Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 170-201).

This thesis describes work done with the thyroid hormone receptor (TR), a nuclear protein which binds to specific DNA sequences and regulates transcription in response to thyroid hormone levels. The studies can be divided into two broad categories: structure/function studies of the TR protein, particularly with regards to DNA binding function; and, structure/function studies of the DNA sequences to which the thyroid hormone receptor binds in order to regulate gene transcription. In order to examine the DNA binding properties of the TR an electrophoretic mobility shift assay (EMSA) was utilized. Conditions of this assay were optimized for the use of in vitro translated TR. Mutant forms of the β-isoform of thyroid hormone receptor were generated using a PCR-based mutagenesis protocol. Each mutant substituted a different residue of the 12 amino acid-long α-recognition helix with alanine. The mutants were analyzed for their abilities to bind to thyroid hormone response elements (TREs), and to activate transcription in transfected eukaryotic cells. The DNA binding results were consistent with a conserved α-helix structure, with conserved function for many residues, that is similar to that of the related receptors for glucocorticoids and estrogen. Only the first of the three non-conserved residues lying in the P-box (EGG), a portion of the recognition α-helix that facilitate differential binding of distinct DNA sequences, disrupted binding when substituted with alanine. The third position of the P-box, when substituted with alanine exhibits an altered ability to bind to certain natural TREs. The mutant form of TR with alanine substituted for the second P-box position displayed only a modest decrease in DNA binding affinity compared to wild-type TR (roughly 3-fold), yet was completely deficient in trans-activation. The structure-function studies of TR binding sites on DNA applied a methylation interference protocol to examine the interactions of TR with direct repeats (DR) of the idealized hexameric sequence, spaced by three to five base-pairs. The interactions of both the TR/TR homodimer and the TR/RXR (9-cis-retinoic acid receptor) heterodimer with the DRs were examined. The methylation interference patterns for the TR/TR homodimer bound to the DR sequences are virtually identical for spacers of four and five base-pairs, but with three base-pairs, there is some evidence that at least one DNA binding domain is misaligned with the DNA to accomodate the unfavourable spacer length. The TR/RXR heterodimer methylation interference pattern is distinct on all three DRs, probably due to the fact that in the heterodimer cooperative intermolecular contacts are made between the DNA binding domains of the two receptors, but only when the spacer distance is four base-pairs. When a poorly conserved everted repeat (EvR) that overlaps the idealized DR is present. the homodimer, but not the heterodimer, binds this cryptic EvR in competition with the DR. The binding modality of the TR homodimer and TR/RXR heterodimer to DRs was reevalutated using point mutants and EMSA. The TR homodimer and TR/RXR heterodimer both bind to idealized direct repeats with DBDs aligned appropriately for a direct repeat; however, evidence is presented that there are certain poorly conserved sequences that are intermediate between DRs and EvRs that are differentially recognized by the TR homodimer and the TR/RXR heterodimer. That is, the homodimer binds with the DBDs aligned appropriately for a EvR, and the heterodimer DBDs are aligned appropriately for a DR. / Graduate

