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<p>5’-Nucleotidases (5’NTs) are catabolic enzymes of the nucleotide metabolism. They catalyze dephosphorylation of deoxyribo- and ribonucleoside monophosphates and constitute an important control point in the regulation of intracellular nucleotide pools for the maintenance of correct DNA and RNA synthesis.</p><p>By removing the alfa-phosphate group from a nucleotide, the 5’NTs release the nucleoside to pass the plasma membrane by facilitated diffusion. Depending on the cellular need for nucleotides, the nucleosides can either exit the cell for reuse elsewhere or be imported and subsequently phosphorylated by nucleoside and nucleotide kinases.</p><p>The knowledge of how nucleotides are metabolized has been used for rational design of nucleoside analogues that are used in treatment of cancer and viral diseases. These drugs are phosphorylated within the cell to become active. Their dephosphorylation by 5’NTs might be one of the mechanisms behind the resistance experienced by patients towards such drugs.</p><p>This thesis describes structure-function studies on four of the seven known human 5’-NTs. The focus of the work is on the substrate specificity and regulation of these enzymes. Inactive variants of the mitochondrial and cytosolic deoxynucleotidases and the cytosolic 5’-nucleotidase II were used to characterize the structural basis for their substrate specificity in high detail.</p><p>Based on structures of the apoprotein and activator/activator+substrate complexes of cytosolic 5’-nucleotidase II, a mechanism for the allosteric activation of this enzyme was presented. In this mechanism, the activator induces a conformational change that involves conserved residues of the active site. The conformational change drastically increases the enzyme affinity for the phosphate moiety of the substrate.</p>

5’-Nucleotidases (5’NTs) are catabolic enzymes of the nucleotide metabolism. They catalyze dephosphorylation of deoxyribo- and ribonucleoside monophosphates and constitute an important control point in the regulation of intracellular nucleotide pools for the maintenance of correct DNA and RNA synthesis. By removing the alfa-phosphate group from a nucleotide, the 5’NTs release the nucleoside to pass the plasma membrane by facilitated diffusion. Depending on the cellular need for nucleotides, the nucleosides can either exit the cell for reuse elsewhere or be imported and subsequently phosphorylated by nucleoside and nucleotide kinases. The knowledge of how nucleotides are metabolized has been used for rational design of nucleoside analogues that are used in treatment of cancer and viral diseases. These drugs are phosphorylated within the cell to become active. Their dephosphorylation by 5’NTs might be one of the mechanisms behind the resistance experienced by patients towards such drugs. This thesis describes structure-function studies on four of the seven known human 5’-NTs. The focus of the work is on the substrate specificity and regulation of these enzymes. Inactive variants of the mitochondrial and cytosolic deoxynucleotidases and the cytosolic 5’-nucleotidase II were used to characterize the structural basis for their substrate specificity in high detail. Based on structures of the apoprotein and activator/activator+substrate complexes of cytosolic 5’-nucleotidase II, a mechanism for the allosteric activation of this enzyme was presented. In this mechanism, the activator induces a conformational change that involves conserved residues of the active site. The conformational change drastically increases the enzyme affinity for the phosphate moiety of the substrate.

Gold(I)phosphines, nucleoside analogues, and azole derivatives have been identified as promising anticancer compounds. The clinical use of these individual compounds is, however, limited due to non-selectivity associated with adverse effects and developed resistance. This study investigated seven novel gold compounds that contain either a nucleoside analogue or an azole, bound via a gold nitrogen bond, which have been designed and synthesized by Dr. Horvath under the supervision of Prof. Raubenheimer from the University of Stellenbosch. The novel compounds are divided into purinecontaining/ nucleoside analogue compounds (UH 86.2, UH 75.1, UH 58.1, UH 145.1) and azole-containing compounds (UH 107.1, UH 126.1, UH 127.1). The anticancer effects of these novel compounds were compared with that of previously described anticancer compounds [Au(dppe)2]Cl and cisplatin. The octanol/water partition coefficients (PC) of the compounds were measured in order to determine whether a correlation between the lipophilicity of the structures and the cytotoxic potency and selectivity exists. This might provide