Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-12-05T17:42:30Z No. of bitstreams: 1 StanrleyBandeira.pdf: 1297237 bytes, checksum: c39acc4db03b1190e402e8b3a4c9d8ff (MD5) / Made available in DSpace on 2017-12-05T17:42:30Z (GMT). No. of bitstreams: 1 StanrleyBandeira.pdf: 1297237 bytes, checksum: c39acc4db03b1190e402e8b3a4c9d8ff (MD5) Previous issue date: 2017-08-28 / FAPEMA / The present master's work was focused on quantum chemistry calculations and based on the Functional Density Theory (DFT) the interaction between platinum compounds used in anti-cancer therapy and DNA. Based on the analysis of the factors that influence the bonding process between the platinum complexes and the DNA. All the nitrogenous bases of the DNA were analyzed, in order to choose the one that would present better interaction with the studied complexes. The theoretical results showed that the different platinum compounds have binders that decrease toxicity, or increase membrane binding. The platinum compounds, Carboplatin, Iproplatin, Nedaplatin and Oxaliplatin, were structured through the GAUSS VIEW 5.0 software. Then, the optimization of these structures was performed using the GAUSSIAN 9.0 program. This optimization used bases extracted from the EMSL BASIS SET EXCHANGE database. As a result of these optimizations the HOMO-LUMO intervals were used to characterize the stability of the molecules, as well as the stability variations that occurred during the hydrolysis reactions, and their connections with the guanine base of the DNA. This observation, as well as the thermodynamic properties, served to describe the toxicities presented in the literature. / O presente trabalho de mestrado foi voltado a examinar por meio de cálculos de química quântica e com base na Teoria do Funcional Densidade (DFT) a interação entre compostos de platina usados na terapia anti-câncer e o DNA. Com base na análise dos fatores que influenciam o processo de ligação entre os complexos de platina e o DNA.Analisou-se todas as bases nitrogenadas do DNA, para a escolha da que apresentaria melhor facilidade de interação com os complexos estudados. os resultados teóricos mostraram que que os diferentes compostos de platina apresentam ligantes que diminuem a toxicidade, ou aumentam a ligação com membranas. Os compostos platínicos,Carboplatina, Iproplatina, Nedaplatina e Oxaliplatina, foram estruturados através do progarama GAUSS VIEW 5.0. Em seguida realizou-se a otimização dessas estruturas utilizando-se o programa GAUSSIAN 9.0. Essa otimização utilizou bases extraídas do banco de dados EMSL BASIS SET EXCHANGE. Como resultado dessas otimizações foram extraídos os intervalos HOMO-LUMO, utilizados para caracterizar a estabilidade das moléculas, bem como, as variações de estabilidade, que ocorriam durante as reações de hidrolise, e respectivas ligações com a base GUANINA do DNA. Essa observação,bem como as propriedades termodinâmicas serviu para descrever as toxicidades, apresentadas em literatura.

