Made available in DSpace on 2015-05-14T13:00:06Z (GMT). No. of bitstreams: 1 parte1.pdf: 2037946 bytes, checksum: 4e39fb4698e718310fa7f174e9a1a6a6 (MD5) Previous issue date: 2008-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The species Bauhinia pentandra (Bong.) Vog. ex Steud (Leguminosae), known as mororó com espinho , is common in Northeast of Brazil. The enzyme DNA topoisomerase II-α (topo II) is responsible for the relaxation of the DNA during the processes of cellular division and is involved in diverse types of cancer, being considered a potential target in the treatment of the cancer. This work aimed to study the chemical composition and antioxidant activity of the bark roots of B. pentandra and the inhibition activity of topo II in natural and semi-synthetic compounds. In the chemical study of the vegetal species was isolated of the cyanogenic glycoside lithospermoside and a mixture of β-sitosterol and stigmasterol. The lithospermoside was acetylated and the structures had been identified through spectral data as IV and RMN1H and 13C including 2D. The Ethanolic extract and the fractions in hexane, AcOEt, MeOH and the substance lihtospermoside were submitted to antioxidant tests (DPPH, ABTS, quelatogenic activity of Fe2+ ion). The determination of total phenolic also was performed with the extract and fractions. All the samples showed antioxidant activity, being the AcOEt with highest activity and higher content of phenolic compounds, then this fraction was analyzed by HPLC-DAD having confirming the presence of phenolic. The inhibition of topoisomerase II-α was analyzed with natural compounds lithospermoside and γ-hydroxyferruginin A, isolated from Vismia guianensis and the EtOH extract of the latex of this species, and with semi-syntetic compounds: peracetyl lithospermoside and ten flavonoids derivated of retusin, isolated from Solanum paludosum. At 220 μM concentration all samples tested were actives, except lithospermoside and its acetylated derivative. In the concentration of 110 μM only two flavonoids did not inhibit the action of topo II. The γ-hydroxyferruginin inhibited the activity of topo II in a minimum concentration of 0,1 μM, being this anthranoid prenilated a promising candidate for posterior therapeutical studies for the treatment of the cancer. / A espécie Bauhinia pentandra (Bong.) Vog. ex Steud (Leguminosae), conhecida como mororó com espinho é comum do Nordeste brasileiro. A enzima DNA topoisomerase II-α (topo II) é responsável pelo relaxamento do DNA durante os processos de divisão celular e está envolvida em diversos tipos de câncer, sendo considerada um alvo potencial no tratamento desta doença. Este trabalho teve como objetivo estudar a composição química e atividade antioxidante das cascas das raízes de B. pentandra e a análise da atividade inibitória da topo II por compostos naturais e semi-sintéticos. O estudo químico da espécie vegetal resultou no isolamento do glicosídeo cianogênico litospermosida e de uma mistura dos esteróides β-sitosterol e estigmasterol. A litospermosida foi acetilada e as estruturas foram identificadas através de dados espectrométricos como IV e RMN1H e 13C, incluindo 2D. O extrato EtOH bruto e as frações hexânica, AcOEt, MeOH e a substância litospermosida foram submetidos aos testes antioxidantes (DPPH, ABTS, atividade quelatogênica do íon Fe2+). A determinação de fenólicos totais também foi realizada com o extrato e com estas frações. Todas as amostras mostraram atividade antioxidante, destacando-se a fração em AcOEt com melhor atividade e maior teor de fenólicos, então esta fração foi analisada por HPLC-DAD confirmando a presença de fenólicos. A atividade inibitória da topoisomerase II-α foi avaliada com as substâncias naturais litospermosida e γ- hidroxiferruginina A, isolado de Vismia guianensis e com o extrato EtOH bruto do látex desta espécie, e com as substâncias semi-sintéticas litospermosida peracetilada e dez flavonóides derivados de retusin, isolado de Solanum paludosum. Na concentração de 220 μM todas as amostras testadas apresentaram atividade, exceto a litospermosida e o seu derivado acetilado. Na concentração de 110 μM apenas dois flavonóides não inibiram a ação da topo II. A γ-hidroxiferruginina A inibiu a atividade da topo II em uma concentração mínima de 0,1 μM, sendo este antronóide prenilado um candidato promissor para estudos terapêuticos posteriores para o tratamento do câncer.