further insight for structural alterations of the compounds in order to improve their anticancer activity. The results from octanol/water PC determinations, revealed that the purine-containing compounds (UH 86.2, UH 75.1, UH 58.1, and UH 145.1), as well as the azole-containing compound, UH 127.1, exhibited hydrophilic properties, while the azole-containing compounds, UH 107.1 and UH 126.1 are lipophilic. In contrast to results by Berners-Price et al. (1999), that reported a direct proportionality between lipophilicity and cytotoxicity, for the current study, involving HeLa cells, CoLo cells, normal resting and PHA stimulated lymphocytes, no correlation was observed. For the Jurkat cell line, however, an increase in lipophilicity for the series of compounds studied was accompanied by an increase in cytotoxicity. The reason for the exception is not yet fully understood. The in vitro tumour specificity of each compound was established with cytotoxicity assays on various cancer cell lines and normal cell cultures. The cancer cell lines included human cervical cancer (HeLa) cells, human colon cancer (CoLo) cells, and human lymphocytic leukaemia (Jurkat) cells. The normal cell cultures included human resting lymphocytes and human phytohemaglutin (PHA) stimulated lymphocytes. From this data, the four most promising novel compounds were identified. Additional tests were performed by adding these four compounds to cancer cells including human breast cancer (MCF-7) cells, and cisplatin sensitive and resistant human ovarian cancer (A2780 and A2780cis) cells as well as normal chicken embryo fibroblasts. The tumour specificity of each compound was determined from the results obtained via the cytotoxicity assays. The compound is more selective the higher the tumour specificity. Cisplatin exhibited the highest tumour specificity, and [Au(dppe)2]Cl, the lowest. The two most promising novelcompounds were identified as UH 126.1 and UH 127.1, which was evidenced by their high tumour specificities. Further experiments were conducted with these two azolecontaining compounds by using Jurkat cells. The possible mechanism by which the novel compounds induce cytotoxicity was investigated with flow cytometric analysis. The effects of the compounds on the cell death pathway, the mitochondrial membrane potential and the cell cycle were determined. These results indicated that the novel compounds, UH 126.1 and UH 127.1 initiate the apoptotic cell death pathway rather than the necrotic cell death pathway. According to results, UH 126.1 and UH 127.1 influenced the status of the mitochondrial membrane potential (MMP) non-selectively and only at high concentrations. Although involvement of mitochondria in the mechanism of action cannot be excluded, results indicated that it is most likely not the primary target. After investigating the effects of the two novel azole-containing compounds on the cell cycle in Jurkat cells, it was detected that these compounds induce cell accumulation in the G1 phase of the cell cycle. It was concluded that UH 126.1 and UH 127.1 might interfere with the cell cycle indirectly, possibly by inhibition of cyclin-dependent kinases and/or other enzymes necessary for DNA replication. In an acute in vivo toxicity test during this study, results revealed drug induced adverse effects (such as significant weight loss, piloerection and diarrhoea), in the mice that received 3 and 6ìmol/kg of both UH 126.1 and UH 127.1. Evidence also revealed signs of nephrotoxicity and epatotoxicity. Due to minimal adverse effects observed in the groups that received UH 126.1 and UH 127.1 at the concentration of 1,5ìmol/kg, this is the suggested maximum tolerated dose (MTD) for these compounds. Further dose-range studies with UH 126.1 and UH 127.1 are, however, needed in order to evaluate clinicalefficacy. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-29T15:07:23Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Ellison Neves de Lima.pdf: 2220112 bytes, checksum: c162c2bc01bd992deeae26d6a7891ccd (MD5) / Made available in DSpace on 2016-06-29T15:07:23Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Ellison Neves de Lima.pdf: 2220112 bytes, checksum: c162c2bc01bd992deeae26d6a7891ccd (MD5) Previous issue date: 2013-02-28 / CNPq / O Papilomavírus humano (HPV) é um vírus causador de verrugas genitais. Estimase que até 80% das mulheres adquiram uma infecção pelo HPV durante sua vida, sendo os vírus tipo 6 e 11 responsáveis por 70% dos casos. A infecção persistente pelo HPV é considerada com a mais importante causa do câncer do colo de útero. O cidofovir é um análogo de nucleosídeo, ativo contra DNA vírus, que inibem a polimerase do DNA viral por seu difosfato metabólito. Estudos “off-label” demonstraram que a aplicação tópica