The Langmuir – Blodgett (LB) technique is about forming insoluble monolayer on the surface of aqueous solution and recently, it has emerged as one of the best method to study floating monolayer at the air – water interface. It has gained popularity after the use of monolayer with chemical complexes as well as biological species, and recently it has been used for the formation of biosensors. Langmuir monolayer arrays the amphiphilic molecules in a fashion where the hydrophobic part points towards the air and the hydrophilic group remains in contact with the aqueous subphase. Due to this property of Langmuir monolayer to orient the molecules at the air – water interface in a particular fashion, it can successfully serve as a template for two – dimensional reactions with restricted freedom. Hence, Langmuir monolayer has been extensively employed to study chemical and biological reactions at the air – water interface. To understand the behavior of Langmuir monolayer, surface pressure – molecular area (P – A) isotherms are studied as these P – A isotherms illustrate general conditions regarding the phase behavior of the two-dimensional Langmuir monolayer. Any change occurring due to the alignment of aliphatic molecules forming the monolayer is reflected by the change in P – A isotherms, which is known as phase transition. The phase transition is the most important element of the P – A isotherms with a characteristic signature of a plateau region in the isotherms. This phase transition point changes with the change of certain external parameters such as temperature, pH, and ionic strength, and as a result gives general information regarding the phase transition behavior. Therefore, with the little change of external parameters, the arrangement of the molecules in the monolayer also changes, which is reflected in the change in the nature of the isotherms. Thus, the system can, in principle, be used to define several physical parameters associated with it. On account of the property of Langmuir monolayer to orient the molecules at the air – water interface with restricted mobility and due to their condensed nature known as solid like phase, it closely mimics the situation inside a biological cell. Hence, we wanted to test whether an artificial nucleus can be generated at LB films. This can be achieved by immobilizing DNA or protein at the air – water interface and then by promoting their biological properties through macromolecular recognition. Here, immobilization of a macromolecule of biological relevance, its interaction with another component of a cell and extracting the thermodynamic parameters utilizing the LB technique will be of significance. This thesis embodies the immobilization of some biologically important proteins then follows their activity as well as DNA recognition properties at the air – water interface. A set of equations are derived here for the two dimensional Langmuir monolayer, which are used to calculate the thermodynamics of the system under study. Chapter 1 outlines the information about Langmuir monolayer and LB films. It sketches the historical background of the Langmuir monolayer and also elucidates the theory behind the same. This chapter cites the technical details of formation of Langmuir monolayer and LB films viś – a – viś other methods available for the fabrication of monomolecular films. It adequately discusses the functional LB films and their utilization for various different purposes. Finally, the role of metal ions in the LB films and in immobilizing biological macromolecules is discussed. Chapter 2 discusses the different techniques employed to perform the experiments described in this thesis. It includes the purification methods for the different proteins and DNA; the details of formation of Langmuir monolayer and fabrication of LB films. This chapter also describes the various techniques used for the characterization of the LB films, i.e Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) spectroscopy. In Chapter 3, immobilization and imaging of protein molecules and protein DNA complexes on a LB substrate have been explored. Firstly, we describe the preparation of a Ni (II) – arachidate (NiA) monolayer and its characterization through P – A isotherm on a LB trough. Then, recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the NiA monolayer through a Ni (II) – histidine interaction. A single molecule of RNA polymerase (RNAP) could be seen through intermittent-contact AFM. Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. In Chapter 4, the interaction between NiA and histidine tagged RNAP (HisRNAP), and RNAP and DNA were studied. LB films of Arachidic acid – NiA, NiA -HisRNAP and NiA – HisRNAP – DNA with different mole fractions were fabricated systematically. P -A isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for FTIR spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the AA monolayer with the change in pH. The increase in mole fraction of RNAP and DNA in the NiA and NiA – RNAP monolayer, respectively, with their increasing concentration in the subphase are also noticed. The system developed here is robust and can be utilized to follow macromolecular interactions. In chapter 5, the Langmuir monolayer has been utilized to array a protein, Dps, specific for Fe (II) and non-specific for DNA. Dps from Mycobacterium smegmatis is known to have a cage like structure, exists in two oligomeric states, trimer and dodecamer, and can accommodate Fe (II) ions in its internal cavity. In addition, it converts Fe (II) to Fe (III), both in trimeric and dodecameric form, whereas the latter species is specific for non-specific DNA binding. We demonstrate here that, histidine tagged Dps in both oligomeric states can be immobilized on NiA LB films, where both ferroxidation and DNA binding ability remained unaffected in the ordered protein assembly. Interestingly, when Fe (II) – arachidate was used to generate a LB layer instead of NiA, Dps protein not only recognizes Fe (II) ion in the monolayer, it also converts it to Fe (III) ion in a time dependent fashion. However, once Fe (III) – Dps complex is formed and arrayed on LB monolayers, it remains very stable.

