Drug development is a long and expensive process. Potential drug candidates can fail clinical trials due to numerous issues, including metabolic stability and efficacy issues, wasting years of research effort and resource. This thesis detailed the development of in silico methods to predict the metabolic stability of structures and their bioavailability. Coralie Atom-based Statistical SOM Identifier (CASSI) is a site of metabolism (SOM) predictor which provides a SOM prediction based on statistical information gathered about previously seen atoms present in similar environments. CASSI is a real-time SOM predictor accessible via graphical user interface (GUI), allowing users to view the prediction results and likelihood of each atom to undergo different types of metabolic transformation. Fast Metabolizer (FAME)1 is a ligand-based SOM predictor developed around the same time by Kirchmair et al. In the course of the evaluation of CASSI and FAME performance, the two concepts were combined to produce FamePrint. FamePrint is a tool developed within the Coralie Cheminformatics Platform developed by Lhasa Limited. which can carry out SOM predictions, as well as bioisosteric replacement identification. Same as CASSI, this is available via the Coralie application GUI. The bioavailability issues caused by the metabolic enzyme, cytochrome P450 3A4, and transporter protein P-gylcoprotein are also investigated in this work, along with the potential synergistic relationship between the two systems. In silico classifiers to distinguish substrates against non-substrates of the two systems are produced and it was envisaged that these classifiers can be integrated into FamePrint as an additional layer of information available to the user when deciding on bioisosteric replacements to use when optimising a compound.

This thesis describes synthetic studies towards the tricyclic terpene framework of the cytotoxic natural product eleutherobin, and the investigation of biocatalytic methodology towards late stage functionalisation of the structure. Chapter 1 provides an introduction to the biomedical significance of the natural product and to the various synthetic studies reported to date. Chapter 2 explores intramolecular Diels-Alder approaches to the construction of the nine- and six- membered rings present in the natural product. Chapter 3 describes alternative approaches to the eleutherobin core structure, leading to a formal synthesis of the natural product. The synthetic sequence developed is applied to the preparation of a small library of furanoid analogues of eleutherobin. Chapter 4 details the development of methodology for the one-pot preparation of (E)-4-methylhexa-3,5-dien-1-ol, and its use in an intermolecular Diels-Alder/lactonisation sequence to achieve enantioselective preparation of an important lactone building block. Chapter 5 describes studies on the mP450-mediated oxidation of various eleuthoside structures obtained. Finally, Chapter 6 details the construction of an electrochemical flow cell, and its use for the anodic oxidation of furfuryl alcohol derivatives.

How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.

High-valent transition metal-oxo intermediates play essential roles as active oxidizing species in enzymatic and biomimetic catalytic systems. Extensive research has been conducted on a variety of transition metal catalysts being studied as models for the ubiquitous cytochrome P450 enzymes. In doing so, the production of enzyme-like oxidation catalysts and probing studies on the sophisticated oxygen atom transfer mechanism are taking place. In this work, visible-light irradiation of highly-photo-labile corrole-manganese(IV) bromates and chlorates was studied in two corrole systems with differing electronic environments, i.e. 5,10,15-trisphenylcorrole (H3TPC) and 5,10,15- tris(pentafluorophenyl)corrole (H3TPFC). In both systems, homolytic cleavage of the O- Br and O-Cl bonds in the ligands was observed to result in one-electron photo-oxidation to afford the corrole-manganese(V)-oxo species as determined by their distinct UV-vis spectra. Kinetics of oxygen atom transfer (OAT) reactions by each photo-generated [MnV(Cor)O] species with various substrates were conducted in two solvents, CH3CN and CH2Cl2. It was found that [MnV(Cor)O] exhibits noteworthy solvent and ligand effects on its reactivity and spectroscopic behavior. In the more electron-withdrawing TPFC species in polar CH3CN solvent, MnV-oxo corrole returned to MnIII corrole following oxidation of substrate. However, the TPFC species in the less polar CH2Cl2 solvent, and in both solvents for the TPC system, MnIV product was formed instead of MnIII. An inverted reactivity pattern, i.e. TPC > TPFC, for the MnV-oxo corroles was observed. These spectral and kinetic results were rationalized by a multiple oxidation pathway model, where either a two-electron oxidation for oxygen atom transfer reactions takes place or a disproportionation reactive takes place forming the elusive manganese(VI)-oxo as the true oxidant. The preferred pathway is highly dependent on the nature of the corrole ligand and the solvent. Furthermore, a variety of [MnIV(Cor)Cl] complexes were investigated as biomimetic catalysts for the selective catalytic oxidation of the organic sulfide with mild sacrificial oxidant PhI(OAc)2. It was found that catalytic activity was affected by the oxidation state and electron environment of the catalyst. It was also found that in the same TPC system, [MnIV(TPC)Cl] was more reactive than [MnIII(TPC)], presumably due to the MnIV-corrole having easier access to the active metal-oxo intermediates than MnIII-corrole. In the same oxidation state, catalytic sulfoxidation of thioanisole resulted in a slower reaction rate for corrole species with more electron withdrawing ligands. In addition to thioanisole, [MnIV(TPC)Cl] was tested for its reactivity under catalytic conditions for eight other substrates. In most cases, quantitative conversions and excellent selectivity for sulfoxide were achieved.

Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. / In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. / In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. / This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.

Les cytochromes P450 (CYP) sont des enzymes responsables de la biotransformation de composés exogènes, aussi bien dans les phénomènes de détoxication que d'intoxication par formation d'entités réactives. La forme hépatique humaine la plus abondante (CYP3A4) est responsable du métabolisme de plus de 60 % des médicaments utilisés actuellement, entraînant de nombreuses interactions médicamenteuses indésirables. La connaissance des mécanismes moléculaires de fonctionnement de ces CYPs au moyen de modèles prédictifs est d'un intérêt primordial pour les industriels. L'obtention de ces modèles par modélisation comparative est toutefois pénalisée par la dispersion en séquences dans cette superfamille. Une méthode originale de reconstruction des CYPs basée sur l'identification au sein de cette famille des blocs structuralement conservés (CSB) est proposée ici. Ces CSBs définissent un repliement commun aux CYPs et sont considérés comme la signature structurale de la superfamille. Les CSBs sont codés en termes d'informations statistiques (profile) puis alignés sur les séquences de CYP de structure inconnue, par un outil d'alignement multiple (Caliseq) créé pour produire l'alignement multiple optimal pour les reconstructions par modélisation comparative. Caliseq sert aussi à détecter des séquences originales de CYP dans une banque ou un génome. Le modèle structural obtenu permet de suggérer des mutations pour observer les modifications du comportement de la protéine vis-à-vis de ses substrats spécifiques. Le cas du CYB2B6 est un exemple concret où le modèle a suggéré des mutations permettant d'augmenter l'affinité de l'enzyme pour un substrat spécifique utilisé en chimiothérapie.

L'infection chronique par le virus de l'hépatite C (VHC) peut s'associer chez les patients à des manifestations extrahépatiques auto-immunes. La question se pose de l'origine de cette auto-réactivité et de ses cibles. Le stress induit par l'infection chronique par le VHC induit la surexpression d'HSP70 dans les tissus hépatiques et dans le sang, phénomène s'accompagnant de l'apparition d'anticorps anti-HSP uniquement chez les patients ayant une dysimmunité associée au VHC. Grâce à un modèle cellulaire exprimant de façon stable une partie de la polyprotéine virale, nous montrons qu'à l'échelon cellulaire, ce stress viral se traduit non seulement par une augmentation du taux d'HSP, mais aussi par la présence de complexes HSP-peptides inhabituels, doués d'antigénicité. Ce stress cellulaire semble être en partie lié à l'activité protéolytique de la protéase NS3. C'est la première fois qu'est mis en évidence le rôle direct d'une protéase virale dans le développement du stress cellulaire secondaire à une infection virale. NS3 semble exercer une activité protéolytique vis-à-vis de plusieurs protéines cellulaires substrats dont le cytochrome P450 2D6, dont des épitopes sont impliqués dans l'hépatite auto-immune associée au VHC. NS3 est donc susceptible de favoriser, par son activité protéase, l'émergence d'épitopes nouveaux. L'interaction de NS3 avec C1Inh suggère un possible contrôle cellulaire de la protéase, qui serait favorisé par l'IFN, cytokine connue pour augmenter la synthèse de C1Inh.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.

