Cytochrome P450 was examined in both insecticide resistant and insecticide susceptible strains of Drosophila melanogaster. Much higher levels were observed in the resistant strain IIID when compared to the susceptible strain Canton S. This increase appeared to be the result of an overproduction of a few existing forms. Two heme-staining microsomal proteins found in strain IIID were identified as putative cytochrome P450 isozymes. Polyclonal antibodies produced against these two proteins were used in the immunoanalysis of microsomal proteins from both strains. A lambda gtll cDNA expression vector library was created by inserting cDNA fragments from a Drosophila lambda gt10 library into lambda gtll arms. The library was screened with the polyclonal antiserum. Three clones were isolated, of which one, gtll-Al, was most highly reactive with the antiserum. Analysis of the gtll-Al lysogen indicated a 130 kd fusion protein was produced of which 16 kd was coded for by the cDNA insert. A .5 kb cDNA insert was isolated from the clone as part of a 1.5 kb Kpnl/EcoRl fragment and was used in the analysis of Drosophila genomic DNA and total RNA. Southern analysis revealed an EcoRl polymorphism existed between strain IIID and Canton S. RNA analysis suggested strain IIID produced more coding message for the Al insert in the larval and adult stages than did Canton S. / Thesis / Master of Science (MSc)

Neste trabalho foi investigado o potencial de modelos metaloporfirínicos em mimetizar a ação do citocromo P450 na oxidação de um herbicida, a atrazina. Foram utilizadas as metaloporfirinas comerciais de segunda geração solúveis em solvente orgânico, cloreto de 5,10,15,20-tetrakis(2,6-diclorofenil)porfirina metal(III) [M(TDCPP)Cl] e cloreto de 5,10,15,20-tetrakis(pentafluorofenil) porfirina metal(III) [M(TFPP)Cl] (metal = ferro e manganês), tanto em solução homogênea como suportadas em montmorilonita K-10 aminofuncionalizadas; e metaloporfirinas solúveis em água, como a cloreto de 5,10,15,20-tetrakis-(N-metil-4-piridil) porfirina ferro(III), [Fe(TMPy)Cl], e cloreto de [5,10,15,20-tetra(4-carboxifenil)porfirina] ferro(III), [Fe(TCPP)Cl]. Os oxidantes testados foram iodosilbenzeno, ácido metacloroperbenzóico e peróxido de hidrogênio em água, metanol e acetonitrila. Os produtos de oxidação da atrazina foram identificados por cromatografia líquida de alta eficiência (CLAE). Os resultados mostraram que as metaloporfirinas foram capazes de oxidar a atrazina, um herbicida com características de persistência no meio ambiente, e mimetizar a ação da enzima in vivo e in vitro com formação de dois metabólitos: DEA e DIA, resultado da N-desalquilação das cadeias etila e propila do substrato, respectivamente. O DEA correspondeu a um dos principais produtos da reação, e formou-se apenas traços de DIA, mostrando a preferência das metaloporfirinas em oxidar a cadeia etila da atrazina. Verificou-se também a formação de cinco produtos desconhecidos, sendo possível a identificação de apenas um deles por espectrometria de massas, devido à baixa concentração dos demais, o qual corresponde à formação de uma amida na cadeia etila da atrazina (COA). Esse composto correspondeu ao produto de maior rendimento na maioria das reações. O monitoramento das reações em diferentes intervalos de tempo e a variação nas condições reacionais mostraram que os principais produtos de oxidação do herbicida, DEA e COA, são formados por mecanismos independentes e por espécies catalíticas distintas. O DEA é formado via espécie ativa Me(V)OP [Mn(V)OP ou Fe(IV)OP+], enquanto o COA é originado via Me(IV)OP [Mn(IV)OP ou Fe(IV)OP]. Estudos de intermediários por UV-Vis e EPR mostraram que a espécie ferril predomina como intermediário de reação para os sistemas Fe(TFPP)Cl/ACN com os dois oxidantes, iodosilbenzeno e ácido metacloroperbenzóico. Para as metaloporfirinas Fe(TCPP)Cl e Fe(TMPy)Cl o estudo da oxidação do herbicida ficou comprometido devido à baixa solubilidade da atrazina em água, o que provocava sua precipitação e destruição do catalisador. Para as metaloporfirinas suportadas em montmorilonita K-10 aminofuncionalizada também não foi observada formação de produtos, resultado atribuído à dificuldade do substrato, considerado bastante inerte, atingir o sítio catalítico. Todos esses resultados mostraram o potencial de aplicação desses modelos biomiméticos em estudos que buscam elucidar o metabolismo de herbicidas in vivo, tendo em vista a dificuldade de se trabalhar com as enzimas in vitro, e resultaram na proposição de um esquema de reação da oxidação da atrazina catalisada pelas metaloporfirinas nas condições estudadas. / In this work we investigated the ability of metalloporphyrin model systems to mimic the action of cytochrome P450 in the oxidation of a herbicide, atrazine. To this end, we employed the second generation commercially available metalloporphyrins metal (III) 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin chloride [M(TDCPP)Cl] and metal (III) 5,10,15,20- tetrakis(pentafluorophenyl)porphyrin chloride [M(TFPP)Cl] (metal = iron or manganese), all soluble in organic solvents, as well as the water soluble metalloporphyrins iron (III) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin chloride [Fe(TMPy)Cl] and iron (III) 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin chloride [Fe(TCPP)Cl]. These metalloporphyrins were used both in homogeneous solution and supported on montmorillonite K-10. Iodosylbenzene, metachloroperbenzoic acid, and hydrogen peroxide were tested as oxidants, using one of the following reaction media: water, methanol, and acetonitrile. Products generated during atrazine oxidation were identified by high performance liquid chromatography. Our results show that the metalloporphyrins are able to oxidize atrazine, a highly persistent herbicide in the environment, as well as mimic the action of P450 enzymes both in vivo and in vitro, with formation of two metabolites, namely DEA and DIA, which result from the N-dealkylation of the ethyl and propyl chains of the substrate, respectively. We also detected the formation of five unknown products, and we were able to identify only one of them by means of mass spectrometry, which corresponds to the formation of an amide on the atrazine ethyl chain (COA) and was the compound obtained in highest yields in most of the reactions. The other four unknown products were obtained in very low concentrations, which prevented us from determining their structures. By monitoring the reactions at different time intervals and varying the reactional conditions, we were able to see that the main herbicide oxidation products, DEA and COA, are generated via distinct mechanisms and different active catalytic species. DEA is formed via the species Me(V)OP [Mn(V)OP or Fe(IV)OP.+], while COA results from the action of the species Me(IV)OP [Mn(IV)OP or Fe(IV)OP]. Studies of the reaction intermediates by UV-VIS and EPR showed that the ferryl species is the main reaction intermediate in the case of Fe(TFPP)Cl/ACN systems and the oxidants, iodosylbenzene and metachloroperbenzoic acid. Studies of herbicide oxidation were difficult to carry out in the case of the metalloporphyrins Fe(TCPP)Cl and Fe(TMPy)Cl due to the low solubility of atrazine in water, which led to its precipitation and catalyst destruction. With respect to the metalloporphyrins supported on montmorillonite K-10, no reaction products were obseved because of the difficult diffusion of the inert substrate into the catalytic site. All these results demonstrate the potential application of these biomimetic model systems in studies that pursue the elucidation of herbicide metabolism in vivo, especially when one bears in mind the difficulty in working with enzymes in vitro. Our data enabled the proposition of a scheme for metalloporphyrin-catalyzed atrazine oxidation under the conditions used herein.

