Yes / The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.

Yes / The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation. / The authors would like to thank Yorkshire Cancer Research (Program grant B381PA) for supporting our work focused on exploring CYPs as targets for prodrug development. The human recombinant CYP1A1 was a gift from Prof Emily E. Scott, University of Michigan; the enzyme was produced via NIH funded grant (R37 GM076343).

TxtE is a protein related to cytochrome P450 enzymes, which catalyze a number of reactions that typically involve oxygen and not nitrogen. It has been discovered that TxtE can nitrate tryptophan through an unusual reaction in which it uses nitric oxide (NO) as a nitrogen donor to install the nitro group despite NO typically being considered toxic to bacteria. This project will determine if all cytochromes P450 can catalyze nitration as long as they are given NO. This will have an impact on understanding drug delivery and metabolism for which nitration is important.

No description available.

No description available.

La géosmine est un terpénoïde, provoquant un goût moisi-terreux associée à des flaveurs atypiques dans l'eau et le vin. Chez les bactéries, la voie de biosynthèse de la géosmine est bien caractérisée, mais peu de connaissance sont disponibles au sujet de sa biosynthèse chez les eucaryotes, en particulier dans les champignons filamenteux. L'origine de la géosmine dans la vigne est en grande partie attribuable à la présence de Penicillium expansum sur les raisins. Dans cette thèse, afin de mieux comprendre la voie de biosynthèse de la géosmine chez Penicillium expansum, nous avons décrit la caractérisation et l'analyse de "gpe1", un gène codant pour une cytochrome P450 monooxygénase impliquée dans la biosynthèse de la géosmine. Nous avons démontré que les deux fragments d'ADN: p450-1 et p450-2 appartiennent à un seul gène du cytochrome p450 (gpe1). La séquence d'acides aminés déduite de gpe1 a une identité moyenne de 40 % avec les enzymes PbP450-2 et P450-4 qui ont été trouvées impliquées respectivement dans la synthèse d'indole diterpène et dans la synthèse des gibbérellines. Les amplifications par PCR effectuée sur quatorze espèces de Penicillium ont montré que seules les espèces producteurices de la géosmine ont donné le même fragment de ~1,2 kb que gpe1. L'analyse du gène gpe1 nous a permis d'identifier la présence de certains domaines conservés de cytochromes P450 monooxygénases. Ensuite, la caractérisation fonctionnelle du gène gpe1 chez P. expansum M2230 a été décrite. Nous avons montré que les mutants de gpe1 ont perdus leur pouvoir de produire la géosmine alors que les révertants de gpe1 ont rétablis leur pouvoir de production. Enfin, nous avons démontré qu'une polykétide synthase putative et une putative NRPS sont présentes sur le côté droit du gène gpe1 proposant que le gène gpe1 pourrait être une partie d'un «Cluster» codant pour la biosynthèse de métabolites secondaires. / Geosmin is a terpenoid, an earthy-musty compound associated with off-flavors in water and wine. In bacteria, the biosynthesis pathway of geosmin is well characterized, but little is known about its biosynthesis in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. In this thesis, we have described the characterization and analysis of "gpe1", a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in P. expansum M2230, in order to better understand of the biosynthesis pathway of geosmin in this species. We demonstrated that the two DNA fragments i.e. p450-1 and p450-2 belong to a single cytochrome p450 gene (gpe1). We showed that the deduced amino acid sequence of gpe1 has an average identity of 40 % with PbP450-2 and P450-4 enzymes which have been found involved in indole diterpene synthesis and in gibberellin synthesis respectively. Then, the results of PCRs performed on the fourteen Penicillium species showed that only Penicillium species which were producers of geosmin gave the same fragment of ~1.2 kb like gpe1. Analysis of the gpe1 gene enabled us to identify the presence of some conserved domains of cytochromes P450 monoxygenases in the amino acid sequence of gpe1. Then, the functional characterization of the gpe1 gene in P. expansum M2230 was described. We illustrated that the mutants of gpe1 lost their potential to produce geosmin whereas the reverse complements of gpe1 restored their potential to produce geosmin. Finally, we demonstrated that a putative polyketide synthase and a putative NRPS-like enzyme are present on the right side of the gpe1 gene suggesting that gpe1 gene might be the part of a gene cluster encoding the biosynthesis of secondary metabolites.