abstract: Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed. / Dissertation/Thesis / Ph.D. Chemistry 2011

Helicobacter pylori est une bactérie à Gram-négatif qui colonise la muqueuse de l’estomac humain. Elle se distingue des autres bactéries par un nombre de gènes très limité et de nombreuses particularités physiologiques et biochimiques. Elle provoque des infections associées à différentes maladies gastro-duodénales (ulcères et cancers). Depuis quelques années, une recrudescence de multi-résistances aux antibiotiques est observée. La transformation naturelle est l’un des processus clés qui les propage. Il s’agit d’un mécanisme de transfert horizontal de gènes qui permet aux bactéries de s’adapter à leur environnement, en internalisant des fragments d’ADN exogène à travers leur membrane, puis en les intégrant dans le chromosome par recombinaison homologue. Mes travaux ont visé à étudier de façon structurale et fonctionnelle trois protéines d’H. pylori décrites comme étant essentielles dans le processus de transformation naturelle: NucT, DprA et ComFc. La première partie de ce travail s’est concentrée sur la nucléase périplasmique NucT, supposée être impliquée dans la transformation chez H. pylori. Cependant, la délétion de son gène a permis de démontrer qu’elle ne joue en fait qu’un rôle mineur dans ce processus. La résolution de sa structure 3D a permis de mieux comprendre sa spécificité pour les acides nucléiques simple brin. Dans la seconde partie, la protéine DprA, responsable du chargement de la recombinase RecA sur l’ADN internalisé, a été étudiée. DprA d’H. pylori n’est composée que de 2 des 3 domaines qui constituent habituellement DprA, et fixe aussi bien l’ADN double brin que l’ADN simple brin mais uniquement via son domaine RF. Malgré son homologie structurale avec le domaine WH de liaison à l’ADN, le domaine C-terminal de HpDprA n’a pas d’affinité pour l’ADN. Nous avons mis en évidence des acides aminés conservés dans ce domaine dont l’étude pourrait permettre de comprendre son rôle. Enfin, une étude structurale de la protéine ComFc dont la délétion du gène entraîne la disparition totale de la capacité de transformation d’H. pylori a été réalisée. L’obtention de sa structure 3D a permis de mettre en évidence la présence d’un domaine catalytique phosphoribosyl-transférase ainsi que d’un domaine en doigt en zinc. Ce dernier pourrait être responsable de la capacité de ComFc à fixer l’ADN. Le substrat naturel de cette enzyme reste à découvrir.L’ensemble de ce travail a permis de contribuer à une meilleure compréhension à l’échelle moléculaire du mécanisme de transformation génétique naturelle d’H. pylori. L’avancement sur ces connaissances pourrait à long terme aider à réduire la propagation des multi-résistances par l’élaboration de nouvelles thérapies.Mots-clés : H. pylori, transformation naturelle, NucT, DprA, ComFc, interaction protéine-ADN / Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus of the human stomach. It is distinguished from other bacteria by a limited number of genes and many physiological and biochemical characteristics. It causes infections associated with various gastro-duodenal diseases (ulcers and gastric cancers). In recent years, an increase in multi-resistance to antibiotics has been observed. Natural transformation is one of the key processes that spreads these multi-resistances. It is a horizontal gene transfer mechanism that allows bacteria to adapt to their environment by internalizing exogenous DNA fragments through their membrane and then integrating them into the chromosome by homologous recombination. My work aimed to study in a structural and functional approach three proteins of H. pylori described as essential in the natural transformation process: NucT, DprA and ComFc. The first part of this work focused on periplasmic nuclease, NucT, which is supposed to be involved in transformation in H. pylori. However, the deletion of its gene has shown that it actually plays only a minor role in this process. The resolution of its 3D structure has led to a better understanding of its specificity for single-stranded nucleic acids. In the second part, the protein