The traditional view of drug design is that a single drug should interact with a single molecular target. As science progressed, there was an understanding that most drugs interact with more than one target and that multiple targets may be responsible for either adverse effects or additional therapeutic effects. The idea of polypharmacology, which suggests that the focus of drug design should shift from a single drug that interacts with a single target to a single drug that can have interactions with multiple targets and multiple therapeutic effects, revolutionized the drug discovery process. Discovering new drugs is a long and costly process with years of research and development and clinical trials required before the drugs reach the market for much needed therapeutic applications. By repurposing drugs that are already on the market for a new therapeutic target, the discovery process is accelerated significantly. One such a target disease, for which there is a great need for new effective therapies, is Parkinson’s disease (PD). PD is a progressive neurodegenerative disease that is caused by the death of dopaminergic neurons in the substantia nigra with the resulting loss of dopamine from the striatum. Degeneration in PD leads to varying degrees of motor difficulty and disability, along with other symptoms. Current therapies are focussed on symptomatic management and an improvement of the quality of life of patients, rather than on a cure. There are several therapeutic targets that are currently used in the treatment of PD. One of those targets is the monoamine oxidase (MAO) enzymes, in particular the MAO-B isoform. The MAO enzymes are responsible for the metabolism of amine neurotransmitters, such as dopamine, and inhibition of MAO-B has proven to be an effective strategy to increase the dopamine levels in the brain. Clinically, selective MAO-B inhibitors are administered concurrently with levodopa (a precursor of dopamine) to increase the levels of dopamine derived from levodopa. This approach prolongs the beneficial effects of levodopa. Because MAO-A is responsible for the breakdown of noradrenalin, adrenalin, serotonin and tyramine, non-selective and selective MAO-A inhibitors have therapeutic applications in other neurological and psychiatric disorders such as depression. MAO-A inhibitors, particularly irreversible inhibitors, are also notable from a toxicological point of view. Irreversible MAO-A inhibitors may lead to potentially dangerous effects when combined with serotonergic drugs and certain foods containing tyramine, such as cheeses and processed meats. Selective MAO-B inhibitors and reversible MAO-A inhibitors appear to be free of these interactions. Based on the considerations above, this study aimed to identify clinically used drugs which also inhibit the MAO enzymes as a secondary pharmacological property. Such drugs may, in theory, be repurposed as MAO inhibitors for therapeutic use in the treatment of PD and depression. The identification of potential MAO-A inhibitory properties among clinically used drugs are of further importance since the irreversible inhibition of MAO-A may lead to dangerous effects when combined with certain drugs and foods. To screen clinically used drugs for potential MAO-A and MAO-B inhibitory activities, a pharmacophore approach was followed. A pharmacophore model is a virtual 3D representation of the common steric and electrostatic features of the interaction between an enzyme and a ligand. By identifying hydrogen bond acceptor, hydrogen bond donor and hydrophobic interactions between a reference ligand and an enzyme, a model is created that can search databases for other molecules that would have similar interactions with the enzyme and arguably also act as ligands. This enables the screening of a large amount of molecules in a short amount of time. To assist in the identification of MAO inhibitors, pharmacophore models of the MAO enzymes were constructed using the known crystallographic structures of MAO-A co-crystallized with harmine, and MAO-B cocrystallized with safinamide. The Discovery Studio® software package (Accelrys) was used for this purpose. In this study, virtual libraries of United States Food and Drug Administration (FDA) approved drugs and the United States Environmental Protection Agency (EPA) maximum daily dose databases were screened with pharmacophore models of MAO-A and MAO-B. Among the hits, 26 drugs were selected on the basis of availability and cost, and were subjected to in vitro bio-assays in order to determine their potencies (IC50 values) as inhibitors of recombinant human MAO-A and/or MAO-B. Among the drugs tested, 6 compounds exhibited inhibitory activity towards the MAO enzymes. Of the 6 compounds, pentamidine (IC50 = 0.61 μM for MAO-A and IC50 = 0.22 μM for MAO-B) and phenformin (IC50 = 41 μM for MAO-A) were selected for further analysis. An examination of the recoveries of the enzymatic activities after dilution and dialysis of the enzyme-inhibitor complexes showed that both pentamidine and phenformin interact reversibly with the MAO enzymes. A kinetic analysis suggests that pentamidine acts as a competitive inhibitor with estimated Ki values of 0.41 μM and 0.22 μM for the inhibition of MAO-A and MAO-B, respectively. An analysis of the available pharmacokinetic data and typical therapeutic doses of phenformin and pentamidine suggests that the MAO inhibitory potencies (and reversible mode of action) of phenformin are unlikely to be of pharmacological relevance in humans. Pentamidine, on the other hand, is expected to interact with both MAO-A and MAO-B at typical therapeutic doses. Because of its MAO-A inhibitory activity, pentamidine may thus, in theory, lead to a tyramine-associated hypertensive crisis when combined with tyramine-containing foods. However, pentamidine is unlikely to inhibit central MAO since it does not appear to penetrate the central nervous system to a large degree. In an attempt to gain further insight into the mode of binding to MAO, pentamidine and phenformin were docked into models of the active sites of MAO-A and/or MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation. The results of this study show that the pharmacophore model approach may be useful in identifying existing drugs with potential MAO inhibitory effects. The search for new therapeutic MAO inhibitors, that can be used in the treatment of certain neurological disorders, including PD and depression, may be accelerated by employing a virtual screening approach. Such an approach may also be more cost effective than the de novo design of MAO inhibitors. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