Cytochrome P450 enzymes (CYPs), found in almost all organisms, are involved in endobiotic metabolism and detoxification of xenobiotic compounds, such as drugs, pollutants, and insecticides. In insects, CYPs play a major role in conferring resistance to various insecticides including DDT. In Drosophila and other insects, DDT-resistant strains exhibit increased expression of multiple P450 genes; however, the mechanism of overexpression is unknown. Since many CYP genes including Cyp6a8 of Drosophila are induced by caffeine and other xenobiotics, these chemicals are used as tools to understand the regulation of these genes. Previously it was shown that the 0.8-kb (-1/-732) and 0.2-kb (-1/-170) upstream DNA of Cyp6a8 of the DDT-resistant 91-R strain support caffeine, DDT, and Phenobarbital induction in adult flies and S2 cells, the 0.2-kb DNA has many transcriptionally important sequence motifs. In the present investigation, site-directed mutagenesis was performed on the putative TATA box and CREB/AP-1 motifs located at the -97/-101, -57/-61, -43/-47, and -6/-10 regions of the 0.2- and 0.8 DNAs to determine their cis-regulatory role in caffeine and PB induction in S2 cells using luciferase reporter system. Results showed that all four deletions in 0.2- and 0.8-kb DNA decreased both basal and caffeine-induced activities, but maximum effect was seen with the -57/-61 deletion. Second, the TATA mutations greatly decreased basal activity, but they did not decrease caffeine-inducibility as much as the -57/-61 mutations. Third, the effects of other three deletions on basal activities were not as pronounced in the 0.8-kb environment as were seen in the 0.2-kb environment. Taken together these results suggest that of all four putative CREB/AP1 sites the one located at -57/-61 region is most important for both basal and caffeine-induced activities. The results also suggest that the additional 600 bases upstream of -1/-170 have distal elements that interact with the proximal promoter in the 0.2-kb DNA and boost basal transcription. A model suggesting interactions of all cis elements with the basal promoter for basal and induced transcription has been proposed.

In order to investigate the structural features responsible for the cytotoxicity of the naturally occurring lignan nordihydroguaiaretic acid, the synthesis of four structural analogues of NDGA is proposed for the purpose of studying their oxidative metabolism. One analogue in particular (1), a mono-catechol analogue, is successfully synthesized employing a double Stobbe condensation approach. Following synthesis of this compound a series of oxidation experiments is performed consisting of: incubation in rat liver microsomes with and without the trapping agent glutathione (GSH), oxidation with mushroom tyrosinase, oxidation with silver oxide, and oxidation with horseradish peroxidase. Results are analyzed via HPLC and UPLC-MS. It is found that 1 does not autoxidize at pH 7.4 as NDGA does. Two products are produced during incubation of 1 in rat liver microsomes with UPLC-ESI(-)-MS results giving m/z of 879.2 and 574.18. This is consistent with 1 plus 2 GSH and 1 plus 1 GSH respectively; confirming 1 will oxidize to an electrophilic moiety. Oxidation with mushroom tyrosinase is found to produce high levels of product two with m/z 574.2. Oxidation with horseradish peroxidase is found to produce high levels of the m/z 879.2 product. Silver Oxide produced multiple products rather than the expected one major product, but most are found to be inconsistent with the products seen during rat liver microsomal incubation, and are not pursued.