in cell lines." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ63179.pdf.
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Bateson, Hannah. "Detection and enrichment of cytochrome P450s using bespoke affinity chromatography and proteomic techniques : development of chemical immobilisation and novel affinity chromatography methods, with subsequent proteomic analysis, for the characterisation of cytochrome P450s important in cancer research." Thesis, University of Bradford, 2012. http://hdl.handle.net/10454/5712.
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Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.
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Swami, Shalini. "Structure and biochemistry of the orphan cytochrome P450s CYP126A1 and CYP143A1 from the human pathogen Mycobacterium tuberculosis." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/structure-and-biochemistry-of-the-orphan-cytochrome-p450s-cyp126a1-and-cyp143a1-from-the-human-pathogen-mycobacterium-tuberculosis(a8dc4eea-678c-419a-a3b3-206253c400b7).html.
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Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.
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Mohamed, Hanafy Mahmoud Ismail. "Developing novel pyrethroid activity based probes to identify cytochrome P450S associated with pyrethroid resistance in disease vectors." Thesis, University of Liverpool, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548796.
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Smith, Susan James. "A resonance Raman and surface enhanced resonance Raman study of cytochrome P450s and their substrate/inhibitor interactions." Thesis, University of Strathclyde, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288604.
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JEAN, PASCALE. "Mecanisme de l'inhibition des cytochromes p450s hepatiques par les 2-aroyl thiophenes et modelisation moleculaire (doctorat : toxicologie)." Paris 5, 1997. http://www.theses.fr/1997PA05N137.
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Xu, Yun. "Mechanistic studies on the differences between in vitro and in vivo inhibition potencies of fluvoxamine towards various cytochrome P450s /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/7972.
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LOUERAT, ORIOU BENEDICTE. "Purification et caracterisation des nadph-cytochromes p450 reductases humaine, de plante et de levure, et analyse de leurs couplages avec les formes purifiees des p450s 1a et 2b." Paris 5, 1998. http://www.theses.fr/1998PA05N116.
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Compagnon, Vincent. "Régulation de l'expression et rôles physiologiques de cytochromes P450s catalysant l'hydroxylation d'acides gras chez Vicia sativa et Arabidopsis thaliana." Université Louis Pasteur (Strasbourg) (1971-2008), 2006. https://publication-theses.unistra.fr/restreint/theses_doctorat/2006/COMPAGNON_Vincent_2006.pdf.
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Les acides gras hydroxylés en position terminale (omega) jouent des rôles très variés dans la plante : comme constituants des enveloppes externes (cutine et subérine), comme messagers secondaires lors d’interactions plantes-pathogènes, ou comme intermédiaires du catabolisme des acides gras. Les isoformes dse cytochromes P450 des familles CYP86 et CYP94 sont susceptibles d’intervenir dans ces réactions. Un des enjeux de ce travail de thèse est d’identifier leur rôle physiologique dans la plante. Chez Vicia sativa, l’expression de CYP94A1 est fortement induite lorsque divers composés (clofibrate, 2,4-D, 2,3-D, AIA, SA, méthyljasmonate) sont appliqués aux plantes à des concentrations élevées (50 à 500 mM). L’analyse du promoteur de ce gène a révélé la présence d’une séquence régulatrice de type as-1, qui, par analogie avec la régulation d’une gluthation-S-transferase, suggère une fonction de détoxication des acides gras libres. CYP86B1 et CYP86B2 d’Arabidopsis thaliana participent à la biosynthèse de subérine dans l’endoderme des racines, et probablement à la production de lipides du manteau pollinique et de la paroi des grains de pollenOmega-hydroxylated fatty acids play diversified roles in the plant : they constitute monomers of the plant polyesters cutin and suberin, second messengers in plant-pathogen interactions, and are intermediates of the fatty acids catabolism. Cytochrome P450 isoforms from CYP86 and CYP94 families could be implicated in these reactions. The identification of some of these isoforms is the aim of our work. In Vicia sativa, high concentrations (50 to 500 µM) of chemicals (clofibrate, 2,4-D, 2,3-D, IAA, SA, methyljasmonate) applied on the seedlings produce a strong induction of CYP94A1. A promotor analysis revealed the occurrence of an as-1 regulatory sequence, which, as in the regulation of isoform 6 of the glutathione-S-transferase, would suggest a role in the detoxification of free fatty acids. CYP86B1 and CYP86B2 from Arabidopsis thaliana are involved in the biosynthesis of suberin monomers in the root endodermis, and probably in the production of lipids for the pollen coat and the wall of pollen grains
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Davies, Adrian M. "Genotoxicity of tamoxifen and structural analogues of tamoxifen : studies on the activation of tamoxifen by cytochrome P450s and peroxidase." Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29637.