Human drug metabolites are frequently biologically active, with many implications for human health. Pharmaceutical companies have become increasingly aware of the need to identify and test these metabolites. The P450 BM3 enzyme from Bacillus megaterium offers substantial advantages to the current methods of metabolite synthesis, as its soluble, catalytically self-sufficient nature, coupled with its high catalytic activity, make P450 BM3 ideal for engineering towards specificity for human drugs. The highly-active I401P BM3 mutant was characterized for its reactivity towards human drugs and for the development of a human P450-like metabolite profile. The I401P mutant exhibits binding to molecules including alkaloids, steroids, and azole drugs, along with many other compounds. I401P binds/oxidizes human CYP substrates, including alosetron, phenacetin, caffeine, nicotine and diclofenac. LC-MS product identification shows that I401P BM3 forms 4OH-diclofenac, the major human metabolite for diclofenac. I401P BM3 also produces nornicotine, the second major human metabolite of nicotine. I401P BM3 also forms theophylline, theobromine and paraxanthine, the three major human metabolites of caffeine. Thermostability (DSC) data show that the I401P mutation destabilizes the BM3 heme domain in both its substrate-free and substrate-bound forms. The I401P heme domain X-ray crystal structure reinforces previous structural observations that the Pro401 mutation causes the BM3 protein to adopt a high-spin, "substrate-bound" state, with a displaced heme iron axial water, producing a "catalytically primed" mutant with greater diversity in substrate selectivity. The destabilisation of the BM3 heme domain structure due to the Pro401 mutation increases conformational plasticity in this mutant, allowing it to function as a platform for future mutagenesis aimed at improved binding and metabolite yield from specific drug substrates. Further proline mutations (A330P, A330P/I401P and A82F/F87V/I401P) were examined for increased affinity for drug substrates. The A330P mutant shows no novel drug substrate specificity, despite its reported affinity for small molecules. The A330P/I401P double mutant demonstrates weak binding to WT BM3 and I401P substrates, but no synergistic effects were obtained by combining the two mutations. The double mutant exhibits very low solvent tolerance and significant structural destabilisation. DSC data confirms this, with the double mutant destabilising the BM3 heme domain by up to 20 °C. Initial work with the A82F/F87V/I401P mutant showed increased affinity for A82F/F87V- and I401P-type substrates, including diclofenac. LC-MS product analysis confirms that the A82F/F87V/I401P mutant oxidises diclofenac into its major human metabolite 4OH-diclofenac. These data indicate that human-like oxidation reactions are feasible with BM3 mutants. In this work, proline insertion mutants were generated that introduced novel affinity for biotechnologically relevant substrates. In particular the I401P mutant offers an excellent platform for future biotechnological engineering.