A greenhouse experiment was carried out to screen for potential effects of transgenic aspen over-expressing a hybrid poplar leaf polyphenol oxidase gene on rhizosphere communities. Heterotrophic plate counts and cultivation-independent methods were used to compare bacterial and fungal populations associated with transgenic PPO over-expressing and unmodified control trees. Total community DNA extracted from rhizosphere soils was used to establish Iibraries containing partial gene sequences that were PCR-amplified from community members, and putative taxonomy was assigned to clones based on similarity to reference sequences. Gene libraries for the bacterial component of the rhizosphere were established using partial 16S rRNA and chaperonin-60 gene sequences, and the fungal community was characterized based on partial 18S rRNA gene sequences. Phylogenetic analysis revealed that bacterial 16S gene libraries were dominated by Alphaproteobacterial sequences, and the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group, illustrating the biases potentially incurred by using a single gene locus to profile microbial diversity. In both CPN-60 and 16S rRNA libraries, only minor components of the bacterial community differed between transgenic and unmodified trees. Comparisons based on library coverage indicated that changes in bacterial community structure between transgenic and unmodified trees were minor in comparison to differences observed between individual trees of the same type, and no significant differences in terms of bacterial species diversity were revealed by the calculated diversity, dominance and evenness indices. In comparison to the bacterial gene libraries, higher coverage of the underlying population was achieved in the fungal 18S libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. Dominant groups of fungi associated with each tree type were highly similar, although there were some qualitative differences in the recovery of less abundant fungi as a result of the underlying heterogeneity of the fungal population. No clear differences in terms of fungal species richness were associated with transgenic or unmodified trees, although control libraries were characterized by a slightly higher level of dominance. In general, the methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees, suggesting that impacts of the transgenic plants on the rhizosphere community were minimal.

Aldehyde oxidase catalysed oxidation of various quinolinium and related cations has been studied in vitro. Oxidation products were identified by comparison of their spectral and chromatographic characteristics with those of authentic compounds. The N-heterocyclic cations and quinolones used required synthesis. Incubation of N-methylquinolinium, N-methyl-7,8-benzoquinolinium and N-phenylquinolinium yielded the corresponding 2- and 4-quinolones simultaneously. The ratio of 2- to 4-quinolone formation was found to be species dependent; the proportion of 4-quinolone was greater with guinea pig enzyme than with rabbit enzyme. Incubation of N-methyl-4-methylquinolinium, N-methyl-4-phenylquinolinium and N-methylphenanthridinium produced the expected 2-quinolones. Cations substituted adjacent to the ring nitrogen, i. e. N-methyl-2- methylquinolinium, N-methyl-2-phenylquinolinium and N-phenyl-2-phenylquinolinium, were oxidised to the corresponding 4-quinolones. Kinetic constants were determined spectrophotometrically. The Km values obtained with rabbit enzyme ranged from 1.6 x 10-3 M for N-methylquinolinium to <10-5 M for N-phenyl-2-phenylquinolinium. Quaternary compounds were found to be better substrates than their non-quaternary counterparts, except for N-methylisoquinolinium and N-methylphenanthridinium. In general, guinea pig aldehyde oxidase was shown to have a greater affinity for N-heterocyclic cations than rabbit enzyme. The substrate binding site has been discussed in the light of the results outlined below. Oxidation of N-methyl-4-phenylquinolinium (to the 2-quinolone) was competitively inhibited by N-methyl-2-phenylquinolinium (which yields the 4-quinolone), indicating that both these cations interact at the same active site. The ratio of 2- to 4-quinolone production from N-methylquinolinium was constant under various conditions, including purification of the enzyme but changed at high pH or in the presence of N-methylphenanthridinium. Inhibition studies indicated that both quaternary and non-quaternary compounds act at the same site on the enzyme. Km and Vmax values for phthalazine, N-methyl-2-phenylquinolinium and N-methylquinolinium were determined over the pH range 5.4 to 10.2. In each case, results indicated that the enzyme has an ionisable group at the active site with a pK ca. 8. Aldehyde oxidase was shown to catalyse the dehydrogenation of the pseudobases 3,4-dihydro-4-hydroxy-3-methyl-2-quinazolinone and 3,4-dihydro- 4-hydroxy-3-methylquinazoline.

