Les ADN-Topoisomérases (Topo) de type I et II sont des enzymes essentielles à la suppression des surenroulements de engendrés par la plupart des transactions de l’ADN. Elles sont des cibles de médicaments anticancéreux très utilisés en clinique. Parmi eux, les inhibiteurs de Topo1 de la famille des camptothécines (CPT) exercent leur cytotoxicité en produisant des cassures double-brin de l’ADN provenant de la collision des fourches de réplications avec les complexes ADN-Topo1 stabilisés par ces inhibiteurs. Les dérivés de CPT sont approuvés pour le traitement des cancers coliques, de l’ovaire et du poumon, mais il existe de multiples mécanismes de résistance à ces agents qui sont à l’origine de l’échec du traitement. Les cancers de la prostate sont réfractaires aux CPT, mais très peu d’études ont été réalisées pour expliquer cette résistance « intrinsèque ». Ce travail de thèse visait à identifier les mécanismes de cette résistance en nous appuyant (1) sur des résultats antérieurs de l’équipe montrant que l’interaction entre la DNA-PKcs, une kinase impliquée dans la réparation de l’ADN par recombinaison non-homologue, et la Topo1 pouvait réguler la sensibilité aux CPT de manière indépendante de la réparation de l’ADN et (2) sur une étude ayant mis en évidence une interaction entre DNA-PKcs et le facteur de transcription ERG dont le gène est remanié dans plus de 50% des tumeurs de prostate. Nos résultats montrent pour la première fois que ERG est effectivement impliqué dans la régulation de la réponse aux CPT dans la lignée VCaP présentant un gène de fusion TMPRSS2-ERG. La répression de ERG dans la lignée VCaP induit une sensibilisation à la CPT mais pas à l’étoposide (un inhibiteur de Topo2) et est accompagnée d’une augmentation du nombre de complexes ADN/Topo1. Ce mécanisme peut-être soit lié à un effet de ERG sur l’interaction DNA-PKcs/Topo1 ou à une régulation transcriptionnelle de gènes impliqués dans la réponse aux CPT incluant la Topo1 elle-même. Nous avons confirmé cette deuxième hypothèse, en démontrant que ERG régule la transcription du micro ARN miR-24 et que l’expression de Topo1 est également sous contrôle de miR-24 dans la lignée VCaP. Des résultats similaires ont été obtenus dans la lignée LNCaP (présentant le gène de fusion TMPRSS2-ETV1) dans laquelle la répression de ETV1 confère aussi une sensibilisation à la CPT. Au cours de notre travail nous avons également recherché des inhibiteurs de l’interaction entre la DNA-PKcs et Topo1 afin de pouvoir utiliser ces composés comme agents de potentialisation des dérivés de CPT en clinique. Les résultats du criblage d’une banque de 320 composés naturels réalisé par la technologie AlphaScreen n’ont malheureusement pu identifier que des inhibiteurs catalytiques de Topo1. Nous avons néanmoins pu montrer que l’un d’entre eux, la mahanimbine, présentait une forte activité cytotoxique vis-à-vis de lignées résistantes aux dérivés de CPT et vis-à-vis de la lignée VCaP ce qui permet d’envisager le développement de nouvelles classes d’inhibiteurs catalytiques Topo1 pouvant contourner la résistance des dérivés de CPT en clinique. / Type I and II DNA Topoisomerases (Top) are essential enzymes involved in the removal of DNA torsional constraints induced by most DNA transactions. They are the targets of various anticancer agents used in the clinic. Among them, Top1 inhibitors from the camptothecins (CPT) family exert their cytotoxicity by producing DNA double-strand breaks that are generated by the collision of advancing replication forks with DNA-Top1 complexes that are stabilized by these inhibitors. CPT derivatives are approved for the treatment of colon, ovary and lung cancers but resistance mechanisms are developed and lead to treatment failure. Prostate cancers are refractory to CPT, but few studies have addressed the mechanisms of such “intrinsic” resistance. This work was aimed at identifying such mechanisms based on (1) previous results from the laboratory showing that interaction of Top1 with DNA-PKcs, a kinase that is essential for non-homologous end-joining, could regulate cell sensitivity to CPT independently of DNA repair and (2) a study that showed an interaction of DNA-PKcs with ERG, a transcription factor from the ETS family which is rearranged in more than 50% of prostate tumors. Our results show for the first time that ERG is indeed involved in the regulation of prostate cancer cell response to CPT as its repression sensitized VCaP cells displaying the TMPRSS2-ERG gene fusion to CPT but not to the Top2 inhibitor etoposide. This effect is accompanied with an increase in Top1-DNA complexes. This could be due to either an effect of ERG