Orientadores: Douglas Soares Galvão, Scheila Furtado Braga Llanes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-10T15:39:34Z (GMT). No. of bitstreams: 1 Marchiori_MarceloAmorim_M.pdf: 2387821 bytes, checksum: 159d3a99481a5ba6c6eb057065531e6e (MD5) Previous issue date: 2007 / Resumo: A Combretastatina é um estilbeno isolado na década de 80, e vem sendo amplamente estudada pela indústria farmacêutica devido à sua promissora ação anticarcinogênica. Como fármaco anticarcinogênico, age interrompendo o ciclo de polimerização e despolimerização dos microtúbulos, componente celular extremamente importante para a motilidade, manutenção estrutural e mitose celular. Sua principal forma de atuação consiste em despolimerizar os microtúbulos estáveis das células endoteliais da vasculatura tumoral, levando ao bloqueio do fluxo sanguineo que alimenta os tumores cancerígenos. Uma das grandes vantagens da Combretastatina, em relação aos demais medicamentos antineoplásicos, é o fato de não levar à resistência medicamentosa no tratamento quimioterápico. Investigamos a estrutura da Combretastatina e 17 derivados por meio de métodos semiempíricos e estudamos a relação entre as propriedades teóricas e a atividade experimental destes compostos, utilizando três metodologias de reconhecimento de padrões: a Metodologia de Índices Eletrônicos (MIE), a Análise de Componentes Principais (PCA) e a Análise Hierárquica de Clusters (HCA). Para cada metodologia construímos regras e padrões, permitindo a classificação dos compostos em ativos e inativos, a partir das propriedades calculadas teoricamente. Os resultados das três metodologias confirmam a aplicabilidade da MIE e reforçam a importância das variáveis eletrônicas para a classificação da atividade biológica das Combretastatinas / Abstract: Combretastatin, a stilbene isolated in 80's, has been widely studied by the pharmaceutical industry due to its promising anticarcinogenic action. As an antineoplastic agent it acts interrupting the polymerization-depolymerization cycle of the microtubules, an important cellular component to motility, strutuctural maintenance and cellular mitosis. Its main feature consists in dissociate the microtubules in endothelial cells of the tumoral vascular system, leading to disruption of the blood ow that feeds the carcinomas. One of the great advantages of Combretastatin, when compared with others compounds, is the fact that it does not lead to drug resistance in chemotherapy treatments. We investigated the structure of Combretastatin and 17 derivatives using semiempirical methods. We performed the study of the relationship between theoretical properties and experimental activity of these molecules using three pattern recognition methodologies: Electronic Index Methodology (EIM), Principal Component Analysis (PCA) and Hierarchical Clusters Analysis (HCA). For each methodology we found rules and patterns capable of classifying our molecules into active or inactive, using the properties theoretically calculated. The results obtained from the three methodologies confirm the applicability of the EIM and reinforce the importance of the electronic variables for the classi cation of the biological activity of Combretastatins / Mestrado / Estrutura Eletrônica de Atomos e Moleculas / Mestre em Física

A formação de complexos antígeno-anticorpo ou imunocomplexos (ICs) na circulação e sua eliminação faz parte dos mecanismos de defesa imune humoral do ser humano. Em algumas patologias, como lupus eritematoso sistêmico, artrite reumatóide e vasculite auto-imune, ocorre um desequilíbrio nesse processo, que leva à deposição dos ICs nos tecidos e ao desencadeamento de uma reação inflamatória. Esta, por sua vez, envolve o recrutamento e ativação de neutrófilos, que têm importante participação na patogênese dessas doenças. A ativação dos neutrófilos pelos ICs, via receptores para a porção Fc de IgG (FcR) e receptores de complemento (CR), desencadeia diversas funções efetoras, tais como fagocitose, desgranulação e o metabolismo oxidativo, com a produção de espécies reativas de oxigênio (EROs). Estas funções estão envolvidas na digestão dos ICs, na morte de microorganismos, e na regulação do processo inflamatório. Entretanto, nas doenças mediadas por ICs, os neutrófilos ativados liberam grandes quantidades de enzimas e EROs para o meio extracelular, contribuindo para a lesão dos tecidos do hospedeiro e a amplificação do processo inflamatório. Neste trabalho foi avaliado o efeito modulatório de vinte derivados de 3-fenilcumarina nas funções de neutrófilos estimulados por ICs de ovalbumina (OVA) e IgG anti-OVA. Além disso, foi feita a investigação mecanismos de ação dessas substâncias e a análise da relação estrutura-atividade. O metabolismo oxidativo dos neutrófilos