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This thesis investigates the activation of tamoxifen and structural analogues of tamoxifen by cytochrome P450s and peroxidase. In human lymphoblastoma derived cell lines expressing cDNAs for different human CYP-isoforms, tamoxifen, toremifene and 4-hydroxytamoxifen were clastogenic. Micronucleus formation was dependent on the dose of drug used and was not seen in cell lines not expressing these CYP isoenzymes. Results showed that CYP3A4 was the most important mono-oxygenase in the activation of these drugs. Clastogenicity was not detected when synthetic -hydroxytamoxifen or clomiphene were used. It was shown that in vitro, a horseradish peroxidase/H2O2 system could catalyse the N-demethylation and N-oxidation of tamoxifen and toremifene. Activation of [14C]tamoxifen or toremifene by this system was demonstrated by their irreversible binding to protein or to DNA. This reaction was catalysed by a number of peroxidases, including prostaglandin synthase. In the presence of DNA, activation of tamoxifen, 4-hydroxytamoxifen or toremifene resulted in the formation of 32P-postlabelled DNA adducts. 4-Hydroxytamoxifen was broken down by peroxidases much faster than tamoxifen. Reaction products, detected by HPLC-electrospray mass spectrometry, had structures consistent with 4-hydroxytamoxifen dimers. Incubation of 4-hydroxytamoxifen with horseradish peroxidase, H2O2, glutathione and the radical trap DMPO leads to the formation of a glutathione thiyl radical that can be detected with electron paramagnetic resonance spectrometry. The presence of the thiyl radical supports the proposal for the formation of a phenoxyl radical from 4-hydroxytamoxifen.
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Shepard, Dale Randall. "The Metabolism of Phenytoin by Human Liver Microsomes and Cytochrome P450s Expressed in Saccharomyces Cerevisiae and COS-1 Cells /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487931512618872.
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Lee, Sungbeom. "EXPLORING THE BIOCHEMICAL AND EVOLUTIONARY DIVERSITY OF TERPENE BIOSYNTHETIC ENZYMES IN PLANTS." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/587.
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Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.
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Abdulmughni, Ammar [Verfasser], and Rita [Akademischer Betreuer] Bernhardt. "Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus megaterium DSM319 for the production of vitamin D3 metabolites / Ammar Abdulmughni ; Betreuer: Rita Bernhardt." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2018. http://d-nb.info/1166140032/34.
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Abdulmughni, Ammar Verfasser], and Rita [Akademischer Betreuer] [Bernhardt. "Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus megaterium DSM319 for the production of vitamin D3 metabolites / Ammar Abdulmughni ; Betreuer: Rita Bernhardt." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2018. http://d-nb.info/1166140032/34.
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Ibrahim, Sulaiman Sadi. "Allelic variation and multigenic metabolic activity of cytochrome P450s confer insecticide resistance in field populations of Anopheles funestus s.s., a major malaria vector in Africa." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2008567/.