on DNA-PKcs/Top1 interaction, or to the transcriptional regulation of genes involved in cell response to CPT, including Top1 itself. We confirmed the latter hypothesis by showing that ERG can regulate the transcription levels of the microRNA miR-24 and that Top1 expression relies, at least in part, on miR24 levels in VCaP cells. We obtained similar results in LNCaP cells (characterized by a TMPRSS2-ETV1 gene fusion), in which ETV1 repression also sensitizes cells to CPT. In parallel, we also searched for inhibitors of DNA-PKcs/Top1 interaction in order to use these compounds to potentiate CPT derivatives in the clinic. We screened a chemical library of 320 natural compounds using the AlphaScreen technology. The results were disappointing as we only identified compounds that are catalytic inhibitors of Top1. Nevertheless, we could show that among them, mahanimbine displayed a potent cytotoxic activity towards CPT-resistant colon cancer cell lines and could efficiently inhibit the growth of VCaP cells that are highly resistant to CPT. This opens new avenues for the development of new classes of Top1 catalytic inhibitors that could be used to circumvent the clinical resistance to CPT derivatives.

INTRODUÇÃO: A endometriose, uma doença benigna, tem características invasivas com potencial proliferativo. O desenvolvimento das lesões pode ocorrer em decorrência de crescimento celular glandular e/ou estromal ou de alterações na cinética celular. Cinética celular refere-se ao equilíbrio entre a morte celular, ou apoptose, e a proliferação celular, que pode ser avaliada pela expressão de fatores de crescimento como, por exemplo, a topoisomerase 2-alfa (TOP2A). Também influenciam a cinética celular oncoproteínas como p53 e c-erB2, conhecidas por interferir na apoptose, podendo resultar em oncogênese. OBJETIVOS: O objetivo principal deste estudo foi comparar a cinética celular da endometriose infiltrativa de retosigmoide com a do endométrio eutópico de pacientes sem endometriose. Para tanto, foi avaliada a expressão de apoptose e de TOP2A bem como das oncoproteínas p53 e c-erB2. MÉTODOS: Foram obtidas amostras de lesões de endometriose envolvendo o reto-sigmoide de 60 mulheres com a doença e amostras de endométrio eutópico de 20 mulheres sem endometriose. A expressão de TOP2A e das proteínas p53 e c-erB2 foram quantificadas por técnica imuno-histoquímica. Método TUNEL foi utilizado para analisar os padrões de apoptose, que resultaram em índice de apoptose (IA). Índice de proliferação celular (IP) foi determinado a partir do nível de expressão de TOP2A. Índice de renovação celular (IRC) foi calculado pela razão entre IP e IA. As análises imuno-histoquímicas foram realizadas tanto no tecido endometrial como um todo, quanto nos componentes estromal e glandular separadamente. Coeficiente de Correlação de Spearman foi aplicado para identificar eventuais correlações entre variáveis clínicas, morfológicas (tamanho, quantidade e nível de invasão das lesões) e experimentais. RESULTADOS: Na análise da amostra do tecido como um todo, não foram evidenciadas diferenças entre os grupos experimental e controle em relação ao IA (p = 0,389). Por outro lado, o IP foi significativamente maior nas amostras-controle (p < 0,001). Na avaliação em que se sepaxii raram as células estromais dos componentes glandulares, tanto o IP quanto o IRC foram significativamente maiores no grupo-controle em comparação com o grupo experimental (IP estromal: p = 0,006; IP glandular: p = 0,001; IRC estromal: p =0,032; IRC glandular: p = 0,007). Nas pacientes com endometriose, foi encontrada correlação entre IP e IRC glandular e o número de lesões (p = 0,003). Também foi observada correlação entre o IRC glandular e o tamanho das lesões (p = 0,006). Não houve diferença entre os grupos no que se refere à expressão de p53 e cerB2. CONCLUSÕES: A cinética celular se mostrou alterada em pacientes com endometriose do reto-sigmoide, conforme demonstrado pela redução nos níveis e na frequência de TOP2A, e pelos IP e IRC mais baixos; entretanto, apoptose e as expressões de p53 e c-erB2 se mostraram inalteradas / BACKGROUND: Endometriosis, a benign disease, has invasive features with its proliferative potential. Development of lesions may occur due to stromal and/or glandular cell growth and to alterations in cellular kinetics. Cellular kinetics involves a balance between the regulation of cell death, or apoptosis, and cell growth, that can be evaluated by the expression of growth factors, such as topoisomerase 2- alpha (TOP2A). Oncoproteins, such as p53 and c-erB2, known to affect apoptosis resulting in oncogenesis, also influence cellular kinetics. OBJECTIVES: The main objective of this study was to compare the cellular kinetics in deep endometriosis involving the recto-sigmoid to eutopic endometrium from patients without endometriosis. Apoptosis and TOP2A expression were primarily evaluated, as well as p53 and c-erB2 expression. METHODS: Study samples were obtained from endometriosis lesions involving the recto-sigmoid in 60 women, and control samples were obtained from eutopic endometrium from 20 women without endometriosis. The expression of TOP-2A, p53 and c-erB2 proteins were quantified using immuno-histochemistry. TUNEL method was used in the analysis of apoptosis patterns, and the apoptosis index (AI) was derived. The proliferation index (PI) was derived from the level of expression of TOP-2A. Cellular renew index (CRI) was calculated from the ratio of the PI and AI. Immunohistochemical analyses were performed in two ways: on the tissue collectively, and on the stromal and glandular components separately. Spearmans correlation coefficient was used to identify the correlation between clinical, morphological (size, number and level of invasion of lesions) and the study variables. RESULTS: When looked at collectively, there was no difference in the AI between study and control groups (p = 0.389). PI, however, was noted to be significantly higher in the control samples (p < 0.001). When evaluating the stromal cells separately from the glandular components, the PI and CRI were both significantly xiv higher in the control group compared to the study group (Study stromal PI vs control stromal PI; p = 0.006; Study glandular PI vs study glandular PI; p = 0.001; Study stromal CRI vs control stromal CRI; p = 0.032; study glandular CRI vs control glandular CRI; p = 0.007). In patients with endometriosis, a correlation was found between glandular PI, CRI and number of lesions (p = 0.003). The same result was observed in the analysis of stromal CRI and lesion size (p = 0.006). There was no difference in expression of p53 and c-erB2 between groups. CONCLUSIONS: Cellular kinetics is altered in endometriosis of the recto-sigmoid, as shown by the decrease in the levels and frequency of TOP2A expression, and lower PI and CRI; however, apoptosis and p53 and c-erB2 expression were unaffected

INTRODUÇÃO: A endometriose, uma doença benigna, tem características invasivas com potencial proliferativo. O desenvolvimento das lesões pode ocorrer em decorrência de crescimento celular glandular e/ou estromal ou de alterações na cinética celular. Cinética celular refere-se ao equilíbrio entre a morte celular, ou apoptose, e a proliferação celular, que pode ser avaliada pela expressão de fatores de crescimento como, por exemplo, a topoisomerase 2-alfa (TOP2A). Também influenciam a cinética celular oncoproteínas como p53 e c-erB2, conhecidas por interferir na apoptose, podendo resultar em oncogênese. OBJETIVOS: O objetivo principal deste estudo foi comparar a cinética celular da endometriose infiltrativa de retosigmoide com a do endométrio eutópico de pacientes sem endometriose. Para tanto, foi avaliada a expressão de apoptose e de TOP2A bem como das oncoproteínas p53 e c-erB2. MÉTODOS: Foram obtidas amostras de lesões de endometriose envolvendo o reto-sigmoide de 60 mulheres com a doença e amostras de endométrio eutópico de 20 mulheres sem endometriose. A expressão de TOP2A e das proteínas p53 e c-erB2 foram quantificadas por técnica imuno-histoquímica. Método TUNEL foi utilizado para analisar os padrões de apoptose, que resultaram em índice de apoptose (IA). Índice de proliferação celular (IP) foi determinado a partir do nível de expressão de TOP2A. Índice de renovação celular (IRC) foi calculado pela razão entre IP e IA. As análises imuno-histoquímicas foram realizadas tanto no tecido endometrial como um todo, quanto nos componentes estromal e glandular separadamente. Coeficiente de Correlação de Spearman foi aplicado para identificar eventuais correlações entre variáveis clínicas, morfológicas (tamanho, quantidade e nível de invasão das lesões) e experimentais. RESULTADOS: Na análise da amostra do tecido como um todo, não foram evidenciadas diferenças entre os grupos experimental e controle em relação ao IA (p = 0,389). Por outro lado, o IP foi significativamente maior nas amostras-controle (p < 0,001). Na avaliação em que se sepaxii raram as células estromais dos componentes glandulares, tanto o IP quanto o IRC foram significativamente maiores no grupo-controle em comparação com o grupo experimental (IP estromal: p = 0,006; IP glandular: p = 0,001; IRC estromal: p =0,032; IRC glandular: p = 0,007). Nas pacientes com endometriose, foi encontrada correlação