Made available in DSpace on 2016-08-10T10:29:36Z (GMT). No. of bitstreams: 1 Lilian dos Santos Castro.pdf: 960889 bytes, checksum: 7169384d60e2113b089f6e13a34dbdb8 (MD5) Previous issue date: 2009-09-10 / Some caffeine analogues present activity against skin cancer. In this work, chemometric methods were applied in the search for building discriminant models between active and inactive analogues, based on the correlations among their biological activities and electronic and geometric molecular descriptors. From 70 descriptors calculated with density functional theory (DFT) with the exchange correlation functional B3LYP and the basis set 6-31G* (Gaussian 03), 10 ones were pre-selected based on their Fisher weights, and finally five descriptors (dR1, dR3, LogPR1, LogPR3 and molar area) were selected for a principal component analysis. This analysis was able to discriminate the analogues in active and inactive by using only one principal component, accounting for 49 % of the total variance. The cluster hierarchical analysis, using the descriptors selected by principal component analysis, shows that the caffeine analogues can be grouped into two major groups: active and inactive. The results of this work suggest that the anticancer activity of those analogues is related with hydrophobic, steric, and electronic properties and can be used to help modeling new caffeine analogues with more pronounced anticancer activity. / Alguns derivados análogos da cafeína apresentam atividade contra o câncer de pele. Neste trabalho, métodos de estatística multivariada foram usados na construção de um modelo capaz de discriminar os derivados análogos da cafeína em ativos e inativos baseando-se na correlação da atividade biológica, descritores geométricos e eletrônicos. Dos 70 descritores calculados usando a teoria do funcional da densidade (DFT), com o funcional de troca e correlação B3LYP e o conjunto de base 6-31G* implementados no programa Gaussian 03, 10 foram préselecionados baseados no peso de Fischer e, finalmente, cinco descritores (dR1, dR3, LogPR1, LogPR3 e área molar) foram selecionados para a construção do modelo de componentes principais. Esse modelo foi capaz de discriminar os análogos em ativos e inativos usando apenas uma componente principal, sendo que esta componente responde sozinha por 49% da variância total dos dados. A análise hierárquica de cluster, usando os descritores selecionados pela análise de componentes principais, mostra que os derivados análogos da cafeína podem ser agrupados em dois grandes grupos: ativos e inativos. Os resultados deste trabalho sugerem que a atividade anticancerígena para o câncer de pele destes derivados está relacionada com as propriedades hidrofóbica, estérica e eletrônica e podem ser utilizados para auxiliar o planejamento de novos derivados de cafeína com atividade anticancerígena mais pronunciada.

Sigma receptors originally thought to be an opioid receptor is now categorized as a distinct class of receptor. There are two main subtypes, the sigma-1 receptor and an uncharacterised binding site, named the sigma-2 binding site. The presence of the sigma-2 binding site shows high correlation with proliferation of cells and is associated with cancer. I have categorized sigma-1 and sigma-2 binding sites in 11 human tumour cell lines. I have demonstrated that tumour cell lines from a range of tissues express both sigma-1 and sigma-2 binding sites. One exception is the MCF7 breast cancer cell line, which lacks sigma-1 receptors. I show that the quantitation of sigma-2 binding sites using the "masking" protocols are flawed, significantly overestimating levels of sigma-2 binding sites. I propose novel protocols to determine levels of sigma-1 receptors and sigma-2 binding sites in cell lines and tissue. Using radioligand binding assays in MCF7 cells, I have characterised novel sigma-2 ligands. These ligands are simple ammonium salts containing a single nitrogen atom. They are simpler than the previously recognised pharmacophore for the sigma-2 site. I have shown that these simple ammonium salts show graded affinity for the sigma-2 binding site. The highest affinity ligands were dihexylammonium (pKi 7.58) and dioctylammonium (pKi 7.9). I have used these ammonium salts and previously characterised ligands to determine sigma-2 binding site biology. I have shown that the biological activity of these drugs is related neither to their hydrophobicity nor their ability to effect calcium signalling in cells. I propose that the Hill slope of binding is inversely related to the efficacy of a ligand to inhibit metabolic activity of cancer cells. Furthermore, I offer an explanation as to why concentrations of sigma-2 ligands far higher than their determined binding affinities are required to inhibit metabolic activity.

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.

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.