ativados por ICs foi medido por ensaio de quimioluminescência dependente de lucigenina ou de luminol (QLlucPMN e QLlumPMN, respectivamente). Observou-se que as 3-fenilcumarinas contendo o grupo substituinte 3,4-metilenodioxi e o grupo substituinte 6,7-orto-diidroxi (C13) ou 6,7-orto-diacetoxi (C13a), bem como a 3-fenilcumarina 6,7,3,4-tetraacetoxilada (C24a), apresentaram atividade inibitória maior que a quercetina (QUER) sobre a QLlucPMN e a QLlumPMN. Para as demais substâncias avaliadas, que foram tão ou menos ativas que a QUER, as características estruturais relacionadas à inibição da QLlucPMN foram um pouco diferentes daquelas relacionadas à inibição da QLlumPMN. Além disso, as 3-fenilcumarinas estudadas e a QUER não apresentaram efeito tóxico sobre os neutrófilos, avaliado pela liberação de lactato desidrogenase e pelo ensaio de exclusão ao corante Azul de Tripan, nas condições empregadas. Para as três 3-fenilcumarinas que apresentaram maior efeito inibitório sobre o metabolismo oxidativo dos neutrófilos (C13, C13a e C24a), o aumento do tempo de pré-tratamento levou a uma tendência de redução do efeito inibitório da substância C24a, mas não influenciou na atividade biológica das substâncias C13 e C13a. Essas três substâncias não interferiram na capacidade fagocítica das células, avaliada por microscopia eletrônica de transmissão. Para todas as 3-fenilcumarinas foi avaliada também a capacidade antioxidante frente ao radical livre 2,2-difenil-1-picril-hidrazil e o efeito inibitório dessas substâncias sobre a quimioluminescência produzida pela reação horseradish peroxidase-H2O2-luminol (QLHRP). Foi observado que a QUER e as 3-fenilcumarinas contendo o grupo substituinte orto-diidroxi (C13, C23, C24) tiveram atividade antioxidante e inibiram a QLHRP, mas suas análogas acetoxiladas (C13a, C23a, C24a), bem como as demais substâncias avaliadas, foram significativamente menos ativas nesses modelos experimentais não celulares. O conjunto de resultados deste trabalho sugere que as atividades biológicas das 3-fenilcumarinas estudadas foram dependentes de suas estruturas químicas, e pequenas modificações nestas podem levar a alterações significativas na magnitude de seus efeitos biológicos. Além disso, tanto a lipofilicidade das substâncias quanto a sua capacidade antioxidante parecem ser relevantes para a modulação eficiente do metabolismo oxidativo dos neutrófilos e da conseqüente lesão tecidual. / Formation and clearance of circulating antigen-antibody complexes or immune complexes (ICs) take part in the humoral immune defense mechanisms. In some diseases, as systemic lupus erythematosus, rheumatoid arthritis and auto-immune vasculitis, an imbalance of this process occurs, leading to the ICs deposition within tissues and triggering an inflammatory reaction. The last one involves the recruitment and activation of neutrophils, which have an important role in the pathogenesis of such diseases. Neutrophil activation by ICs, via receptors for the Fc portion of IgG (FcgR) and complement receptors (CR), triggers a sort of effector functions, such phagocytosis, degranulation and the oxidative metabolism, which produces reactive oxygen species (ROS). These functions are involved in the ICs digestion, microbial killing and the inflammatory process regulation. However, the activated neutrophils release large amounts of enzymes and ROS to the extracellular milieu, contributing to the tissue damage and amplification of the inflammatory process in the IC-mediated diseases. In this work, we evaluated the modulatory effect of twenty 3-phenylcoumarin derivatives in the neutrophil functions stimulated by ICs of ovalbumin (OVA) and IgG anti-OVA. In addition, the mechanisms of action of these compounds were investigated, and the structure-activity relationship was analyzed. The IC-activated neutrophil oxidative metabolism was measured by lucigeninor luminol-dependent chemiluminescence assay (CLlucPMN and CLlumPMN, respectively).It was observed that the 3-phenylcoumarins bearing a 3,4-methylenodioxy and the 6,7-orto-dihydroxy (C13) or the 6,7-orto-diacetoxy (C13a) group, as well as the 6,7,3,4-tetraacetoxylated 3-phenylcoumarin (C24a), inhibited CLlucPMN and