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Malaria control relies heavily on the use of insecticides, especially the pyrethroids, for control interventions such as Long Lasting Insecticide Nets (LLINs) and Indoor Residual Spraying (ITNs). However, widespread resistance to insecticides in major malaria vectors, such as An. funestus is threatening to derail these control tools. To design and implement suitable resistance management strategies which will ensure the continued effectiveness of these control tools it is necessary to elucidate the molecular basis of the resistance. In An. funestus, resistance is mainly metabolic with the duplicated P450s CYP6P9a and CYP6P9b implicated as the major pyrethroid resistance genes. Despite the detection of these key resistance genes the detailed molecular mechanisms through which they confer pyrethroid resistance remain uncharacterised. Because CYP6P9a and CYP6P9b were shown to exhibit significant allelic variation between resistant and susceptible mosquitoes, we hypothesised that this allelic variation is potentially a key mechanism conferring pyrethroid resistance. Here, I characterised the role of these genes in the resistance to pyrethroids and identified other candidate genes which confer cross-resistance to non-pyrethroid insecticides. The role of allelic variation in pyrethroid resistance was investigated using polymorphism survery and in silico prediction of activity. Metabolic activities and efficiencies of allelic variants of CYP6P9a and CYP6P9b were investigated using fluorescent probes, metabolism assays and transgenic expression in D. melanogaster system. Pyrethroid resistance causative mutations were detected using the site-directed mutagenesis. Other candidate P450s that confer cross-resistance to pyrethroids carbamates and organochlorines were identified and characterised. This study revealed that CYP6P9a and CYP6P9b from resistant populations of An. funestus are undergoing directional selection with reduced genetic diversity and beneficial mutations selected, compared to the alleles from susceptible strain (FANG), which exhibited high genetic variation. Modelling and docking simulations predicted the alleles of CYP6P9a and CYP6P9b from the resistant strains all across Africa to metabolise pyrethroids with high efficiency while the susceptible alleles FANGCYP6P9a and FANGCYP6P9b were predicted to have low activity toward pyrethroids. Validation of the docking predictions with probes and metabolism assays established that the resistant alleles of CYP6P9a and CYP6P9b possess high activities toward pyrethroids with kinetic profiles significantly different (high affinity and catalytic efficiency) from those obtained from the FANG, indicating that allelic variation is playing a major role in pyrethroid resistance. These findings were further strengthened by results from transgenic expression with GAL4/UAS technology showing that flies expressing the resistant alleles of both genes were significantly more resistant to pyrethroids than those expressing the susceptible alleles. Using mutagenesis, three key residues (Val109, Asp335 and Asn384) from the resistant allele of CYP6P9b were established as the important amino acid changes responsible for resistance with impact on substrate channelling, possible enhancement of interaction with redox partners and inter-molecular hydrogen bonding interactions, respectively conferring high metabolic efficiency. The finding of these resistance markers make it possible to design a diagnostic tool that can allow detection and tracking of the resistant alleles in the field population of An. funestus across Africa. Other up-regulated P450s in multiple resistant populations from southern Africa were also characterised, revealing that pyrethroid resistance is mediated by other P450s as well: CYP6M7, CYP6Z1, CYP9J11 and CYP6AA4 all of which metabolise pyrethroids. CYP6Z1 and CYP9J11 are cross-resistance genes which metabolise bendiocarb, while CYP6Z1 metabolise DDT in addition. In conclusion, allelic variation is a key mechanism conferring pyrethroid resistance in An. funestus s.s. from sub-Saharan Africa. Key amino acid changes control pyrethroid resistance factors and these molecular markers can be used to design DNA-based diagnostic tests which will allow tracking of the resistance alleles in the field. Pyrethroid resistance is multi-genic in the field populations of An. funestus with other P450s involved apart from CYP6P9a and CYP6P9b. The finding of cross-resistance P450s is of concern to resistance management and should be taken into account when designing resistance management strategies.
