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.

Notre objectif était d’identifier des cytochromes P450 capables d’oxygéner des mono et sesquiterpènes, pour produire des molécules aux propriétés organoleptiques intéressantes labellisés « naturelles » par l’industrie des arômes et du parfum.Nous avons identifié 7 couples P450-substrat catalysant une conversion in vitro supérieure ≥ 45 % et/ou formant un produit attendu. Les quantités de produit obtenu par bioconversion dans la levure restent insuffisantes pour un procédé industriel. Les facteurs limitants ont été identifiés dans le cas du valencène comme : 1) la toxicité induite par les produits, 2) l’accumulation du β-nootkatol dans les membranes, 3) l’inhibition de l’enzyme par les produits réactionnels. Trois cytochromes P450 d’Arabidopsis thaliana impliqués dans le métabolisme indolique oxydent activement le limonène. Ils s’expriment dans les inflorescences et constituent le premier exemple de P450s suceptibles de participer à deux voies métaboliques indépendantes chez les plantes. / Our aim was to identify cytochromes P450 catalyzing hydroxylation of mono-and sesquiterpenes to produce functionalized "natural" compounds with interesting organoleptic properties for the flavor and fragrance industry. We identified 7 P450-substrate pairs showing . 45 % in vitro conversion and/or forming an expected product. The amounts of products resulting from yeast bioconversion were however too low for implementation of an industrial process. Factors limiting the nootkatone production from the P450-dependent bioconversion of valencene were identified : 1) toxicity for yeast of the ƒÀ-nootkatol and nootkatone products, 2) ƒÀ-nootkatol accumulation in endomembranes, 3) products inhibition of valencene hydroxylation. Three previously characterized P450s from Arabidopsis thaliana in indolic metabolism were shown to actively oxidize limonene. They are expressed in inflorescences and may provide the first demonstrated case of multifunctional P450s involved in independent plant pathways.

Les cytochromes P450s (CYPs) sont des hémoprotéines intervenant dans la fonction de détoxication cellulaire. Le site actif des CYPs est enfoui dans la protéine, mais accessible aux ligands par des canaux. A l'aide d'une méthode récente basée sur la triangulation de Delaunay de la protéine, et implémentée dans le logiciel CCCPP, nous avons modélisé géométriquement ces canaux pour plusieurs isoformes humaines, dont le 3A4, présent au niveau du foie humain et responsable de la métabolisation d'un nombre important de médicaments, afin de constituer un filtre stérique destiné au criblage virtuel rapide de chimiothèques. Cette approche nous a permis d'obtenir des informations sur les mécanismes d'ouverture et de fermeture des canaux, permettant d'expliquer comment des ligands volumineux peuvent accéder au site actif. Ces résultats confirment et étendent ceux de la littérature, et peuvent contribuer à l'élaboration de médicaments nouveaux ou ayant moins d'effets secondaires. / The cytochromes P450s (CYPs) are hemoproteins involved in the cellular detoxification function. The CYPs active site is buried inside the protein, but it can be accessed by the ligands through channels. With a recent method based upon the Delaunay triangulation of the protein, and implemented in the CCCPP software, we modelized geometrically these channels for several human isoforms, including the 3A4, located in the human liver and responsible of the metabolization of an important number of drugs, in order to build a sterical filter devoted to high throughput virtual screening of chemical libraries. This approach let us to get information on mechanisms of opening and closing of the channels, allowing to explain how large ligands can access to the active site. These results are in agreement and extend those found in the literature, and can contribute to the design of new drugs or of drugs having less side effects.

Ce travail présente l'étude de la modulation des canaux K[indice inférieur Ca] des muscles lisses des voies respiratoires par les EETs. Nous avons démontré que l'isomère 11,12-EET active directement les canaux K[indice inférieur Ca], sans l'implication d'une protéine G. Des concentrations de 0.9-3.0 [mu]M multiplient par 1.2-4 la probabilité d'ouverture des canaux K[indice inférieur Ca] sans affecter leur conductance. De plus, les effets du 11,12-EET sont présents lorsque la molécule exogène est ajoutée du côté extracellulaire (cis), et non du côté cytoplasmique (trans) du canal. Le précurseur du 11,12-EET, l'acide arachidonique, induit aussi une activation des canaux K[indice inférieur Ca], mais à des concentrations beaucoup plus élevées. Par contre, les platelet-activating factors (PAF) n'ont aucun effet direct sur le canal, même à des concentrations de 10 [mu]M. Le 11,12-EET n'induit pas une plus grande activation des canaux K[indice inférieur Ca] avec la présence de GTP du côté cytoplasmique, et donc l'hypothèse de la double activation de ces canaux par le 11,12-EET n'a pas été retenue. Finalement, le 11,12-EET induit une relaxation des muscles lisses des bronches de cobayes contractés avec le carbamylcholine, un agoniste muscarinique".--Résumé abrégé par UMI.