entre IP e IRC glandular e o número de lesões (p = 0,003). Também foi observada correlação entre o IRC glandular e o tamanho das lesões (p = 0,006). Não houve diferença entre os grupos no que se refere à expressão de p53 e cerB2. CONCLUSÕES: A cinética celular se mostrou alterada em pacientes com endometriose do reto-sigmoide, conforme demonstrado pela redução nos níveis e na frequência de TOP2A, e pelos IP e IRC mais baixos; entretanto, apoptose e as expressões de p53 e c-erB2 se mostraram inalteradas / BACKGROUND: Endometriosis, a benign disease, has invasive features with its proliferative potential. Development of lesions may occur due to stromal and/or glandular cell growth and to alterations in cellular kinetics. Cellular kinetics involves a balance between the regulation of cell death, or apoptosis, and cell growth, that can be evaluated by the expression of growth factors, such as topoisomerase 2- alpha (TOP2A). Oncoproteins, such as p53 and c-erB2, known to affect apoptosis resulting in oncogenesis, also influence cellular kinetics. OBJECTIVES: The main objective of this study was to compare the cellular kinetics in deep endometriosis involving the recto-sigmoid to eutopic endometrium from patients without endometriosis. Apoptosis and TOP2A expression were primarily evaluated, as well as p53 and c-erB2 expression. METHODS: Study samples were obtained from endometriosis lesions involving the recto-sigmoid in 60 women, and control samples were obtained from eutopic endometrium from 20 women without endometriosis. The expression of TOP-2A, p53 and c-erB2 proteins were quantified using immuno-histochemistry. TUNEL method was used in the analysis of apoptosis patterns, and the apoptosis index (AI) was derived. The proliferation index (PI) was derived from the level of expression of TOP-2A. Cellular renew index (CRI) was calculated from the ratio of the PI and AI. Immunohistochemical analyses were performed in two ways: on the tissue collectively, and on the stromal and glandular components separately. Spearmans correlation coefficient was used to identify the correlation between clinical, morphological (size, number and level of invasion of lesions) and the study variables. RESULTS: When looked at collectively, there was no difference in the AI between study and control groups (p = 0.389). PI, however, was noted to be significantly higher in the control samples (p < 0.001). When evaluating the stromal cells separately from the glandular components, the PI and CRI were both significantly xiv higher in the control group compared to the study group (Study stromal PI vs control stromal PI; p = 0.006; Study glandular PI vs study glandular PI; p = 0.001; Study stromal CRI vs control stromal CRI; p = 0.032; study glandular CRI vs control glandular CRI; p = 0.007). In patients with endometriosis, a correlation was found between glandular PI, CRI and number of lesions (p = 0.003). The same result was observed in the analysis of stromal CRI and lesion size (p = 0.006). There was no difference in expression of p53 and c-erB2 between groups. CONCLUSIONS: Cellular kinetics is altered in endometriosis of the recto-sigmoid, as shown by the decrease in the levels and frequency of TOP2A expression, and lower PI and CRI; however, apoptosis and p53 and c-erB2 expression were unaffected

Les ADN topoisomérases régulent la topologie de l’ADN lors des processus cellulaires tels que la réplication, la transcription ou la ségrégation des chromosomes au cours de la division cellulaire. Leur rôle crucial dans le maintien de l’intégrité du génome et dans la transmission des gènes en fait des cibles privilégiées pour le développement de molécules thérapeutiques. Cependant, les inhibiteurs utilisés actuellement comme agents anti-tumoraux contre les topoisomérases humaines sont peu spécifiques et entrainent de nombreux effets secondaires. De même, l’utilisation massive d’antibiotiques à large spectre contre les topoisomérases bactériennes entraine l’apparition de mutations et le développement de souches résistantes. L'amélioration des traitements dépend de notre compréhension du mécanisme catalytique, des fonctions cellulaires précises, des architectures tridimensionnelles et des processus d'inhibition de ces ADN topoisomérases. Nous nous sommes tout d’abord consacrés à l’étude structurale de l’interaction entre la Coumermycine-A1, un antibiotique de la famille des aminocoumarines, avec le domaine ATPase de l’ADN Gyrase, une topoisomérase 2A bactérienne. Les résultats obtenus ont révélé un mode d’inhibition inédit et ouvrent la voie à la conception de nouveaux dérivés de cet antibiotique, ciblant plus spécifiquement les