Doenças tropicais negligenciadas compartilham características que permitem que elas persistam nas condições de pobreza, onde se aglomeram e se sobrepõe frequentemente. Mais de um bilhão de pessoas sofrem de uma ou mais doenças tropicais negligenciadas. Entre estas está a doença de Chagas, resultante da infecção pelo protozoário parasito hemoflagelado Trypanosoma cruzi, tendo insetos triatomíneos como vetores. Topoisomerases são enzimas envolvidas na regulação do superespiralamento do DNA resolvendo problemas topológicos associados com replicação, transcrição, recombinação e reparo. As topoisomerases do tipo II são essenciais para tripanossomatídeos por atuarem na replicação e organização do DNA contido em uma região especializada da mitocôndria denominada cinetoplasto. O estudo da Topoisomerase II Mitocondrial (TcTopoIImit) foi realizado através de análises sequenciais e estruturais de outras topoisomerases para a determinação de construções para a clonagem. As construções para o domínio N-terminal foram clonadas no vetor pTZ57R/T e subclonadas em vetores do sistema pET para expressão proteica em E. coli. Experimentos de expressão foram realizados em diferentes cepas, vetores, soluções tampão e outros parâmetros variáveis visando a obtenção dos produtos recombinantes na forma solúvel, porém, tais resultados não foram obtidos. A flavoproteína Old Yellow Enzyme de Trypanosoma cruzi (TcOYE) é uma oxidoredutase que usa NAD(P)H como cofator. Esta enzima é clinicamente relevante devido ao seu papel no mecanismo de ação de alguns agentes tripanocidas, utilizados no tratamento da doença de Chagas, por produzirem espécies reativas de oxigênio. Neste trabalho, a enzima recombinante TcOYE foi produzida, purificada, cristalizada pelo método de difusão de vapor e sua estrutura cristalográfica, resolvida por substituição molecular e refinada. A TcOYE foi cristalizada nas formas cristalinas P212121 e P21 e os dados de difração foram coletados para o máximo de resolução de 1,27 e 2,00 Å, respectivamente. As coordenadas atômicas e fatores de estrutura das estruturas da TcOYE nas formas cristalinas P212121 e P21 foram depositados no Banco de Dados de Proteínas com os códigos de acesso 4E2B e 4E2D, respectivamente. A TcOYE apresenta um enovelamento canônico em um barril (α/β)8 com o grupo prostético localizado na cavidade maior do sítio ativo. Após a obtenção dos modelos cristalográficos análises sequenciais e estruturais foram realizadas para a TcOYE e outras OYEs. A região 141-156 do subdomínio capping em TcOYE, assim como em outras OYEs, é intrinsecamente desestruturada exibindo altos valores de fatores B. A região mais divergente entre as OYEs é o loop estendido (289-297), que pode variar em comprimento e composição mudando o volume e a acessibilidade ao sítio ativo. / Neglected tropical diseases share features that allow them to persist in conditions of poverty, which clump together and frequently overlap. More than 1 billion people suffer from one or more neglected tropical diseases. Among them is the Chagas disease is an important parasitic disease resulting from infection by the flagellate protozoan parasite Trypanosoma cruzi, with triatomine insects as vectors. Topoisomerases are ubiquitous enzymes involved in the regulation of DNA supercoiling and overcoming topological barriers during replication, transcription, recombination and repair. The type II topoisomerases are essential for trypanosomatids since, in addition to their role in nuclear DNA metabolism, these enzymes might also play an important role in the replication and organization of the DNA contained in the specialized region of the mitochondrion known as the kinetoplast. The study of Mitochondrial Topoisomerase II from T. cruzi (TcTopoIImit) was performed by sequence and structural analyses into topos members for determination of domains constructions for cloning. Constructions for the N-domain were then cloned in pTZ57R/T vector and subcloned into pET system vectors for protein expression in E. coli. The protein expression experiments were performed by different strain cells, vectors, buffers solutions and others adjustable parameters for improve the solubility but recombinant products in soluble form were not obtained. The flavoprotein Old Yellow Enzyme from Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced, purified, crystallized by the vapour diffusion method and its crystallography structure was solved for molecular replacement and refined. TcOYE was crystallized in two crystalline forms, P212121 and P21 and diffraction data were collected to a maximum resolution of 1.27 and 2.00 Å, respectively. The atomic coordinates and structure factors of the TcOYE structure in P212121 and P21 crystalline forms have been deposited in the Protein Data Bank with the accession codes 4E2B and 4E2D, respectively. TcOYE displays a canonical (α/β)8-barrel fold with a FMN prosthetic group located at the large active-site cavity. Afterwards, sequential and structural analyses were carried out for TcOYE and others OYEs. The region 141-156 of the capping subdomain in TcOYE as well as in others OYEs is intrinsically flexible exhibiting high B-factor values. The most divergent region among these OYEs is the extended loop (289-297), which can vary in length and composition changing the volume and accessibility to the active site.