L'épilepsie idiopathique généralisée avec absences (EIGA) est une des formes les plus fréquentes d'épilepsie chez l'enfant. Elle survient chez les enfants exempts de maladie avec un pic d'incidence autour de 5 ans. Des statistiques mondiales indiquent que les crises d'absences sont contrôlées par l'Acide Valproïque (AV) chez 75 % des enfants. Pour les autres, la médication est inefficace ou elle entraîne des effets secondaires qui limitent son utilisation. Certaines études ont identifié des facteurs cliniques associés à l'épilepsie réfractaire mais elles incluent tous les types d'épilepsie et tous les traitements. De plus, un nombre croissant de recherches démontrent que la réponse aux médicaments pourrait être grandement influencée par des facteurs génétiques, plus particulièrement par les polymorphismes du cytochrome P450 (CYP). Il faudrait donc ajuster le traitement en fonction du profil génétique de chaque patient. Dans cette étude, nous avons voulu identifier des facteurs cliniques, sociodémographiques et génétiques qui pourraient influencer la réponse à l'AV chez des enfants diagnostiqués d'EIGA. Méthodes : Nous avons tout d'abord identifié des patients diagnostiqués d'EIGA qui étaient suivis au CHU Sainte-Justine de Montréal et traités à l'AV pendant au moins deux mois. Par la suite, une même personne (MLO) a réalisé la revue de tous les dossiers médicaux afm [i.e. afin] d'extraire l'information concernant la réponse à l'AV et les données cliniques et sociodémographiques qui pourraient influencer celle-ci. Une collecte d'échantillons d'ADN des patients toujours suivi à la clinique a ensuite été effectuée afm d'évaluer l'impact de polymorphismes dans deux enzymes du CYP (CYP2C9 et CYP2C19) sur la réponse à l'AV chez ces patients. L'évaluation des polymorphismes s'est faite par hybridation avec oligonucléotides spécifiques pour des allèles (ASO). Des analyses bivariées ont été effectuées afin d'identifier toutes les variables qui différaient entre les répondants (R) et les non-répondants (NR) à l'AV. Par la suite, nous avons effectué une analyse de régression logistique afm d'éliminer les variables qui cessaient d'être significatives en présence des autres. Résultats : Nous avons révisé 190 dossiers (112 R et 68 NR) pour l'analyse des facteurs cliniques et sociodémographiques. Trois variables étaient significativement différentes entre les deux groupes soit l'âge au diagnostic (R=7,55 ans vs NR=6,11 ans, p,001), la présence de crises généralisées tonico-cloniques (R=13,4 % vs NR=33,8 %, p=0,001) et fmalement [i.e. finalement] une fréquence élevée de crises avant le début du traitement (R=27,0 % vs NR=52,9 %, p<0,001). Ces trois variables continuaient d'être significatives dans le modèle de régression logistique. Pour la partie génétique du projet, un échantillon d'ADN a été prélevé sur 83 patients (46 R et 37 NR). Les NR présentaient significativement plus de polymorphismes du CYP2C19 (43,2 % vs 17,4 %, p=0,010) avec un rapport de cotes de 3,62. Lorsqu'on incluait ce polymorphisme dans le modèle de régression logistique, seuls l'âge au diagnostic et le polymorphisme du CYP2C19 demeuraient significatifs. Conclusions : Dans cette étude, nous avons pu identifier certains facteurs cliniques et génétiques qui influencent la réponse à l'AV chez les enfants diagnostiqués d'EIGA. Les résultats montrent qu'il y a un certain profil de patient plus à risque de ne pas répondre au traitement. Donc, si un patient ne semble pas répondre à l'AV et qu'il présente ces facteurs, le neurologue devrait considérer un traitement alternatif plutôt que d'augmenter la dose d'AV et ainsi risquer l'augmentation des effets secondaires dus au traitement.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / This study describes: 1. The preparation of chloroform, methanol and aqueous extracts of unfermented and fermented rooibos (Aspalathus linearis). 2. The chromatographic fractionation of aqueous rooibos extracts and an investigation into the polyphenol content and antioxidant activity of the fractions. 3. The preparation of ovine adrenal microsomes containing active steroidogenic P450 enzymes, including cytochrome P450 17a-hydroxylase, CYP17, and cytochrome P450 steroid 21-hydroxylase, CYP21. 4. An investigation into the influence of chloroform and methanol extracts of rooibos on the binding of steroid substrates, progesterone and 17-hydroxyprogesterone, to CYP17 and CYP21.

Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant inter individual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mouse model, indicating attenuation of the activity of Cyp1a2 and Cyp2c29, respectively. Decreased mRNA expression of Cyp1a2 and Cyp2c29, as well as an overall decrease in CYP protein expression, was found in the diabetic NASH mice. Overall, these data suggest that the diabetic NASH model only partially recapitulates the human ex vivo CYP alteration pattern. Therefore, in vivo determination of the effects of NASH on CYP activity should be conducted in human, and more appropriate models are required for future drug metabolism studies in NASH.

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.

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.

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.