ADN Gyrases de pathogènes. La seconde partie de la thèse a consisté en l’étude de l’architecture complète de l’ADN Gyrase et de l’isoforme alpha de la Topo II humaine par Cryo-microscopie électronique. Les données structurales obtenues révèlent pour la première fois l’architecture complète de ces topoisomérases à haute résolution. L’ensemble des résultats apporte de nouveaux éléments pour la compréhension des mouvements allostériques des topoisomérases 2A lors du cycle catalytique. Ces données, jusque-là inaccessibles par les méthodes structurales conventionnelles, sont essentielles pour le développement de nouvelles molécules inhibitrices pouvant cibler spécifiquement différents états catalytiques. / DNA topoisomerases regulate DNA topology during cellular processes such as replication, transcription or chromosome segregation. Their crucial role in maintaining the integrity of the genome and in genetic transmission makes them prime targets for the development of therapeutic molecules. However, the inhibitors currently used as anti-tumor agents against human topoisomerases are not very specific and cause many side effects. Similarly, the massive use of broad-spectrum antibiotics against bacterial topoisomerases leads to the appearance of mutations and the development of resistant strains. The optimization of current therapeutics depends on our understanding of the catalytic mechanism, the precise cellular functions, the complete three-dimensional architectures and the inhibition processes of these topoisomerases. This study first focused on the structural characterization of the interaction between Coumermycin-A1, an aminocoumarin antibiotic, with the ATPase domain of DNA Gyrase, a bacterial Type 2A topoisomerase. The results obtained revealed an unprecedented mode of inhibition and pave the way to the design of new variants of this antibiotic, specifically targeting pathogenic DNA Gyrases. The second part of the thesis consisted in the study of the complete architecture of the DNA Gyrase and the human Topo II alpha isoform by Cryo-electron microscopy. The structural data obtained reveal for the first time the complete architecture of these topoisomerases at high-resolution. The results shed light on the finely-tuned allosteric movements of topoisomerases 2A during the catalytic cycle. These information, until now out of reach using the conventional structural methods, are essential for the development of new inhibitory molecules that can specifically target different catalytic states.

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation. / Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta. / In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

Maintenance of a topological homeostasis by introduction and removal of the supercoils to relieve excessive strain on the DNA is a hallmark of topoisomerase function in the cell. The requirement of the topoisomerases during DNA transaction processes marks a ubiquitous presence of the enzymes in all the life forms. Different reactions carried out by the enzymes include relaxation of positive and negative supercoils required majorly during DNA replication and transcription, decatenation at the end of DNA replication to separate the daughter chromosomes and removal of lethal knots generated in the circular chromosome. In eubacteria, the enzymes introduce negative supercoils to facilitate easier strand separation for DNA transaction processes. However, in thermophiles, a different enzyme maintains the genome in a positively supercoiled form to protect from denaturation by excessive heat. These varied functions are carried out by different topoisomerases. Therefore, each organism maintains a minimum required set of the enzymes and the absence of a certain enzyme may be compensated for by topoisomerases with dual functions. For example, Mycobacterium tuberculosis and many other slow growing mycobacteria do not possess topoisomerase IV or its homologs. In these organisms, the DNA gyrase is suggested to carry out both negative supercoiling and decatenation reactions. Therefore, the mycobacterial DNA gyrase must be able to manage between both the functions in vivo. In contrast, Mycobacterium smegmatis and few other mycobacteria contain an additional type II topoisomerase which does not resemble any known type II enzyme but could catalyze relaxation and decatenation reactions. Importantly, the enzyme displays a unique ability to introduce limited positive supercoils and may have certain functions inside the cell which remains to be studied. Owing to the indispensability for bacterial survival topoisomerases present themselves as important drug