CLlumPMN more than quercetin (QUER). Regarding the other evaluated compounds, whose inhibitory effects were similar to or lower than QUER, the structural features related to the CLlucPMN inhibition were different from those related to the CLlumPMN inhibition. Moreover, the studied 3-phenylcoumarins and QUER had no toxic effects on neutrophils, as evaluated by lactate dehydrogenase release and Trypan Blue exclusion, under the assessed conditions. With respect to the three 3-phenylcoumarins that had the highest inhibitory effects on the neutrophil oxidative metabolism (C13, C13a and C24a), the increase of the cell pre-treatment period showed a tendency to decrease the inhibitory ability of compound C24a, but did not influence the biological activity of compounds C13 and C13a. These three compounds did not interfere in the neutrophil phagocytic ability, as evaluated by transmission electron microscopy. The antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl free radical and the inhibitory effect in the chemiluminescence generated by the horseradish peroxidase-H2O2-luminol reaction (CLHRP) were evaluated for all 3-phenylcoumarins. It was found that QUER and those 3-phenylcoumarins bearing the orto-dihydroxy group (C13, C23, C24) had antioxidant activity and inhibited the CLHRP. However, their acetoxylated analogues (C13a, C23a, C24a) and the other evaluated compounds were significantly less active on these cell-free experimental models. Taken together, the results of the present work suggest that the biological activities of the 3-phenylcounarins here investigated were dependent on their chemical structures, and small changes on the molecule can lead to significant changes on the magnitude of their biological effects. Moreover, both lipophilicity and antioxidant capacity of these compounds seem to be relevant to an efficient modulation of the neutrophil functions and the consequent tissue damage.

Os neutrófilos são células fagocíticas do sistema imune inato com mecanismos especializados de digestão de patógenos, complexos imune e detritos celulares, que são mediados principalmente por espécies reativas de oxigênio (EROs) e enzimas proteolíticas. Entretanto, a ativação maciça de neutrófilos leva a uma liberação exacerbada de enzimas e EROs para o meio extracelular, o que pode ultrapassar a capacidade de defesa tecidual, composta por antioxidantes e antiproteinases, e lesar o tecido, bem como amplificar o processo inflamatório observado em algumas doenças inflamatórias, autoimunes e infecciosas. O envolvimento de neutrófilos na fisiopatologia de tais doenças tem atraído o interesse na pesquisa de novas substâncias com propriedades antioxidantes e imunomodulatórias. Neste trabalho, foi avaliado o efeito modulatório de onze derivados de cumarinas hidroxiladas e acetoxiladas sobre duas funções efetoras de neutrófilos humanos (produção de EROs e desgranulação), bem como a citotoxicidade dessas substâncias. Além disso, a relação estrutura-atividade foi analisada. Para tais investigações, o sangue venoso foi coletado de voluntários saudáveis e os neutrófilos foram isolados pelo método da gelatina. O metabolismo oxidativo dos neutrófilos foi desencadeado por zimosan opsonizado com soro humano normal (ZIops) ou forbol-12-miristato-13-acetato (PMA), e a resposta celular foi avaliada pelos ensaios de quimioluminescência dependente de lucigenina (QLluc) ou de luminol (QLlum). Para a realização desses ensaios, foram padronizadas as seguintes condições experimentais: concentração das sondas luminol e lucigenina; concentração do solvente dimetilsulfóxido; tempo de leitura da QLuc e QLlum. Posteriormente, a atividade antioxidante das cumarinas frente ao radical 2,2-difenil-1-picrilhidrazil (DPPH) foi avaliada espectrofotometricamente em 510 nm. Além disso, foi avaliado o efeito das cumarinas na desgranulação dos neutrófilos induzida por n-formil-metionil-leucil-fenilalanina (fMLP), utilizando-se a enzima elastase como marcador, bem como o efeito dessas substâncias na atividade da elastase liberada pelos neutrófilos. Ambos os ensaios foram realizados através da quantificação de p-nitroanilina liberada após a quebra de um substrato específico para essa enzima, em 