Cette thèse est dédiée à l'approche pharmacogénétique des effets anti-inflammatoires de la thérapie par les thiénopyridines. Prenant en compte que les plaquettes activées jouent un rôle central dans les états inflammatoires et que des polymorphismes du cytochrome P450 (CYP) 2C19 ont été montré responsable de différences inter individuelle dans la réponse de l'effet antiplaquettaire de thiénopyridines, nous avons émis l'hypothèse que CYP2C19 *2 ou *17 sont également associés à la variabilité interindividuelle du potentiel antiinflammatoire des thiénopyridines. Les marqueurs d'inflammation utilisés pour suivre l'effet des thiénopyridines sont : la CRP, l'haptoglobines et l'orosomucoïde. Nous avons démontré que pour interpréter les valeurs de l'haptoglobine il était nécessaire de tenir compte du statut génétique et obtenir des valeurs de référence stratifiés. D'abord dans une population saine, nous n'avons pas trouvé d'association entre les valeurs de base des marqueurs inflammatoires et les polymorphismes fréquents de CYP époxygenases. Dans une population après intervention coronarienne percutanée qui était composée de 1128 sujets traités par clopidogrel ou prasugrel, le niveau de CRP observé a montré une interaction significative entre le tabac et le polymorphisme de CYP 2C19 ; cet effet est indépendant du niveau d'agrégation plaquettaire. Dans une 3ème population, sur plus de 1000 sujets hospitalisés à Coimbra, nous avons identifié une interaction entre le clopidogrel CYP2C19 et les médicaments bloqueurs des canaux calciques. En résumé, tous ces résultats obtenus sur plusieurs populations laissent envisager que les marqueurs d'inflammation pourraient être un moyen intéressant de suivi des patients lors de la thérapeutique par les thiénopyridines / The main part of the thesis is devoted to pharmacogenetic approach to the anti-inflammatory effects of thienopyridine therapy. Taking into the account that activated platelets play a central role in the inflammatory responses and that CYP2C19 gain- and loss-of-function polymorphisms (*2 and *17) are sources of inter-individual difference in response to the anti-platelet effects of thienopyridines, we hypothesized that *2 and/or *17 alleles are also associated with inter-individual variability in the potential inflammation-reducing effects of thienopyridines. The following markers were used to test the hypothesis: CRP, haptoglobin and orosomucoid acid. To be reliably interpretable in daily medical practice, genetic status should be considered for partitioning the reference values of haptoglobin. In a small healthy population, no significant association was observed between *2 allele and changes in levels of inflammatory markers from baseline to 7 days after administration of clopidogrel and our findings did not support the notion that the genetic variations of CYP epoxygenases are associated with the level of inflammatory markers. Also, in post-PCI population consisting of 1128 on-clopidogrel or on-prasugrel patients, CRP levels were observed to be regulated with a significant interaction between smoking and CYP2C19 polymorphisms; this effect was independent to the level of platelet aggregation. Additionally, in a large population of 1000 on-clopidogrel patients, whether there is a potential interaction between clopidogrel and calcium channel blockers. Collectively, we demonstrated in this thesis that inflammatory markers might be alternative tools for the prediction of response to thienopyridines

Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Heme oxygenases (HO) catalyze the degradation of heme into biliverdin, carbon monoxide (CO) and free iron. The two major isoforms, HO-2 (constitutive) and HO-1 (inducible by various stressors such as heavy metals and reactive oxygen species) are involved in a variety of physiological functions, including anti-inflammation, antiapoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (CYPs), soluble guanylyl cyclase (sGC), and nitric oxide synthase (NOS). Our laboratory has been able to successfully synthesize a series of small molecule non-porphyrin HO inhibitors (QC-xx) that have had little or no effect against sGC and NOS; however, their effects on various CYP isoforms has yet to be fully elucidated. In order to determine the effects on CYP enzyme activity, microsomal preparations of two CYP isoforms (2E1 and 3A1/3A2) were incubated with varying concentrations of HO inhibitor and the activity was determined via spectrophotometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of CYP2E1 and/or CYP3A1/2, while some others did inhibit these CYP isoforms. Four regions of interest were analyzed further and several structural changes were identified as conferring increased HO inhibition and decreased effect on both CYP2E1 and 3A1/2. Based on the information obtained, three putative compounds were designed and it is hypothesized that these compounds will be selective inhibitors for HO-1 over HO-2 and will display little effect on either CYP2E1 or 3A1/2 activities. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-11-20 11:19:48.841