targets. A large number of inhibitors have been found to inhibit the enzyme and thereby killing the bacterial. Among these, quinolones are successfully being used as broad spectrum antibacterial drugs. Although the commonly used quinolones inhibit many bacterial pathogens, a reduced susceptibility is exhibited by some of the pathogens e.g. Mycobacterium tuberculosis. To circumvent the lower efficacy of existing drugs, new and modified quinolones have been developed which are highly effective against mycobacteria. The difference in the susceptibility may be conferred by a difference in the chemical property of the drug and the interacting residues present in the enzyme. In the present thesis efforts have been made to understand the mechanism of the type II topoisomerases from mycobacteria and drug action on these enzymes. The thesis is divided into four chapters. In Chapter I of the thesis an introduction is provided on the topoisomerases, their classification and different reactions catalyzed by these enzymes. As the work in present thesis has been carried out with type II topoisomerases, introduction of type II enzymes, their structure and mechanisms is elaborated. DNA gyrase, its mechanism of reaction and in vitro and in vivo functions are explained in great detail. DNA gyrase and topoisomerase IV are targeted by a range of different inhibitors. These different classes of inhibitors and their mechanism of action are described. Finally, the mechanism of mycobacterial DNA gyrase with structural information and the current understanding of quinolone action on the enzyme are explained. The chapter ends with the objective of the study in the present thesis. In chapter II, the studies are aimed at understanding the molecular basis for decatenation carried out by mycobacterial DNA gyrase. Previous work from the laboratory showed that the enzyme can carry out decatenation more efficiently than its homolog from E. coli. It was shown that the mycobacterial enzyme binds two DNA molecules in trans in a length dependent manner. The ability to bind the second DNA is conferred upon the holoenzyme by ATPase subunit (GyrB) subunit which alone can bind DNA. Similar studies using topo IV from E. coli, the strongest known decatenase showed binding of two DNA molecules and the second DNA binding by ATPase (ParE) subunit. However, GyrB subunit from E. coli DNA gyrase, a weaker decatenase, does not bind second DNA molecule efficiently. The results provide a general mechanism for decatenation by type II enzymes in which efficient binding of second DNA is important. In Chapter III, studies have been carried out using topo NM, an atypical type II topoisomerase from Mycobacterium smegmatis. The enzyme has been characterized previously in the laboratory. In addition to efficient decatenation and relaxation, the enzyme exhibits a unique ability to introduce positive supercoils into the DNA. As demonstrated for the mycobacterial DNA gyrase and topo IV in the Chapter II, the ATPase subunit (Topo N) of topo NM, binds second DNA efficiently. The binding of both gate and transport segments increases with the length of the DNA. Binding of two DNA molecules by the holoenzyme appears to be a cumulative effect of DNA binding to individual subunits. In the absence of any inhibitor, the enzyme accumulates cleaved DNA products with shorter DNA but not with larger DNA. The cleavage of the shorter DNA is supported only in the presence of Mg2+ and Mn2+. Another important property of the enzyme is to introduce positive supercoils which appears to be due to its efficient utilization of ATP and a high rate of reaction. Chapter IV deals with the interaction of mycobacterial gyrase with fluoroquinolones (FQs). Although DNA gyrase is the sole target of the FQs in M. tuberculosis, the lower susceptibility to commonly used FQs have led to the studies to find out more effective quinolones. Previous studies from the laboratory showed a lower susceptibility of the mycobacterial gyrase to ciprofloxacin, but moxifloxacin could inhibit the enzyme efficiently. The better inhibition by moxifloxacin appears to be due to efficient trapping of the enzyme-DNA covalent complex. Both ciprofloxacin and moxifloxacin bind the DNA gyrase from mycobacteria, E. coli and E. coli topo IV, independent of DNA. The extent of binding also correlates with the inhibition potential of the drug against a given enzyme. A general model of quinolone enzyme interaction is provided wherein the quinolones are shown to interact with GyrA subunit or holoenzyme or the enzyme- DNA complex which would finally result in the trapping of the covalent complex.

No description available.