405 nm. A toxicidade das cumarinas sobre os neutrófilos foi avaliada pela exclusão do azul de tripan e pela medida da liberação de lactato desidrogenase. Observou-se que, tanto para as células estimuladas por ZIops quanto por PMA: (i) a maioria das cumarinas inibiu a QLluc e a QLlum de maneira dependente da concentração, sendo que as mais ativas (C, D) possuíam grupos orto-diidroxi; (ii) quatro cumarinas (A, B, F, G) inibiram a QLluc, mas aumentaram a QLlum; (iii) a cumarina não-substituída (K) não teve efeito modulatório significativo sobre a QLlum ou QLluc; (iv) ordem de efeito inibitório das demais substâncias (E, H, I, J) foi dependente do número e posição dos grupos substituintes, bem como do tipo de sonda quimioluminescente (luminol ou lucigenina) e do estímulo utilizado. Verificou-se também que três cumarinas (C, D, H) tiveram atividade antioxidante significante frente ao radical livre DPPH. Além disso, quatro das substâncias avaliadas (A, B, E, G) inibiram a desgranulação dos neutrófilos, mas nenhuma delas (A - K) interferiu na atividade da elastase. A análise do conjunto de resultados obtidos sugere que o número e posição dos grupos hidroxil e acetil no esqueleto cumarínico foram importantes para a modulação do metabolismo oxidativo de neutrófilos humanos, sendo que, dependendo do tipo de EROs medida, tais características estruturais podem levar a um efeito anti- ou pró-oxidante. Além disso, o efeito modulatório das cumarinas sobre as funções efetoras dos neutrófilos foi dependente o tipo de estímulo utilizado, e não foi mediado pela toxicidade dessas substâncias, nas condições ensaiadas. Sendo assim, os resultados obtidos podem auxiliar no entendimento dos requisitos estruturais das cumarinas necessários para uma modulação eficiente das funções efetoras dos neutrófilos envolvidas em doenças inflamatórias. / Neutrophils are phagocytic cells from the innate immune system with highly developed mechanisms for intracellular digestion of pathogens, immune complexes and cell debris, which are mainly mediated by reactive oxygen species (EROs) and proteolytic enzymes. However, massive neutrophil activation lead to release of large amounts of enzymes and EROs to the extracellular milieu, that may overpower the tissue defense systems, composed of antioxidants and antiproteinases, and damage the tissue, as well as contribute to the amplification of the inflammatory process found in some inflammatory, autoimmune and infectious diseases. The involvement of neutrophils in the physiopathology of such diseases has attracted the interest in the search of new compounds with antioxidant and immunomodulatory properties. In this work, we evaluated the modulatory effect of eleven hydroxylated and acetoxylated coumarin derivatives in two effector functions of human neutrophils (EROs production and degranulation) as well as the cytotoxic effects of these compounds. In addition, the structure-activity relationship was analyzed. Venous blood was collected from healthy volunteers and neutrophils were isolated by the gelatin method to perform our experiments. The neutrophil oxidative metabolism was triggered by normal human serum-opsonized zymosan (ZIops) or phorbol-12-myristate-13-acetate (PMA), and the cellular response was evaluated by the lucigenin (QLluc)- or luminol (QLlum)-amplified chemiluminescence assays. In order to conduct this study, the following experimental conditions had to be established: concentration of the chemiluminescence probes luminol and lucigenin; concentration of the solvent dimethylsulfoxide; the QLluc and QLlum reaction time. Afterwards, the antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was evaluated spectrophotometrically at 510 nm. Moreover, the effect of coumarins in the n-formyl-metionyl-leucyl-phenylalanine (fMLP)-induced neutrophil degranulation was evaluated by using elastase as marker, and the effect of these compounds in the elastase activity were also evaluated. Both assays were performed by measuring the elastase-mediated p-nitroaniline release from a specific substrate (405 nm). Toxicity of coumarins to the neutrophils was evaluated by trypan blue exclusion and measurement of lactate dehydrogenase