Synthetic cathinones are stimulant drugs of abuse that act at monoamine transporters e.g. the dopamine transporter (DAT) as releasing agents or as reuptake inhibitors. More than >150 new synthetic cathinones have emerged on the clandestine market and have attracted considerable attention from the medical and law enforcement communities. threo-Methylphenidate (tMP) is an FDA approved drug for the treatment of ADHD and narcolepsy, which also acts as a DAT reuptake inhibitor and is widely abused. tMP and synthetic cathinones share some structural similarities and extensive structure-activity relationship (SAR) studies on tMP have been conducted. However, much less is known about the SAR of synthetic cathinones, and the available MP literature might assist in understanding it. The main focus of this research was to compare SAR between methylphenidate-cathinone hybrids and available methylphenidate SAR in order to identify some guiding principles that might allow
us to predict their abuse potential and to identify which cathinones should be
targeted for more extensive evaluation. In the present study, we evaluated eight 2-benzoylpiperidine analogs and a descarbonyl analog to determine if tMP SAR can be applied to cathinone SAR. We conducted molecular modeling and docking studies and predicted the order of potency to be tMP > 2-benzoylpiperidine > 2-benzylpiperidine based on the number of hydrogen bonds. The synthesized analogs were evaluated in a competition assay using live-cell imaging against APP+ in HEK293 cells stably expressing hDAT. All compounds were found to be DAT reuptake inhibitors and, as the modeling studies predicted, the order of potency in our functional studies was also found to be tMP > 2-benzoylpiperidine > 2- benzylpiperidine. A significant correlation was obtained between the potency of the benzoylpiperidines and tMP binding data (r = 0.91) suggesting that the SAR of tMP analogs might be applicable to the synthetic cathinones as DAT reuptake inhibitors.

Transient receptor potential (TRP) channels comprise a superfamily of cation-selective ion channels that are largely calcium (Ca2+) permeable and that play diverse physiological roles ranging from nociception in primary afferent neurons to the absorption of dietary Ca2+. The 28 mammalian TRP channels are categorized into 6 subfamilies. The vanilloid subfamily is named for its founding member, TRPV1, the capsaicin receptor, and has 6 members. TRPV1-4 are all heat sensitive ion channels whereas TRPV5 and TRPV6 are involved in renal Ca2+ reabsorption and Ca2+ absorption in the intestine, respectively. In our structural studies, we have focused on TRPV3 and TRPV6. TRPV6 is a highly Ca2+ selective TRP channel (PCa/PNa ~ 130) that functions in active Ca2+ absorption in the intestine. Its expression is upregulated by vitamin D and is, on the molecular level, regulated by PIP2 and calmodulin (CaM). Previously, the structure of TRPV6 was solved using X-ray crystallography. Using the crystal structure, a negatively charged extracellular vestibule was identified and anomalous diffraction was used to identify ion binding sites in the pore. Also, at the top of the selectivity filter, four aspartates were identified that coordinate Ca2+ entering the pore and confer to TRPV6 its selectivity for Ca2+. However, only the structure of the rat orthologue was solved and only in the closed, apo state. We used cryo-electron microscopy (cryo-EM) to solve structures of the human orthologue of TRPV6 in the open and closed (we used the mutation R470E to close the channel) states. The closed-to-open TRPV6 transition is accompanied by the formation of short π-helices in the middle of the pore-lining S6 helices, which in turn results in their turning and a different set of residues facing the pore. Additionally, the formation of the π-helices results in kinking of the S6 helices, which further widens the pore. TRPV6 is constitutively active when expressed heterologously. In other words, the addition of external stimuli is not necessary for the activation of the channel. Therefore, its activity needs to be regulated to prevent toxic Ca2+ overload. One mechanism by which this occurs is through its regulation by CaM. CaM has been shown to bind TRPV6 and regulate its function, however, the way it binds to and regulates TRPV6 remained unknown. To uncover this mechanism, we solved the structure of TRPV6 bound to CaM. We found that CaM binds TRPV6 in a 1:1 stoichiometric ratio and that CaM directly blocks the TRPV6 pore by inserting a positively charged lysine into a tera-tryptophan cage at the bottom of the pore. As a result, the channel adopts an inactivated conformation; although the pore-lining S6 helices still contain local π-helices, they are pulled closer together, narrowing the pore and further blocking it with hydrophobic side chains. We have also conducted studies of TRPV3. Unlike TRPV6, TRPV3 is a heat-activated vanilloid TRP channel. TRPV3 is expressed highly in keratinocytes where it has been implicated in wound healing and maintenance of the skin barrier, and in the regulation of hair growth. We solved the structure of apo TRPV3 in a closed state, and the structure of a TRPV3 mutant bound to 2-APB in an open state. Like TRPV6, the opening of TRPV3 is accompanied by the formation of local π-helices in the middle of the pore-lining S6 helices. The formation of the π-helices results in the lining of the ion permeation pathway with a different set of residues, resulting in a largely negatively charged pathway. Unlike TRPV6, TRPV3 is only slightly selective for Ca2+ and correspondingly, during gating state transitions, rearrangements were not only observed only in its pore-lining helices, but also in the cytosolic domain and the selectivity filter. Based on a comparison of our structures, we proposed a model of TRPV3 regulation by 2-APB. Together, our studies provide insight into the regulatory and gating mechanisms of the vanilloid subtype TRP channels and can provide the foundation for future studies.