do cidofovir inibiu ou preveniu o desenvolvimento de papilomas. Sabendo que não há padronização da concentração do cidofovir ou forma farmacêutica para aplicação tópica, este trabalho objetiva estudar o desenvolvimento de formas farmacêuticas emulsionadas e microemulsionadas contendo ou não promotores de permeação, realizar a caracterização físico-química destas formulações e realizar estudos de liberação utilizando membrana sintética e permeação cutânea através da pele de orelha de porco. Nas preparações desenvolvidas avaliou-se o pH, viscosidade, aspecto organoléptico, tamanho de gotículas e exclusivamente para as microemulsões: condutividade, índice de refração, potencial zeta e microscopia de luz polarizada. Para a quantificação das amostras nos estudos de liberação e permeação utilizou-se as técnicas de espectrofotometria por ultravioleta e cromatografia líquida de alta eficiência com detecção por ultravioleta, respectivamente. Nos estudos de liberação e permeação observou perfis semelhantes entre as formulações, porém notou-se que as formulações microemulsionadas tiveram melhor retenção tanto no estrato córneo quanto na derme e epiderme. Constatou-se que a utilização dos promotores de permeação não incrementaram as quantidades permeadas e retidas do fármaco na pele. / The human papillomavirus (HPV) is a virus that causes genital warts. It is estimated that up to 80% of women acquire an HPV infection during their lifetime, virus type 6 and 11 are responsible for 70% of cases. Persistent infection with HPV is considered the most important cause of cancer of the cervix. Cidofovir is a nucleoside analogue, active against DNA viruses, which inhibit viral DNA polymerase by its diphosphate metabolite. Studies "off-label" demonstrated that topical application of cidofovir inhibited or prevented the development of papillomas. Knowing that there is no standardization of the concentration of cidofovir or pharmaceutical form for topical application, this paper aims to study the development of dosage forms and emulsified microemulsion with or without permeation enhancers, perform physical-chemical characterization of these formulations and release studies using membrane synthetic skin permeation through pig ear skin. In preparations developed evaluated the pH, viscosity, sensory aspect, droplet size and solely for microemulsions: conductivity, refractive index, zeta potential and polarized light microscopy. To quantify the samples studied release and permeation techniques was used for ultraviolet spectrophotometric and high performance liquid chromatography with ultraviolet detection, respectively. In studies of release and permeation observed similar profiles among formulations, however noted that microemulsion formulations were much better retention in the stratum corneum and in the epidermis and dermis. It was found that the use of permeation enhancers did not increase the amount of drug permeated and retained on the skin.

Student Number: 0420652W - MSc(Med) Dissertation - School of Pathology - Faculty of Health Sciences / Candida species are opportunistic yeasts that cause infections in immunocompromised individuals such as HIV and cancer patients. Recent studies show that 5fluorouracil, a nucleoside analogue used for cancer treatment, increases Candida cell virulence. The aim of this study is to determine the effects of commonly used antiHIV nucleoside analogue drugs on the virulence of Candida albicans, the predominant species associated with oral candidiasis. Oral swabs were collected from antiretroviralnaïve HIVpositive individuals. C. albicans was characterised from 39 of these swabs using standard microbiological techniques and polymerase chain reaction. The effect of nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine, stavudine, didanosine and lamivudine, at predicted drug peak concentrations in patients, as well as half and double these concentrations on select virulence factors of C. albicans isolates were studied. In addition, antifungal susceptibility to amphotericin B was assessed. Not all 39 isolates were used in the assays because of delays in obtaining reagents from respective manufacturers. Results show no change in the adherence and biofilm formation of 29 isolates upon exposure to NRTIs. In contrast, a steady increase in the number of viable cells was observed upon exposure to double the peak concentration of lamivudine to 23 of the clinical isolates. All 31 isolates tested were susceptible to amphotericin B (MIC£1mg/ml). Although these results suggest that NRTIs may have little effect on the virulence of C. albicans it is postulated, that, in a dosedependent manner, cytidine analogues act similarly to 5FU by activating a signaltransduction pathway which stimulates proliferation.