release. Considering both, ZIops and PMA-stimulated neutrophils, we observed that: (i) most of coumarins inhibited the QLluc and the QLlum in a concentration-dependent manner, being the orto-dihydroxylated (C, D) the most active ones; (ii) four coumarins (A, B, F, G) inhibited the QLluc but increased the QLlum; (iii) the unsubstituted coumarin (K) had no significant modulatory effect on QLlum or QLluc; (iv) the rank order of inhibitory effect among the other compounds (E, H, I, J) was dependent on the number and position of substituents, as well as the type of chemiluminescent probe (luminol or lucigenin) and stimulus used. In addition, we observed that three coumarins (C, D, H) had a significant antioxidant activity against DPPH, and four compounds (A, B, E, G) inhibited the neutrophil degranulation, but none of the tested coumarins (A - K) interfered in the elastase activity. Taken together, our results suggest that the number and position of the hydroxyl and acetyl groups in the coumarin moiety were important to modulate the human neutrophil oxidative metabolism. Depending on the type of EROs measured, such structural features can lead to an anti- or pro-oxidant effect. Furthermore, the modulatory effect of coumarins on the neutrophil effector functions was dependent on the type of stimulus used, but it was not mediated by toxicity of these compounds to the neutrophils, under the assessed conditions. Therefore, the results described herein may be helpful to understand the structural requirements of coumarins to reach an efficient modulation of the neutrophil effector functions involved in inflammatory diseases.

De nouveaux antiviraux à large spectre, agissant comme intercalant d'acides nucléiques, ont été identifiés par criblage virtuel et grâce à des cartes de l’espace chimique. La 1ère partie de la thèse présente le modèle QSPR pour la solubilité aqueuse des molécules organiques dans une grande gamme de températures. Ce modèle a été utilisé pour l'évaluation de la solubilité des composés antiviraux. Dans la 2ème partie de cette thèse, les filtres structuraux, les modèles QSAR et pharmacophores sont présentés. Leur utilisation pour cribler une base de données contenant plus de 3,2 M de composés est ensuite décrite. Cette étape a conduit à sélectionner 55 touches qui ont été synthétisées et testées expérimentalement. Parmi eux, deux composés ont révélé une activité élevée contre le Vaccinia virus et une faible toxicité. Dans la 3ème partie de la thèse, l'approche par Cartes Topographiques Génératives (GTM) a été utilisée pour construire des cartes 2D de l'espace chimique des composés antiviraux. Les structures chimiques et les données expérimentales des composés antiviraux, présents dans la base de données ChEMBL, ont été extraites de la base, examinées et annotées avec les principaux Genus des virus. Ce jeu de données a été utilisé pour construire des cartes sur lesquels tous les autres composés de la base de données ChEMBL ont été projetés. L'analyse de ces cartes révèle des motifs structuraux caractérisant des types particuliers d'antiviraux. / Virtual screening and cartography of chemical space approaches have been used for design of broad-spectrum antivirals acting as nucleic acids intercalators. The 1st part of thesis reports QSPR model for aqueous solubility of organic molecules within the wide temperature range. This model was later used for solubility assessment of antiviral compounds. In the second part of work, structural filters, QSAR and pharmacophore models were developed then used to screen a database containing some 3.2 M compounds. This resulted in 55 hits which were synthesized and experimentally tested. Two lead compounds displayed high activity against Vaccinia virus and low toxicity. In the 3d part of the thesis, Generative Topographic Mapping (GTM) approach was used to build 2D maps of chemical space of antiviral compounds. Experimental data on antiviral compounds were extracted from ChEMBL database, curated and annotated by major virus Genus. Selected dataset was used to build maps on which all other ChEMBL compounds were projected. Analysis of the maps revealed structural motifs characterizing particular types of antivirals.