Fluoroquinolones, broad-spectrum bactericidal antibiotics, exert their effects by inhibiting type II topoisomerases through the formation of a fluoroquinolone-DNA-topoisomerase ternary complex. Recently, newer, structurally unique fluoroquinolones have been shown to kill bacteria by promoting chromosomal fragmentation in the presence and absence of protein synthesis, thus allowing fluoroquinolones to potentially be used in the treatment of microorganisms that go into a dormant state. There is a need to further understand the structure activity relationships (SAR) of fluoroquinolones to develop new antibiotics that can kill dormant bacteria and are active against current resistant strains. The hypothesis that structurally unique fluoroquinolones interact with the DNA- fluoroquinolone-topoisomerase ternary complex in a unique way that leads to different killing pathways is the basis of this work. The first approach to understand SAR for fluoroquinolones to kill non-growing bacteria was to evaluate the effect of modifications at the C-8 and C-5 positions on lethality. Novel, synthetically-derived and commercially-available fluoroquinolones were evaluated for ability to kill Escherichia coli in the presence and absence of chloramphenicol, a known protein synthesis inhibitor used to simulate non-growing bacteria. The second study was to understand SAR of fluoroquinolone-class agents necessary to maintain antibacterial activity against common fluoroquinolone resistance-causing bacterial mutations on topoisomerase IV. A panel of novel fluoroquinolones, 2,4-quinazoline diones, and fluoroquinolone-like analogues with unique substitution combinations at C-8 and C-7 was synthesized and evaluated for ability to poison wild-type and mutant Bacillus anthracis topoisomerase IV. The third study to understand the contribution of SAR of fluoroquinolone-class agents to novel killing mechanisms was to evaluate the binding interaction of fluoroquinolones to double-stranded and nicked DNA. Binding affinities of fluoroquinolones to DNA were determined; fluoroquinolones were found to bind different DNA types with varied affinities. The ability of a series of C-8 and C-7 modified fluoroquinolones to stabilize or destabilize DNA was assessed. The results of these studies also add broadly to the understanding of SAR associated with fluoroquinolone-class antibiotics for killing in the presence and absence of protein synthesis, maintaining activity in the presence of resistance-causing mutations in the target enzymes, and increasing binding interactions with different types of DNA.

Abstract not available

Copies of author's previously published articles inserted / Describes the exploratory clinical development of tucaresol, consisting of three studies performed on humans and subsequent in vitro and animal studies investigating the possible effects on the immune system. Demostrates that rational drug design may be an efficient way of selecting potential therapeutic candidates. / xxi, 265 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995

Hepatitis C is a blood-borne disease affecting 130-170 million people worldwide. The causative agent, hepatitis C virus (HCV), infects the liver and is the major reason for chronic liver disease worldwide. The HCV NS3 protease, a key enzyme in the virus replication cycle, has been confirmed to be an important target for drug development. With the recent release of two HCV NS3 protease inhibitors onto the market and an arsenal of inhibitors in clinical trials, there are now hopes of finally combating the disease. However, the success of treatment relies heavily on the ability to overcome the emergence of drug-resistant forms of the protease. The main focus of this thesis was on designing and synthesizing novel inhibitors of the NS3 protease with a unique resistance profile. Efforts were also made to decrease the peptide character of the compounds, with the long-term goal of making them into more drug-like compounds. Special attention was devoted to developing inhibitors based on a phenylglycine in the P2 position, instead of the highly optimized and commonly used P2 proline. Around ninety acyclic and macrocyclic inhibitors have been synthesized and biochemically evaluated. P2 pyrimidinyloxy phenylglycine was successfully combined with an aromatic P1 moiety and alkenylic P1´ elongations, yielding a distinct class of HCV NS3 protease inhibitors. Macrocyclization was performed in several directions of the inhibitors via ring-closing metathesis. Only the macrocyclization between the P3-P1´ residues was successful in terms of inhibitory potency, which suggests that the elongated P1-P1´ residue is oriented towards the P3 side chain. The metathesis reaction was found to be significantly more dependent on the substrate than on the reaction conditions. It was also found that the P3 truncated inhibitors were able to retain good inhibitory potency, which initiated the synthesis and evaluation of a series of P2-P1´ inhibitors. The potential of the P3-P1´cyclized inhibitor and the smaller, acyclic P2-P1´ as new potential drug leads remains to be determined through pharmacokinetic profiling. Gratifyingly, all the inhibitors evaluated on A156T and D168V substituted enzyme variants were able to retain inhibitory potency towards these as compared to wild-type inhibition.