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-10-24T13:09:10Z No. of bitstreams: 1 PDF - Rawny Galdino Gouveia.pdf: 21187770 bytes, checksum: 320540e5f560ea3cbfd7e23034f4444d (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-10-26T16:55:41Z (GMT) No. of bitstreams: 1 PDF - Rawny Galdino Gouveia.pdf: 21187770 bytes, checksum: 320540e5f560ea3cbfd7e23034f4444d (MD5) / Made available in DSpace on 2017-10-26T16:55:41Z (GMT). No. of bitstreams: 1 PDF - Rawny Galdino Gouveia.pdf: 21187770 bytes, checksum: 320540e5f560ea3cbfd7e23034f4444d (MD5) Previous issue date: 2017-02-24 / Among the main alternatives for cancer treatment, chemotherapy is one of them, but most chemotherapeutic agents act nonspecifically, damaging several types of body cells. DNA intercalators are standout among the existing chemotherapeutics, for example, among these compounds, acridine derivatives have inhibitory activity against nuclear regulatory enzymes, such as topoisomerase, and there is a strong interaction with pairs of DNA bases. Therefore, researchers are seeking to find new candidates intercalators of DNA drugs and a significant number of molecules have been evaluated for their antitumor and intercalators properties. This paper proposed the synthesis of novel spiro-acridines derivatives; it was conducted to evaluate their interaction with BSA, DNA, and inhibition of the enzyme topoisomerase IIα. Thus, eleven new spiro-acridínicos compounds were synthesized and were determined their physicochemical characteristics and demonstrated the possible reaction mechanisms for obtaining these compounds. The compounds were obtained in parallel and convergent manner, initially the diphenylamine suffered an acylation followed by cyclization, leading to 9-metilacridina that by successive reactions led to 9-carboxaldehyde acridine. In parallel, 2- cianoacetohidrazina reacted with aromatic aldehydes substituted in acid and ethanol, leading to N-acylhydrazones intermediates substituted, finally, the two intermediates were condensed, through a Knovenagel reaction and suffered spontaneous cyclization, leading to spiro-acridine derivates. The compounds show yields ranging from 24% to 86.75%, and melting points ranging between 3 and 5 °C. Most of the compounds were characterized and had their chemical structures elucidated by spectroscopic H-NMR and C-NMR; IR and MS. By 1^H NMR analysis were observed singlets referring to NH, protons coming from the formation of the C = N bond (imino carbon) and C = C (vinyl carbon) corroborating the structural elucidation of all compounds. The 13^C NMR spectra showed the absorptions of carbon atoms characteristic of spiro-acridine derivatives, especially the chemical shift showed by the quaternary carbon evidenced on the signals from 69.33 to 71.59, demonstrating the occurrence of spontaneous cyclization of spiro-acridine derivatives. The compounds evaluated against the enzyme Topoisomerase IIα, presented different degrees of inhibition when compared with the mAMSA standard, and AMTAC 06 presented the best capacity for inhibition. The compounds that interacted with the DNA showed binding constants (Kb) 10^5M^-1, thus suggesting that the likely mechanism of action of these compounds may be related to the formation of a ternary complex between compound, DNA and Topoisomerase. The interactions with the BSA showed that all compounds evaluated were able to cause fluorescence extinction in a dose-dependent manner, and AMTAC 14 showed the highest fluorescence extinction capacity, probably due to its electronic effects. / Uma das principais abordagens para o tratamento do câncer é a quimioterapia, porém a maioria dos agentes quimioterápicos atua de forma não específica, lesando vários tipos de células do organismo. Entre os quimioterápicos existentes, destacam-se os intercaladores de DNA, como por exemplo, os derivados de acridina que possuem atividade inibitória frente a enzimas reguladoras nucleares, como as topoisomerase, além de uma forte interação com pares de bases do DNA. Por isto, pesquisadores vêm buscando a descoberta de novos candidatos a fármacos intercaladores do DNA e um número expressivo de moléculas tem sido avaliado quanto às suas propriedades antitumorais e intercaladoras. Este trabalho propôs síntetizar novos derivados espiro-acridínicos, analisar a interação destes com o BSA, DNA e a inibição da enzima topoisomerase IIα. Assim, onze novos compostos espiro-acridínicos foram sintetizados e determinados as suas características físico-químicas e demonstrados os possíveis mecanismos reacionais para a obtenção destes compostos. Os compostos foram obtidos de forma paralela e convergente, onde inicialmente a difenilamina sofreu uma acilação seguido de ciclização, obtendo-se a 9-metilacridina que por sucessivas reações foi obtido a 9-carboxialdeido acridina. Em paralelo, a 2-cianoacetohidrazina reagiu com aldeídos aromáticos substituídos em meio ácido e etanólico, conduzindo aos intermediários N- acilidrazônicos substituídos, por fim os dois intermediários foram condensados, através de uma reação de Knovenagel e ciclizaram espontaneamente, conduzindo aos derivados espiro- acridínicos. Os compostos apresentaram rendimentos que variaram de 24% a 86,75% e as faixas de fusão oscilaram entre 3 e 5ºC. A maioria dos