Neste trabalho foi investigado o potencial de modelos metaloporfirínicos em mimetizar a ação do citocromo P450 na oxidação de um herbicida, a atrazina. Foram utilizadas as metaloporfirinas comerciais de segunda geração solúveis em solvente orgânico, cloreto de 5,10,15,20-tetrakis(2,6-diclorofenil)porfirina metal(III) [M(TDCPP)Cl] e cloreto de 5,10,15,20-tetrakis(pentafluorofenil) porfirina metal(III) [M(TFPP)Cl] (metal = ferro e manganês), tanto em solução homogênea como suportadas em montmorilonita K-10 aminofuncionalizadas; e metaloporfirinas solúveis em água, como a cloreto de 5,10,15,20-tetrakis-(N-metil-4-piridil) porfirina ferro(III), [Fe(TMPy)Cl], e cloreto de [5,10,15,20-tetra(4-carboxifenil)porfirina] ferro(III), [Fe(TCPP)Cl]. Os oxidantes testados foram iodosilbenzeno, ácido metacloroperbenzóico e peróxido de hidrogênio em água, metanol e acetonitrila. Os produtos de oxidação da atrazina foram identificados por cromatografia líquida de alta eficiência (CLAE). Os resultados mostraram que as metaloporfirinas foram capazes de oxidar a atrazina, um herbicida com características de persistência no meio ambiente, e mimetizar a ação da enzima in vivo e in vitro com formação de dois metabólitos: DEA e DIA, resultado da N-desalquilação das cadeias etila e propila do substrato, respectivamente. O DEA correspondeu a um dos principais produtos da reação, e formou-se apenas traços de DIA, mostrando a preferência das metaloporfirinas em oxidar a cadeia etila da atrazina. Verificou-se também a formação de cinco produtos desconhecidos, sendo possível a identificação de apenas um deles por espectrometria de massas, devido à baixa concentração dos demais, o qual corresponde à formação de uma amida na cadeia etila da atrazina (COA). Esse composto correspondeu ao produto de maior rendimento na maioria das reações. O monitoramento das reações em diferentes intervalos de tempo e a variação nas condições reacionais mostraram que os principais produtos de oxidação do herbicida, DEA e COA, são formados por mecanismos independentes e por espécies catalíticas distintas. O DEA é formado via espécie ativa Me(V)OP [Mn(V)OP ou Fe(IV)OP+], enquanto o COA é originado via Me(IV)OP [Mn(IV)OP ou Fe(IV)OP]. Estudos de intermediários por UV-Vis e EPR mostraram que a espécie ferril predomina como intermediário de reação para os sistemas Fe(TFPP)Cl/ACN com os dois oxidantes, iodosilbenzeno e ácido metacloroperbenzóico. Para as metaloporfirinas Fe(TCPP)Cl e Fe(TMPy)Cl o estudo da oxidação do herbicida ficou comprometido devido à baixa solubilidade da atrazina em água, o que provocava sua precipitação e destruição do catalisador. Para as metaloporfirinas suportadas em montmorilonita K-10 aminofuncionalizada também não foi observada formação de produtos, resultado atribuído à dificuldade do substrato, considerado bastante inerte, atingir o sítio catalítico. Todos esses resultados mostraram o potencial de aplicação desses modelos biomiméticos em estudos que buscam elucidar o metabolismo de herbicidas in vivo, tendo em vista a dificuldade de se trabalhar com as enzimas in vitro, e resultaram na proposição de um esquema de reação da oxidação da atrazina catalisada pelas metaloporfirinas nas condições estudadas. / In this work we investigated the ability of metalloporphyrin model systems to mimic the action of cytochrome P450 in the oxidation of a herbicide, atrazine. To this end, we employed the second generation commercially available metalloporphyrins metal (III) 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin chloride [M(TDCPP)Cl] and metal (III) 5,10,15,20- tetrakis(pentafluorophenyl)porphyrin chloride [M(TFPP)Cl] (metal = iron or manganese), all soluble in organic solvents, as well as the water soluble metalloporphyrins iron (III) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin chloride [Fe(TMPy)Cl] and iron (III) 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin chloride [Fe(TCPP)Cl]. These metalloporphyrins were used both in homogeneous solution and supported on montmorillonite K-10. Iodosylbenzene, metachloroperbenzoic acid, and hydrogen peroxide were tested as oxidants, using one of the following reaction media: water, methanol, and acetonitrile. Products generated during atrazine oxidation were identified by high performance liquid chromatography. Our results show that the metalloporphyrins are able to oxidize atrazine, a highly persistent herbicide in the environment, as well as mimic the action of P450 enzymes both in vivo and in vitro, with formation of two metabolites, namely DEA and DIA, which result from the N-dealkylation of the ethyl and propyl chains of the substrate, respectively. We also detected the formation of five unknown products, and we were able to identify only one of them by means of mass spectrometry, which corresponds to the formation of an amide on the atrazine ethyl chain (COA) and was the compound obtained in highest yields in most of the reactions. The other four unknown products were obtained in very low concentrations, which prevented us from determining their structures. By monitoring the reactions at different time intervals and varying the reactional conditions, we were able to see that the main herbicide oxidation products, DEA and COA, are generated via distinct mechanisms and different active catalytic species. DEA is formed via the species Me(V)OP [Mn(V)OP or Fe(IV)OP.+], while COA results from the action of the species Me(IV)OP [Mn(IV)OP or Fe(IV)OP]. Studies of the reaction intermediates by UV-VIS and EPR showed that the ferryl species is the main reaction intermediate in the case of Fe(TFPP)Cl/ACN systems and the oxidants, iodosylbenzene and metachloroperbenzoic acid. Studies of herbicide oxidation were difficult to carry out in the case of the metalloporphyrins Fe(TCPP)Cl and Fe(TMPy)Cl due to the low solubility of atrazine in water, which led to its precipitation and catalyst destruction. With respect to the metalloporphyrins supported on montmorillonite K-10, no reaction products were obseved because of the difficult diffusion of the inert substrate into the catalytic site. All these results demonstrate the potential application of these biomimetic model systems in studies that pursue the elucidation of herbicide metabolism in vivo, especially when one bears in mind the difficulty in working with enzymes in vitro. Our data enabled the proposition of a scheme for metalloporphyrin-catalyzed atrazine oxidation under the conditions used herein.