No description available.

The isolation and antimycobacterial activity of several naphthoquinones from Euclea natalensis were previously reported and initiated this study into the occurrence, chemistry and biological activity of this class of compounds. The structure activity relationship of the isolated naphthoquinones, and commercially available derivatives were also studied. Several plant species were investigated to establish a possible link between their traditional use for chest related symptoms (including tuberculosis infection) and the occurrence of 7-methyljuglone in these plants. The plants were extracted and tested qualitatively with the use of three analytical tools for the presence of 7-methyljuglone or related naphthoquinones. Due to its commercial unavailability, the chemical synthesis of two of these naphthoquinones, 7-methyljuglone and diospyrin, was attempted with varying degrees of success. The Friedel-Crafts acylation method was used to synthesise 7-methyljuglone from m-cresol and maleic anhydride as starting material. The optimisation of the synthesis was also investigated. Through a two-step pathway of epoxidation and steam distillation, diospyrin was successfully synthesised albeit in small quantities. During the attempts to synthesise diospyrin, two other related compounds were also synthesised. These compounds, neodiospyrin and mamegakinone, are structural isomers of diospyrin. The stability of some of the naphthoquinones was tested in various carriers in an attempt to explain the influence this will have on the obtained antituberculosis and toxicity data. The BACTEC vial solution, which is widely used to determine potency against Mycobacterium tuberculosis, was analysed with HPLC to determine the stability of these compounds in it. In addition the stability in organic solvents especially DMSO, was also tested as this is the solvent of choice for hydrophobic compounds in almost all bioassays. The antituberculosis activity and/or toxicity of 7-methyljuglone was investigated with three bioassays, to broaden our knowledge on the mechanism of action of naphthoquinones. Vero cells were employed to determine the inhibitory concentration (IC50) of most of the naphthoquinones. Mice experiments were carried out to determine the toxicity of 7-methyljuglone and diospyrin in vivo. In addition the lead compound, 7-methyljuglone, was tested on Musca domestica (house fly) to establish its toxicity on this organism. In order to find the pharmacophore of this class of compounds, a preliminary structure-activity relationship was conducted. During this study the active site in the compounds which confers potency and toxicity was partly established. The mode of action of some of the naphthoquinones was investigated and it was established that the compounds might interfere with the mycobacterial electron transport chain. A fluorinated 7-methyljuglone stops the production of menaquinone which transports electrons from the NADH dehydrogenase complex to the cytochrome bc complex and effectively kills the mycobacterium. / Thesis (PhD (Botany))--University of Pretoria, 2007. / Plant Science / unrestricted

Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.

The goal of this work is to demonstrate the utility of using systematically formatted mixture based libraries as part of the drug discovery processes. While there are a number of different valid approaches for identifying hit and tool compounds, systematically formatted mixture based libraries, such as those described in this study, offer the ability to develop a significant amount of structure activity relationship data from the testing of very few samples. In support of this claim a review of recent developments in the area of systematically formatted mixture based libraries as well as three case studies are presented. The three case studies provide the detailed approach and results obtained from using systematically formatted mixture based libraries in programs focused on identifying broad spectrum antibiotics, therapeutics to treat leishmaniasis, and inhibitors of palmitoylation. In each of these three cases approximately 200 samples were utilized to survey millions of compounds in order to develop a series of hit and tool compounds as well as significant structure-activity relationship (SAR) data around the compounds identified. This information will be utilized in future studies to potentially uncover novel mechanisms of action for treating infections and diseases as well as developing therapeutics to treat the patients affect by them. So while systematically formatted mixture based libraries are not the only option for identifying hit or tool compounds they do provide a very efficient method that can be adapted to a variety of assay formats and therefor should be considered when conducting a screening campaign.