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Crocoll, Christoph [Verfasser], Jonathan [Akademischer Betreuer] Gershenzon, Christian [Akademischer Betreuer] Hertweck, and Harro [Akademischer Betreuer] Bouwmeester. "Biosynthesis of the phenolic monoterpenes, thymol and carvacrol, by terpene synthases and cytochrome P450s in oregano and thyme / Christoph Crocoll. Gutachter: Jonathan Gershenzon ; Christian Hertweck ; Harro Bouwmeester." Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2011. http://d-nb.info/1016391315/34.
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Fraser, Emily Anne. "Investigating the role of CAR and PXR in the regulation of cytochrome P450s and other drug metabolising enzymes by anti-cancer drugs using novel humanised mouse models." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/53400dff-cf5e-49a6-99ca-9bd95b2326bd.
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Nuclear receptor activation, particularly that of the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), is increasingly recognised as a key determinant in the development of drug-drug interactions (DDIs) as a result of their key role in the transcriptional regulation of numerous drug metabolizing enzymes and drug transporters. PXR and CAR involvement in the regulation of the cytochrome P450 enzymes is of greatest concern, since these enzymes metabolise the majority of currently available therapeutics. Various methods are available to investigate the activation of these receptors in response to drug challenge, including reporter gene assays, primary human hepatocytes and transgenic mouse models. However, these models lack the sophistication to effectively assess receptor cross-talk, a key regulatory mechanism in the control of drug metabolism with the potential to impact the development of DDIs. Using a novel panel of PXR & CAR transgenic mouse models this study was designed to investigate the role of cross-talk between PXR and CAR in the metabolism and pharmacokinetics of commonly available pharmaceuticals, with particular emphasis on species-specific regulation. This study has identified potential interactions with PXR and CAR following treatment of wild-type mice with cyclophosphamide, gefitinib, anastrozole and letrozole. Data from the PXR/CAR transgenic mouse panel has also provided evidence that the aromatase inhibitors, anastrozole and letrozole, interact with PXR and CAR in a species- and gender-specific manner. Cross-talk between these receptors plays a key role in the regulation of P450 expression and drug pharmacokinetics following treatment by these agents, although the elimination of these drugs appears to be primarily renal, in contrast to data derived from humans. Of particular note is the aromatase inhibitor-induced up-regulation of Cyp2b10 expression and activity observed in all models possessing a functional CAR moiety. A corresponding induction in CYP2B6 transcriptional activation has been confirmed in a novel reporter mouse model, indicating a potential DDI risk if coadministered with a drug requiring CYP2B6 for its metabolism, i.e.cyclophosphamide. These data therefore support the use of these models as a tool to dissect the regulatory cross-talk of these receptors in the control of drug metabolism, and thus to improve the assessment of DDI risk in the development of therapeutics.
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Sarkar, Md Raihan. "Application of the Monooxygenase Enzymes CYP101B1 and CYP101C1 from Novosphingobium aromaticivorans for Selective and Efficient Functionalisation of Inert C-H bonds." Thesis, 2019. http://hdl.handle.net/2440/119892.