compostos tiveram suas estruturas químicas caracterizadas e elucidadas através de técnicas espectroscópicas de RMN de 1^H RMN de 13^C ; IV e EM. Através da análise do RMN de 1^H foram observados singletos referentes ao grupo NH, prótons oriundos da formação da ligação C=N (iminico) e C=C (vinílico) corroborando para a elucidação estrutural de todos os compostos. Os espectros de RMN de 13^C evidenciaram as absorções dos átomos de carbono característicos dos derivados espiro-acridinicos, principalmente o deslocamento que mostrou o carbono quaternário evidenciado nos sinais entre 69.33 a 71.59 o qual demonstra a ocorrência da ciclização espontânea dos derivados espiro-acridinicos. Os compostos avaliados frente a enzima Topoisomerase IIα, apresentaram diferentes graus de inibição quando comparados com o padrão mAMSA, sendo o AMTAC 06 o que apresentou a melhor capacidade de inibição. Os compostos que interagiram com o DNA apresentaram constantes de ligação (Kb) 10^5M^-1, sugerindo dessa forma que o provável mecanismo de ação desses compostos pode estar relacionado a formação de um complexo ternário entre composto, DNA e Topoisomerase. As interações com a BSA, mostraram que todos os compostos avaliados foram capazes de causar a extinção da fluorescência de forma dose-dependente, sendo o AMTAC 14 o que apresentou a maior capacidade de extinção da fluorescência, provavelmente devido aos seus efeitos eletrônicos.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T15:47:17Z (GMT). No. of bitstreams: 1 Lapis_MireleSanchesFernandes_M.pdf: 6468600 bytes, checksum: 6af92e23ef4b2fdd6be4740a8ba9f443 (MD5) Previous issue date: 2005 / Resumo: Este trabalho teve como objetivo elucidar algumas interações ecológicas de espécies de Orchidaceae e Guttiferae. No primeiro capítulo, é descrita a determinação da composição química das fragrâncias florais de cinco espécies de orquídeas pertencentes ao complexo Maxillaria madida (M. juergensii, M. madida, M. vernicosa, M. ferdinandiana e M. pachyphylla), através da técnica de headspace dinâmico e análise por CG/EM. Monoterpenóides e sesquiterpenóides não oxigenados são os principais compostos voláteis emitidos e provavelmente responsáveis pela atração dos insetos polinizadores. No segundo capítulo, demos continuidade ao estudo da composição química do látex dos frutos de C. grandiflora iniciado por Cláudio A. G. da Câmara. Este estudo resultou no isolamento de 3 compostos, sendo 2 inéditos e derivados do ácido cis-d-tocotrienólico. Estes compostos quando submetidos ao teste em CCD com b-caroteno, apresentaram propriedades antioxidantes. No último capítulo deste trabalho, é apresentada a análise da composição química das resinas florais do híbrido interespecífico de C. weddelliana X paralicola, comparada com a das resinas florais de seus parentais. Observou-se que todos os compostos isolados também estão presentes nas espécies genitoras, variando apenas suas abundâncias. Finalmente, foi avaliada a atividade antineoplástica das resinas florais de C. grandiflora, C. hilariana, C. renggerioides, C. fluminensis, C. spiritu-sanctensis, C. lanceolata, C. nemorosa hermafrodita e C. paralicola, em 8 diferentes linhagens de células. Os resultados revelaram que os metabólitos secundários (benzofenonas poliisopreniladas) presentes nestas espécies são eficientes agentes antitumorais e o mecanismo de ação destes compostos envolve a inibição de topoisomerases. / Abstract: The aim of this research was the elucidation of ecological interactions in Orchidaceae and Guttiferae species. The first chapter describes the floral fragrance chemical compositions of five orchid species belonging to the Maxillaria madida group (M. juergensii, M. madida, M. vernicosa, M. ferdinandiana and M. pachyphylla) obtained by applying dynamic headspace sampling technique and GC/MS analysis. Monoterpenoids and sesquiterpenoids without oxygen in its structure are the main emitted volatile compounds and probably responsible for the pollinator attractions. In the second chapter the investigation of Clusia grandiflora fruit latex chemical composition initiated some years ago by Dr Cláudio A. G. da Câmara was concluded with the isolation of novel compounds derived from tocotrienolic acid. Two of which (methyl-(2Z, 6E, 10E)-13-(6-hydroxy-2,7,8-trimethyl-3, 4-dihydro-2H-2chromenyl)-2,6,10-trimethyl-2,6,10-tridecatrienoate and methyl-(2Z, 6E,10E)-13-(6-formyl-5-hydroxy-2,8-dimethyl -3, 4-dihydro-2H-2-chromenyl)-2,6,10-trimethyl-2,6,10-tridecatrienoate) are novel secondary metabolites which have antioxidant properties revealed by b-carotene TLC test. Evaluation of the interespecific hybrid of Clusia weddelliana x paralicola floral resin and revealed that the chemical constituents in the parents and hybrid species were identical with different relative abundances. Finally, we have evaluated the anticancer activity of the floral resins of C. grandiflora, C. hilariana, C. renggerioides, C. fluminensis, C. spiritu-sanctensis, C. lanceolata, C. nemorosa hermafrodita and C. paralicola in eigth cell lines. The results showed that the secondary metabolites (polyisoprenylated benzophenones) were efficient antitumor agents by topoisomerase inhibition. / Mestrado / Quimica Organica / Mestre em Química

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No description available.