Les nanoMOFs – nanoparticles poreuses hybrides ont récemment été introduites dans le domaine de la vectorisation des médicaments afin de combiner les avantages de systèmes purement organiques ou inorganiques. Les nanoMOFs biodégradables et biocompatibles à base de trimesate de fer (MIL-100) ont été étudiées. Une méthode de synthèse «verte» a été développée et validée, ouvrant la voie à la production à grande échelle et à l’utilisation de ces matériaux pour des applications biologiques. Par la suite, les MIL-100 nanoMOFs ont été proposées comme potentiels vecteurs pour l’administration de médicaments hydrophiles antirétroviraux et anti-cancéreux, tels que les analogues nucléosidiques azydothimidines mono et triphosphates ou encore le Topotécan. Finalement, une nouvelle stratégie de modification de surface des nanoMOFs par leur recouvrement avec une couronne à base de dérivés de la β cyclodextrine, a été developpée. / Hybrid porous materials, as Metal Organic Frameworks (MOF) have been recently introduced in the drug delivery field in the attempt to combine advantages of the conventional “purely organic” or “purely inorganic” nanocarriers, such as important loading capability and controlled release.In this work the potential of biodegradable and biocompatible MOF nanoparticles made of iron trimesate (MIL-100 nanoMOF) has been investigated. A “green” synthetic procedure has been developed and validated, opening the way to the scale up synthesis of these materials for biological applications. MIL-100 nanoMOFs have been further applied to the delivery of hydrophilic drugs such as antiretroviral nucleoside analogues mono and triphosphate (azydothimidine mono and triphosphate) and the anticancer drug topotecan. Finally a new method of nanoMOFs surface modification, based on the nanoparticles coating with a β cyclodextrin-based extrenall shell, has been developed

Les principes actifs, dans leur chemin vers leur cible thérapeutique, rencontrent une ou plusieurs membranes biologiques (plasmique, intracellulaire). Les interactions entre un principe actif et ces membranes sont importantes : d’une part les propriétés pharmacocinétiques de la molécule active (transport, distribution, accumulation) en dépendent, d’autre part le principe actif peut modifier les propriétés structurales des membranes. L’étude de ces interactions est rendue difficile par la complexité des membranes en termes de composition (lipidique et protéique) et de structure (hétérogénéité de l’organisation). Par conséquent, l’utilisation de systèmes modèles simplifiés est nécessaire. Au cours de ce travail de thèse nous avons cherché à réaliser des bicouches lipidiques modèles dont les caractéristiques se rapprochaient de celles des membranes biologiques en complexifiant progressivement leur composition lipidique. Nous avons ensuite étudié l’interaction d’une molécule anti-VIH squalénisée, le ddC-SQ, avec nos modèles de membrane.Un des rôles essentiels des membranes biologiques étant de séparer deux milieux aqueux de composition ionique différente, nous avons étudié dans un premier temps l’influence de la nature du milieu d’hydratation sur les propriétés thermiques et structurales des bicouches lipidiques. A pH physiologique, nous avons mis en évidence que seuls les ions divalents (à faibles concentrations) induisaient de profondes modifications structurales en provoquant la formation de vésicules unilamellaires dans les systèmes simples. Une seconde partie de nos travaux a consisté à étudier l’interaction d’un antiviral squalénisé, le ddC-squalène (ddC-SQ), avec nos différentes bicouches modèles. Cet analogue nucléosidique a été associé de manière covalente à une chaîne de squalène afin d’améliorer ses propriétés pharmacocinétiques. Cette squalénisation confère à la molécule la capacité de s’auto-assembler en nanoparticules présentant une structure cubique bicontinue. Les résultats obtenus ont révélé que le principe actif squalénisé interagissait fortement avec les membranes à l’inverse de la molécule native. L’organisation structurale des systèmes modèles est profondément modifiée par l’insertion du ddC-SQ, ce qui pourrait influer sur l’activité du composé. / Drugs must cross one or more biological membranes (plasma membrane, intracellular membrane) to reach their intracellular target. Interactions between drug and membranes play a significant role in the pharmacokinetic properties of drug such as transport, distribution, accumulation. Moreover, drugs may alter membrane properties. The complexity of the composition (protein and lipid) and the structural properties (heterogeneity) of membranes leads to a difficult investigation of these interactions. Consequently, use of simplified model membranes is needed. In this work, model lipid bilayer systems in which the lipid organization mimics the arrangement of lipids in natural membrane have been developed. In this way, the complexity of lipid composition mixtures has been progressively increased. The primary function of membrane is to physically separate aqueous compartments from their surroundings. The intracellular and extracellular fluids differ in ionic composition. This study firstly consists to estimate the influence of aqueous medium nature on the thermodynamic and structural properties of these model membranes.In physiological conditions (pH 7.4, ionic strength 150 mM), the most significant change was obtained in the presence of divalent ions. Markedly change in lipid organization was observed and the formation of unilamellar vesicles has been evidenced (at low concentrations) in simple model bilayers. Interactions of an antiretroviral nucleoside analogue, the SQddC, with lipid systems constitute the second part of our work. Squalene has been covalently coupled to ddC, in order to improve its therapeutic index. Squalenoylation leads to amphiphilic prodrugs which self-organize as nanoparticles. ddC weakly interacts with lipid membranes while SQddC-SQ can insert into membranes between hydrophobic alkyl chains and induce disruption of lipid organization. Consequently, the efficacy and/or toxicity of this drug could change.