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The cytochrome P450 enzymes CYP101B1 and CYP101C1, which are from the aromatic hydrocarbon degrading bacterium Novosphingobium aromaticivorans DSM12444, can hydroxylate norisoprenoids with high activity and selectivity. With the aim of further understanding their substrate range, a selection of cyclic alkanes, ketones and alcohols were studied. Cycloalkanes were oxidised, but both enzymes displayed low binding affinity and productive activity. The presence of a ketone moiety in the cycloalkane skeleton significantly improved the substrate binding affinity and the oxidation activity. CYP101C1 catalysed the oxidation of the cycloalkanones at the C-2 position with high regioselectivity. The regioselectivity of CYP101B1 was different. It oxidised cycloalkanones at positions remote from the carbonyl group. This indicated that the binding orientation of the cyclic ketones in the active site of each enzyme must be different. Cyclic alcohols and cyclohexylacetic acid showed little to no activity with either enzyme. The introduction of an ester protecting group to these substrates significantly enhanced the monooxygenase activity. These substrates were oxidised regioselectively on the opposite side of the ring system to the ester directing group. For example, both enzymes preferentially oxidised the C-H bond at the C4, C5 and C7 position of the cyclohexyl, cyclooctyl and cyclododecyl ester compounds, respectively. In addition, certain linear ketones and esters were also found to be suitable substrates for these biocatalysts. CYP101B1 mediated metabolism of the tricyclic compounds adamantane, 1‐ and 2‐ adamantanol and 2‐adamantanone proceeds with low oxidation activity and multiple metabolites were identified. Insertion of a directing group (acetate/isobutyrate) at the alcohol of these adamantanols significantly increased the affinity, activity and coupling efficiency (productive use of reducing equivalents) of CYP101B1 compared to the parent compounds. This substrate engineering approach with these adamantyl derivatives led to a 65 to 122-fold higher product formation activity. The turnovers were also regioselective and in some instances stereoselective. Additionally, the amide N‐(1‐adamantyl)acetamide was oxidised efficiently by CYP101B1, whereas 1‐adamantylamine was not. Whole-cell biotransformation systems were used to generate the metabolites in good yield (g/L scale). Overall, the use of ester directing groups and the modification of the amine to an amide enabled CYP101B1 to oxidise the adamantane skeleton more efficiently and selectively. Wild-type (WT) CYP101B1 can catalyse the oxidation of aromatic substrates such as alkylbenzenes, alkylnaphthalenes and acenaphthene, but the binding affinities and the oxidation activities were low. Both the binding affinity and product formation activity of this enzyme for these hydrophobic substrates were enhanced using site-directed mutagenesis. The Histidine 85 (H85) of CYP101B1 aligns with tyrosine 96 of CYP101A1 (P450cam), which, in the latter enzyme forms the only hydrophilic interaction with its natural substrate, camphor. The H85 residue of CYP101B1 was therefore replaced with phenylalanine (F), and this H85F variant exhibited greater affinity and activity towards hydrophobic substrates. For instance, the product formation activity of the H85F variant for acenaphthene oxidation was increased sixfold to 245 nmol.nmol-CYP–1.min–1. This indicated that this residue is in the substrate binding pocket or the access channel of the enzyme. Methylcubanes have been used as mechanistic probes to differentiate between radical and cationic pathways in cytochrome P450 oxidation. A series of methylcubanes were designed which would place the methyl group close to reactive heme iron centre of CYP101B1. CYP101B1 efficiently oxidised the substituted methylcubane derivatives yielding the equivalent cubylmethanol in 93 ± 7 % yield. The cube was found to be intact in all the turnover products, and no methylcubanols or any other rearranged metabolites containing homocubyl were detected. These results were consistent with a rapid radical rebound step in these oxidations and argued against the involvement of any carbocation-based intermediates during the oxidation. The CYP101B1 system, which also combines a FAD-containing ferredoxin reductase and a [2Fe-2S] ferredoxin, was investigated with oxygenated aromatics including naphthols, naphthoquinones, dihydroxynaphthalene and phenols. In vitro NADH oxidation rates in both the presence and absence of CYP101B1 were fast with these substrates (≥800 min-1). Minimal metabolite formation was detected, and the majority of reducing equivalents were transformed into hydrogen peroxide. Large amount of H2O2 in these reactions in the absence of P450 indicated that the ferredoxin (Arx) and ferredoxin reductase (ArR) catalysed futile redox cycling with naphthoquinones giving rise to the uncoupling of the reducing equivalents. Further examination of naphthols and naphthoquinones together with 2-adamantyl acetate in the fully reconstituted CYP101B1 turnovers demonstrated that the presence of naphthoquinones led to diminished product formation as they interfere with the electron transfer process. This type of uncoupling in the bacterial P450 electron transfer partners containing ferredoxin system would be considered an additional form of uncoupling over those which arise in the P450 active site.Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2019
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Fu, Chung-Nan, and 傅正男. "Effect of Carbendazim on Cytochrome P450s in Rats." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/24962781166715390064.