Genomic clones were isolated and characterized using the human monoamine oxidase A (MAOA) cDNA to screen a phage library, constructed from a human 4X cell line (48, XXXX). The genomic contig derived from overlapping phage clones showed that the size of the MAOA gene is over 80 kb. Exon-containing fragments from these phage clones were subcloned and sequenced. The data from this showed that the MAOA gene consists of 15 exons. A YAC (yeast artificial chromosome) isolated using the MAOA cDNA was characterized. This YAC was found to contain both the MAOA and the MAOB genes. Using PFGE (pulsed-field gel electrophoresis) to investigate the YAC, it was found that the MAOA and the MAOB genes are located within 50 kb and adjacent to each other. The two genes are localized in a 3'-to-3' fashion, suggesting their expression may be regulated independently. The analysis of the homology shown by the two genes clearly demonstrated that they were derived from duplication of a common ancestral gene. A CpG island was discovered to be associated with the 5' end of both genes. A restriction map of -2.5 Mb of genomic DMA around the MAO genes was generated by PFGE. Long-range mapping defined the physical relationship between the marker L1.28 and the MAO genes as L1.28_MAOA_MAOB. A number of genetic diseases have been linked to the Xp11.3 region. Strong linkage was known to exist between the Norrie disease locus and L1.28. Studies showed that some of the Norrie patients have deletions encompassing the region which contains L1.28 as well as the MAO genes. Another YAC isolated by using L1.28 as the probe was also characterized. A phage library was constructed from the L1.28 YAC and the end clones were isolated. Studies on some of the Norrie deletion patients showed that the proximal end clone of the YAC was retained in one of the deletion patients. Previous studies had shown that the Norrie disease locus was also localized proximal to the 5' end of the MAOB gene. The combined information placed the disease locus to an interval of 240 kb within the YAC. More phage clones were characterized in order to define further the region for the Norrie locus which was finally localized within 160 kb. A YAC fragment of 160 kb was isolated and used to screen two human retinal cDNA libraries. Among the cDNAs isolated, one group was found to be deleted in some of the Norrie patients previously without any known deletion, which established their candidacy as the transcripts of the Norrie disease locus. Further characterization of the candidate gene showed that it is conserved across species. The expression of the gene was detected in various tissues. The homology shared between the NDP gene and some of the growth factor binding proteins suggests its role in neural cell proliferation and differentiation.

This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450_cam. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an in vitro reconstituted P450_cam system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the in vitro reconstituted P450_cam system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450_cam hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450_cam system were constructed and expressed in E. coli. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450_cam enzyme and other substrates with the Y96A mutant. The in vivo catalytic system was then used to screen many P450_cam mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450_cam mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Digestive proteases were isolated from the pancreas of the stomachless cunner fish (Tautogolabrus adspersus) and characterized in terms of their physicochemical properties, their ability to hydrolyze native pectin methylesterase (PME) from orange and polyphenol oxidases (PPO) from mushroom and the ability of the cunner enzyme(s) to maintain the stability of orange juice cloud. / The cunner trypsin fraction exhibited exceptional capacity to hydrolyze native proteins versus the bovine trypsin. Incubation of native PME with cunner or bovine trypsin resulted in a loss of 75% or 35% in PME activity respectively. Similarly, a 75% or 55% loss in PPO activity was observed after treatment with cunner and bovine trypsin respectively. Bovine trypsin, however, hydrolyzed the heat-denatured PME and PPO better than the cunner trypsin. Also, there was no reactivation of both PME and PPO activity after treatment with either the cunner or bovine enzyme during storage at 4$ sp circ$C for 3 weeks. However, PPO retained up to 20% or 50% of the initial activity after treatment with cunner or bovine trypsin, respectively. / A 3 x 3 factorial design involving the factors of temperature, enzyme concentration and incubation time carried out gave an r$ sp2$ of 0.92 and 0.95 for cunner and bovine trypsin treated PME respectively. On the other hand, an r$ sp2$ of 0.91 and 0.94 was obtained for the combined effects using cunner and bovine trypsin for PPO inactivation. Validation of the model of PME inactivation measured as the % cloud remaining revealed that the cunner trypsin fraction upheld the cloud stability of cloud juice better than bovine trypsin, with cunner trypsin retaining more than 90% of the cloud whereas the juice treated with bovine trypsin only resulted in a 70% retention of the juice cloud. (Abstract shortened by UMI.)

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Ruh Sağlığı ve Hastalıkları Anabilim Dalı, 2006. / Bibliyografya var.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.