L'exposition humaine aux contaminants environnementaux est inévitable du fait de leur présence dans l'eau, l'air et l'alimentation. La plupart d'entre eux sont reconnus comme étant mutagènes et/ou carcinogènes chez l'animal mais ils sont souvent seulement suspectés de l'être chez l'Homme. Le manque de connaissance vis-à-vis des substances chimiques a conduit l'UE à lancer le programme REACH avec l'objectif d'évaluer la toxicité d'environ 30 000 molécules. Cette évaluation nécessiterait l'utilisation de plus de 4 millions d'animaux et la pertinence controversée de ces modèles pourrait aboutir à des conclusions discutables. Les méthodes in vitro sont considérées comme une alternative potentielle à l'expérimentation animale. Néanmoins, le choix du modèle cellulaire et des conditions expérimentales restent à préciser. Les hépatocytes humains en culture primaire représentent le modèle le plus pertinent en toxicologie malgré de nombreuses contraintes (variabilité inter-individuelle, changements phénotypique précoces, obtention aléatoire). La lignée HepaRG constitue une alternative intéressante puisque ces cellules peuvent proliférer de manière illimitée et expriment les EMXs à des niveaux proches des hépatocytes humains. L'expression de ces enzymes restant stable pendant plusieurs semaines, ce modèle permet l'évaluation du risque lié à une exposition chronique aux contaminents environnementaux, essentielle en génotoxicité. Il reste cependant nécessaire de caractériser plus amplement cette lignée vis-à-vis des EMXs et de l'adapter aux tests de toxicologie actuels. Dans ces travaux, nous avons développé un test haut débit utilisant la quantification in situ des histones phosphorylées γH2AX avec l'objectif de pouvoir évaluer le risque génotoxique d'une exposition unique ou répétée aux contaminants environnementaux. Ce test a été validé avec succès par l'évaluation de la génotoxicité associée à une exposition de 1, 7 et 14 jours pour 10 polluants. Nous avons ensuite généré des lignées recombinantes biosenseurs, dérivées du modèle HepaRG et permettant d'identifier les xénobiotiques altérant l'expression transcriptionnelle des EMXs. Par transfection transitoire, nous avons dans un premier temps validé à l'aide d'inducteurs prototypiques et de nos 10 contaminants nos constructions contenant le gène rapporteur de la luciférase sous le contrôle des promoteurs de plusieurs EMXs. Ensuite, nous avons généré des lignées stables exprimant la GFP comme gène rapporteur et permettant une détection rapide des xénobiotiques capables d'induire l'expression des EMXs. Parmi les EMXs, le CYP2E1 joue un rôle important en santé humaine. En effet, cette enzyme induite dans certaines conditions physiopathologiques comme le diabète et l'obésité est responsable de l'activation de nombreux procarcinogènes et est à l'origine d'une production d'EROs. Les cellules HepaRG pourraient constituer un modèle pertinent pour l'étude du CYP2E1. Cependant, l'expression et l'activité de cette enzyme au sein de ce modèle nécessitent d'être mieux caractérisées en regard des données discordantes de la littérature. A l'aide de la chlorzoxazone, un marqueur spécifique de l'activité du CYP2E1, nous avons démontré l'influence du métabolisme de phase II sur l'activité apparente du CYP2E1. Nous proposons ici quelques recommandations afin de mieux quantifier l'activité du CYP2E1 sur les hépatocytes humains et sur le modèle HepaRG à l'aide la chlorzoxazone. / Human exposure to toxic chemicals is virtually unavoidable due to contamination of air, water and food. A number of environmental contaminants are recognized as mutagenic and/or carcinogenic in animal but they are often only suspected to have similar effects in Humans. The lack of knowledge on the effects of most industrial-made chemicals has led the EU to launch the REACH program with the aim of evaluating the toxicity of more than 30.000 molecules. Such evaluation would require the use of at least 4 millions of animals for an estimated cost of 2.8 billions €. While the relevance of these in vivo models remains controversial.

Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...

Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...

Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...

No / Duocarmycin natural products are promising anti-cancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies, however to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. While enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726 and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. Significance Statement Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme. / National Institutes of Health and Yorkshire Cancer Research Program Grant (B381PA)

(1) Ten monoclonal antibodies have been raised against human hepatic microsomal proteins, seven of which recognised a purified human liver cytochrome P450, P450hA7. (2) Two of the seven anti-P450hA7 monoclonal antibodies, when blotted against control and induced rat liver microsomes, recognised a male-specific, weakly expressed constitutive protein that showed marked induction by pregnenolone-16-carbonitrile (PCN) but not by phenobarbitone (PB). No such P450 has been previously reported. (3) Another of the anti-P450hA7 monoclonal antibodies recognised a protein that was not expressed in control, male or female, rat liver microsomes but was strongly expressed in both PCN- and PB-induced microsomes. This protein may correspond to cytochrome P450PCN1 which exhibits identical induction characteristics. (4) A third rat protein, strongly and constitutively expressed in male liver microsomes, is recognised by other anti-P450hA7 antibodies. This protein may be induced by PCN. (5) The hepatic microsomal level of immunoreactive P450hA7 in fifteen adult individuals showed marked interindividual variation and was approximately ten times higher in an epiletic chronically treated with the drugs PB, phenytoin, carbamazipine and valproate. (6) Foetal human liver microsomes contained a protein that was immunochemically similar, but not identical, to adult P450hA7. The foetal protein exhibited a slightly greater molecular mass than the adult form. The switch from the foetal to the adult form of P450hA7 occurred shortly after birth. (7) P450hA7 was immunochemically detected in HEP G2 human hepatoma cells. The cytochrome was constitutively expressed being present in cells treated with PB, PCN and benzanthracene as well as untreated cells. (8) The anti-P450hA7 antibodies have been put to a variety of applications including the immunohistochemical localisation of cytochrome P450hA7 in human tissue and the screening of human hepatic cDNA libraries in gtll. (9) These monoclonal antibodies constitute a precise and powerful tool for the further characterisation of the human cytochromes P450.