Full textAbstract:
碩士國立臺灣大學
毒理學研究所
95
Carbendazim (methyl-2-benzimidazole carbamate) is a broad spectrum and systemic fungicide for the control of molds, rots, and blight. Carbendazim and related benzimidazoles show marked reproductive toxicity and endocrine-disrupting activity in rats. Cytochrome P450 (CYP) is the primary enzyme system in metabolism and is responsible for the metabolism of drugs, carcinogens, steroid hormones, and environmental pollutants. CYP genes are markedly responsive to the stimulatory and inhibitory effects of xenobiotics and provide a powerful tool to investigate gene-environment interaction. The major objective of the present study was to investigate the ability of carbendazim to modulate CYP-dependent monooxygenases and antioxidant enzymes in rat liver, kidney, lung, and testis. Treatment of male rats with 10, 50, and 100 mg/kg carbendazim intraperitoneally once daily for 4 days decreased testis spermatid density dose-dependently. In liver microsomes, the carbendazim treatment increased P450 content, NADPH-cytochrome c reductase, 7-ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), pentoxyrsorufin O-dealkylase, and 7-ethoxycoumarin O-deethylase (ECOD) activities. In kidney microsomes, carbendazim increased P450 content and EROD and ECOD activities. In lung microsomes, the treated increased EROD activity. In testis microsomes, the treatment increased NADPH-cytochrome c reductase activity. Carbendazim increased glutathione S-transferase (GST) and catalase activities in liver cytosol and GST and superoxide dismutase activities in testis cytosol. The fungicide decreased glutathione content in the kidneys and lipid peroxidation in the testes. Oral administration of 400 mg/kg carbendazim for 7 days increased MROD activity in liver microsomes. The results of immunoblot and RT-PCR analyses showed that carbendazim induced CYP1A1/2 and CYP2B proteins and mRNA in the liver and CYP1A1 protein and mRNA in kidneys and lungs. These present findings show that carbendazim is an inducer of CYP1A1/2 and CYP2B in rats. The significance of carbendazim induction of CYP in metabolic activation warrants further investigations.
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Armstrong, Catherine. "Characterizing the Expression of Cytochrome P450s in Breast Cancer Cells." Thèse, 2011. http://hdl.handle.net/1866/9017.
Full textAbstract:
Une résistance aux agents anticancéreux utilisés dans le traitement du cancer du sein est souvent associée à un échec de traitement. Des variations dans le devenir des agents anticancéreux dans l’organisme, sont des facteurs pouvant expliquer des phénomènes de résistance. Notre but était d’évaluer l’impact des isoenzymes du CYP450s, dans le métabolisme local des agents anticancéreux.
Notre premier objectif était de valider un gène rapporteur pour nos analyses de PCR en temps réel. Pour ce faire, nous avons criblé l’expression de 6 gènes rapporteurs dans 23 lignées cellulaires. NUP-214 a été démontré comme étant le gène rapporteur le plus stable avec un écart-type de seulement 0.55 Ct.
Notre deuxième objectif était de déterminer le niveau d’expression des ARNm de 19 isoformes du CYP450 dans plusieurs lignées cellulaires du cancer du sein. Les ARNm des CYP450s ont démontré une très grande variabilité entre les lignées cellulaires. Les isoformes CYP1B1 et CYP2J2 démontrent l’expression la plus importante pour la majorité des lignées.
Notre troisième objectif était d’évaluer la corrélation entre l’expression des isoformes des CYP450s et leur activité métabolique en utilisant les substrats spécifiques du CYP1B1 et 2J2, 7-éthoxyrésorufine et ébastine, respectivement. Une forte corrélation (r2=0.99) fut observée entre l’activité métabolique vis-à-vis l’ébastine et l’expression du CYP2J2. De même, le métabolisme du 7-éthoxyrésorufine était fortement corrélé (r2=0.98) avec l’expression du CYP1B1.
