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.

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.

Pre-systemic elimination by local enzymatic degradation can play a key role in limiting the bioavailability of intranasally administered drugs. Despite remarkable advancement in the characterization of the nasal biotransformative enzymes, knowledge of the role of the nasal mucosa in limiting bioavailability of therapeutic agents is still inadequate. The aim of this work was to evaluate the expression and substrate biotransformation activity of cytochrome P450 enzymes in the nasal mucosa using bovine olfactory and respiratory explants as in vitro models. Gene expression and localization of major CYP450 isoforms in the nasal mucosa were examined using RT-PCR and immunohistochemistry. The bovine nasal mucosa showed abundant expression of CYP2A6 and 3A4 genes whereas 1A1, 1A2, 2C9, and 2C19 isoforms were expressed at much lower levels. The CYP450 proteins were observed to be present in the epithelial layer and in submucosal glandular cells. The diffusion of melatonin, a CYP1A2 substrate, and the appearance of 6-hydroxymelatonin, its primary metabolite, across bovine olfactory and respiratory explants was measured, and nasal olfactory and respiratory microsomal preparations were used to quantify the kinetic parameters for melatonin 6-hydroxylation. Results indicated that bovine olfactory and respiratory CYP450 isoforms were metabolically active towards melatonin metabolism, and the respiratory mucosa demonstrated the greatest melatonin 6-hydroxylation activity. Numerical simulations were used to probe the effects of the relative magnitudes of the permeability coefficient and enzymatic parameters on net substrate mass transfer across nasal mucosal tissues. The simulations indicated that the concentration gradient of the drug coupled with its permeability coefficient were the most significant factors controlling the transport of drugs across the mucosal tissue. Enzymatic degradation decreased the flux of drugs across the mucosa and had the greatest impact on low permeability compounds. The results from these studies show that the bovine nasal mucosa possesses significant metabolic activity, and the flux of a metabolically labile substrate across the nasal mucosa can be significantly reduced by its enzymatic degradation within the tissue. Use of kinetic modeling to characterize of the extent of biotransformation in the nasal mucosa enables the identification of metabolism-limited bioavailability of intranasally administered drug compounds.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Thesis (M.Sc.)--Memorial University of Newfoundland, 1997. / Bibliography: leaves 50-51.