DNA topoisomerase I (TopoI) is an essential enzyme involved in the relief of DNA supercoiling during replication. TopoI plays important role in various DNA events, however the recognition that it is the target of anticancer drug camptothecins (CPTs) led to the rapid growth in this field. CPTs inhibit TopoI during S phase and cause double stranded DNA lesions in rapidly dividing cells. This class of drug is used extensively in oncology clinical settings worldwide. However, resistance to this type of therapy has been found in approximately 70% of the patient population. Current evidence supports that degradation of TopoI by the Ubiquitin Proteasomal Pathway (UPP), and consequent compensation by Topoisomerase II expression may be involved in imparting drug resistance, but this mechanism requires much greater understanding. Protein-protein interaction studies have indicated that, BRCA1 is the E3 ligase for TopoI ubiquitination in response to CPT. BRCA1 impaired cells fail to ubiquitinate and degrade TopoI and are sensitive to CPTs. It is important to note that triple negative breast cancer patients have impaired BRCA1 function, higher mutation rate and/or a lower expression of BRCA1. The Bharti lab has shown that TopoI associates with BRCA1. Our work attempts to elucidate the nature of the interaction between BRCA1 and TopoI in the hope of better understanding the mechanism of resistance to camptothecin therapy in TNBC.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Medicinal Chemistry." Includes bibliographical references (p. 198-218).

Ethonafide is an anthracene-containing derivative of amonafide that belongs to the azonafide series of anticancer agents. The lack of cross-resistance in MDR-expressing cancer cell lines and the absence of a quinone and hydroquinone moiety make ethonafide a possible less-cardiotoxic replacement for existing anthracene-containing anticancer agents, such as mitoxantrone and doxorubicin. For this study, we investigated the anticancer activity and mechanism of action of ethonafide in human prostate cancer cell lines. Ethonafide was cytotoxic against three human prostate cancer cell lines at nanomolar concentrations. Ethonafide was also found to be better tolerated and more effective at inhibiting tumor growth compared to mitoxantrone in DU 145 prostate cancer-bearing mice. Mechanistically, we found that ethonafide inhibits topoisomerase II activity in human prostate cancer cell lines and equally inhibits purified topoisomerase IIα and recombinant topoisomerase IIβ. The inhibition of topoisomerase II activity was due to stabilization of the cleavable complex, involving both topoisomerase IIα and β. By creating stable DU 145 cell lines with decreased expression of either topoisomerase IIα or β, we found that topoisomerase IIα is necessary for ethonafide-induced cytotoxicity. The decrease in sensitivity to ethonafide was due to a decrease in DNA damage and an increase in DNA repair as measured by the neutral comet assay. Additionally, ethonafide induces potent G₂ cell cycle arrest in the DU 145 human prostate cancer cell line. Ethonafide also induces apoptosis as measured by procaspase and PARP cleavage. In conclusion, we have identified ethonafide as a topoisomerase II poison and determined that it is topoisomerase IIα-specific in the DU 145 human prostate cancer cell line. Due to ethonafide’s activity in vitro and in vivo, decreased toxicity in mice compared to mitoxantrone, and its activity in multi-drug resistant cancer cell lines, ethonafide may be a suitable replacement to mitoxantrone for the treatment of hormone-refractory prostate cancer.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.