En résumé, ces résultats suggèrent que le métabolisme local des agents anticancéreux pourrait significativement moduler le devenir des agents anticancéreux dans l’organisme, et pourrait être ainsi, une source de résistance.Several types of cancer cells have shown an innate or accute resistance to anti-cancer agents which in turn causes a failure in treatment. This resistance has been suggested to be caused by the expression of membrane transporters in cancer cells, as well as inter-individual variability in metabolism. Our interest was to evaluate the implication of CYP450 enzymes in the local metabolism of cancer cells. Our first objective was to screen the expression level of six housekeeping genes (HKG) using 23 different cell lines to determine which gene was the most stable. We found that NUP-214 was the most stable HKG across the panel of cell lines tested, with a standard deviation of only 0.55 Ct. Our second objective was to determine the expression level of 19 CYP450 mRNA isoforms in various breast cancer cell lines by RT-PCR. The CYP450 mRNAs showed a large variability between the different cell lines analyzed, where CYP1B1 and 2J2 were strongly expressed in most cell lines. Our third objective was to determine if measurable metabolic activity was present and correlates with mRNA expression in these same breast cancer cell lines using the specific substrates 7-ethoxyresorufin and ebastine for CYP1B1 and 2J2 activities, respectively. The metabolism of 7-ethoxyresorufin showed an excellent correlation of 0.98 with CYP1B1 expression while ebastine demonstrates a strong correlation (r2=0.99) with 2J2 expression. Overall, these results suggest that local metabolism of anti-cancer agents could significantly affect drug disposition and be a source of chemoresistance.
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Munday, Samuel David. "Investigations and applications of self-sufficient cytochrome P450 monooxygenases." Thesis, 2016. http://hdl.handle.net/2440/100734.
Full textAbstract:
The cytochrome P450 superfamily catalyses the oxidation of a vast array of organic molecules. Most commonly, this oxidation process ensues by the insertion of a single oxygen atom from dioxygen into an unreactive C-H bond. There is a high degree of interest for this reaction type in conventional synthesis, but it is difficult to achieve high levels of selectivity and is often performed under harsh conditions. CYP102A1 or P450Bm3 from Bacillus megaterium however, can perform this oxidative process under physiological conditions and so researchers have a strong interest in exploiting the potential benefits of this enzyme. The natural substrates of P450Bm3 are fatty acids but this thesis will address both modern and classical techniques to improve catalytic performance with a variety of non-natural substrates. The first two results chapters of this thesis (Chapters 3 and 4) describe the effect of decoy molecules on non-natural substrate oxidation with the aim of improving rates of product formation while maintaining the selectivity of the enzyme. Analysis of the oxidation of these substrates by wild-type P450Bm3 and the variant KT2 showed substantial increases in product formation rate while maintaining the regioselectivity. As a rigorous test of regioselectivity, a selection of xylenes were used that have previously been shown to generate multiple products upon P450Bm3 oxidation. Retention of enantioselectivity was also assessed by using prochiral substrates that have stereocentres introduced upon P450Bm3 oxidation. Chiral chromatography analysis of these turnovers showed that in most cases, the enantioselectivity of the enzyme was either maintained or marginally improved. Knowing that xylenes give a range of oxidation products upon P450Bm3 activity, a wider range of disubsituted benzene compounds were also analysed (Chapter 5). These substrates were chosen to resemble potential xenobiotic compounds in order to assess what metabolites may be produced by P450Bm3 and therefore other P450 systems. These substrates were analysed with several P450Bm3 variants and significantly improved rates of product formation were observed, enabling identification of the likely metabolites. Chapter 6 describes an investigation into two potential CYP102 family members from the bacterium Ktedonobacter racemifer DSM44963 (Krac0936 and Krac9955). Their sequenced genes show similarities to P450Bm3, which encouraged the investigation of a range of fatty acid substrates with these two enzymes. Although their product distributions differed, both Krac0936 and Krac9955 were active with straight-chain saturated and unsaturated fatty acids.Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2016.
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Chang, Chiao-Chia, and 張巧佳. "Role of Human Hepatic Cytochrome P450s in Territrem B and C Metabolism." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/78550460089862607345.
Full textAbstract:
碩士國立臺灣大學
毒理學研究所
93
Territrem A, B and C, the structure related tremogenic mycotoxins isolated from the chloroform extracts of rice culture of Aspergillus terreus 23-1. The previous study on metabolism of Territrem A (TRA) by liver microsome from Wistar rats showed that three metabolites, MA1, MAX and MA2 were formed in male rats, but only one metabolite, MA1 in female rats. The studies were further carried with specific chemical and antibody inhibitors for CYP isoforms and the sypersomes expressed with specific type of CYP isoforms. The results indicated that CYP3A1, which is dominated in female rat liver, plays a mean role in metabolic pathway from TRA to MA1 and that CYP3A2, which is dominated in male rat liver, plays the mean role in metabolic pathway of TRA to MA1, MAX and MA2. On the other hand, four metabolites, such as MB1, MB2, MB3 and MB4 (which structure is the same as Territrem C (TRC) ) were formed from Territrem B (TRB) and one metabolite, MC (which structure is the same as MB4) from Territrem C respectively by liver microsome from each of male and female rats. In order to elucidate the role of human hepatic CYP isoforms in TRB and TRC, the experiments were performed with several enzyme sources such as from different age and sex, V79MZh3A4 cell line derived from Chinese hamster, in which human CYP3A4 were expressed, and several supersomes having enzyme or different tyoe of CYP isoforms. The following are the resultes obtained： (1) In human liver microsome, MB2, and MB4 were main products from TRB and MC from TRC. There were no age and gender difference in ability to metabolize TRB and TRC. (2) CYP3A4/5 had the major role in metabolism of TRB and TRC. (3) The rate of production of MB2 from TRB or MC from TRC were competitively inhibited by the present of testosterone. However the rate of the production of 6β-hydroxytestosterone from testosterone were inhibited by the present of TRB or TRC in mixed type of inhibition (non competitive and non uncompetitive inhibition). The rate of the production of MB4 from TRB was also inhibited by the present of testosterone in mixed type of inhibition. (4) It suggested that CYP3A4 had the major role in 4β-C hydroxylation of TRB, and CYP3A5 had the major role in O-demethylation of TRB.
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Behan, Rachel Koren. "Spectroscpic characterization of high-valent intermediates in Cytochrome P450s and other heme enzymes." 2008. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-2438/index.html.
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Green, Robert. "Investigation of Cytochrome P450 Monooxygenases in S. homoeocarpa for Chlorothalonil Biotransformation." 2017. https://scholarworks.umass.edu/masters_theses_2/506.
Full textAbstract:
Sclerotinia homoeocarpa (F.T. Bennett) is one of the most economically important pathogens on high amenity cool-season turfgrasses where it causes dollar spot. Due to decades of over-reliance and repeated chemical treatments, S. homoeocarpa has developed resistance and insensitivity to multiple classes of fungicides. To understand the genetic mechanisms of fungicide resistance, the whole genomes of two strains with varying resistance levels to fungicides, were sequenced. In unpublished data (Sang et al.), a RNA-sequencing analysis revealed three CYP450s that were validated to play a functional role in S. homoeocarpa’s resistance against different fungicide classes. We also identified CYP450 metabolic action on the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been known to be persistent in soils. Yet, S. homoeocarpa resistance to chlorothalonil has not been reported in the field. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP450 overexpression strains when compared to the wild-type. We show by GC-MS that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloroisophthalonitrile is produced by CYP450s’ metabolism.