DprA, responsible for loading RecA recombinase onto internalized DNA, was studied. HpDprA is composed of only 2 of the 3 domains that usually constitute DprA, and binds both double-stranded and single-stranded DNA but only via its RF domain. Despite its structural homology with the WH DNA binding domain, the C-terminal domain of HpDprA has no affinity for DNA. We have identified conserved amino acids in this domain that could be studied to understand its role. Finally, a structural study of ComFc, whose deletion of the gene leads to the total disruption of the transformation capacity of H. pylori, has been carried out. The acquisition of its 3D structure has highlighted the presence of a phosphoribosyl transferase catalytic domain as well as a zinc finger domain. The latter could be responsible for capacity of ComFc to bind DNA. The natural substrate of this enzyme remains to be discovered.All this work has contributed to a better knowledge at the molecular level of the natural genetic transformation mechanism of H. pylori. Advancing this knowledge could in the long term help to reduce the spread of multiresistance through the development of new therapies.Keywords: Helicobacter pylori, natural transformation, NucT, DprA, ComFc, protein-DNA interaction

The thesis deals with physico-chemical properties of the MADS box, binding domain of transcription factors, which are important for the formation of complexes with the DNA regulatory segment bearing the CArG box. The study was performed also on model oligopeptides, selected segments of the MADS box and their analogues with a point mutation. A wide range of spectroscopic techniques was employed, namely absorption, circular dichroism, fluorescence and Raman spectroscopies. Advanced approaches including multivariate methods were used for data processing. The three tyrosines of the MADS box located in amino-acid vicinities of different charge and hydrophobicity, were used as intrinsic spectroscopic probes. The obtained characteristics of the MADS box and its segments structural arrangement, flexibility and acid-base equilibria are the main results of the work.

El cisplatí, PtCl2(NH3)2, ha estat una de les drogues més utilitzades en la quimioteràpia del càncer des del descobriment de la seva activitat. Però degut a la seva alta toxicitat i greus efectes secundaris, s'han sintetitzat nous compostos amb la finalitat de reduir aquests inconvenients.En aquest sentit, el treball desenvolupat en aquesta tesi doctoral ha estat la síntesi i caracterització de tretze complexos de Pt(II) amb la finalitat d'estudiar llur activitat antitumoral. Aquests complexos presenten unes característiques estructurals comunes: geometria cis, dos lligands làbils de tipus clorur i un lligand diaminoquelatant derivat dels àcids d,l-2,3-diaminopropiònic (Hdap) i d,l-2,4-diaminobutíric (Hdab). S'han dissenyat unes estratègies sintètiques a partir de les quals els lligands han estat funcionalitzats amb diferents grups de tipus éster, aminoàcid i peptídic: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], essent Metala= éster metílic de L-alanina, phe= L-fenilalanina, Mettrp= éster metílic del L-triptofà. Aquests lligands diaminoquelatants s'han utilitzat per sintetitzar els corresponents complexos de Pt(II): PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).A través de diferents tècniques i assaigs biològics (dicroisme circular, electroforesi en gel d'agarosa, microscopia de forces atòmiques, citometria de flux, assaigs de proliferació cel·lular) s'ha pogut demostrar l'activitat antitumoral d'aquests compostos. A través de la tècnica de dicroisme circular (DC) s'ha pogut demostrar que els lligands lliures no interaccionen covalentment amb el DNA de Calf Thymus i no modifiquen l'estructura secundària de la doble hèlix. En canvi, els respectius complexos han demostrat tenir capacitat per interaccionar amb el DNA i modificar la seva estructura secundària. Els complexos PtCl2(Hdap), PtCl2(Hdab) i PtCl2(dab-phe) mostren un comportament similar al cisplatí, generant adductes cis-bifuncionals que distorcionen la doble hèlix de forma no desnaturalitzant amb obertura de la doble cadena. Els complexos PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2) quan interaccionen amb el DNA generen un canvi en la conformació del DNA de la forma B a la forma C, produint-se un augment de la curvatura de l'hèlix per rotació de les bases nitrogenades. En aquests estudis s'ha comprovat que l'estructura del complex influeix en l'efecte generat sobre l'estructura secundària de l'àcid nucleic. En primer lloc, existeix una diferència en el comportament en funció del tamany del lligand diaminoquelatant, de manera que els complexos amb el lligand (dab) provoquen un efecte més remarcable. També s'observa aquest canvi de comportament al passar dels complexos que tenen el grup funcional esterificat als que el tenen protonat. D'aquesta manera, s'observa un major efecte sobre l'estructura secundària del DNA en aquells complexos que tenen el lligand diaminoquelatant de tres metilens (dab) i amb el grup carboxilat terminal protonat.Per tal de modelitzar la interacció d'aquests complexos amb el DNA, s'ha estudiat la interacció d'aquests compostos de Pt(II) amb 5'-GMP a través de RMN-1H, observant la variació dels senyals corresponents al H8 de 5'-GMP. Així s'ha pogut demostrar que aquests compostos interaccionen amb la 5'-GMP a través d'un enllaç covalent Pt-N7, de la mateixa manera a com interacciona el cisplatí.A través d'electroforesi en gel d'agarosa i microscopia de forces atòmiques (AFM) s'ha pogut determinar l'efecte que generen els lligands lliures i els respectius complexos de Pt(II) sobre l'estructura terciària del plasmidi pBR322. Els lligands provoquen un augment de l'agregació de les molècules de DNA i un lleuger augment de la compactació de l'estructura terciària. Aquests resultats s'atribueixen a la capacitat d'aquests compostos a interaccionar per pont d'hidrogen amb el DNA. Els corresponents complexos de Pt(II) provoquen un augment de l'agregació i una important compactació, degut per una banda a la capacitat de l'àtom de Pt a interaccionar covalentment amb el DNA, i per altra banda, a la capacitat del lligand a interaccionar per pont d'hidrogen amb l'àcid nucleic.Finalment s'ha estudiat l'activitat citotòxica d'aquests complexos de Pt(II) en diferents línies cel·lulars: A431 (línia de carcinoma epidermoide), HeLa (línia de carcinoma de coll d'úter) i HL-60 (línia promielocítica de leucèmia). Els complexos moderadament solubles en aigua, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), han demostrat ser actius. L'activitat depèn de la concentració de complex, del temps d'incubació i de la línia cel·lular. Per temps d'incubació alts i concentracions de complex elevades s'observa la màxima activitat. Els complexos de l'alanina, PtCl2(dap-ala) i PtCl2(dab-ala), són els que mostren més activitat, mentre que els compostos de la fenilalanina són els menys actius, degut probablement a la voluminositat del lligand, la qual pot impedir o dificultar el transport del compost a través de la membrana cel·lular.L'activitat citotòxica dels complexos insolubles en aigua, PtCl2(Etdap) i PtCl2(Etdab), queda bloquejada per l'elevada concentració de DMSO (12%) necessària per solubilitzar els compostos. Aquests resultats permeten deduir que la presència d'un 12% de DMSO anul·la l'activitat d'aquests complexos, ja que el DMSO pot coordinar-se amb el Pt ocupant les posicions làbils del complex i evitant que es pugui coordinar amb el DNA.Els assaigs de proliferació cel·lular del complex PtCl2(dap-ASTTTNYT-NH2) i del pèptid lliure ASTTTNYT-NH2 han demostrat que ambdós compostos són actius. Tot i això, l'activitat del complex és superior a la del pèptid lliure, ja que el Pt pot interaccionar covalentment amb el DNA i augmentar l'efecte citotòxic. Per tant, el complex presenta un lligand portador biològicament actiu que pot transportar el metall a través de la membrana cel·lular i facilitar així la seva interacció amb el DNA.A través de la tècnica de citometria de flux s'ha comprovat que en tots els casos la mort cel·lular produïda pels complexos ha estat per apoptosi.Per últim, s'ha sintetitzat i caracteritzat un complex trinuclear de Pt(II), {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2}, essent Me2Bpy= 4,4'-dimetil-2,2'-dipiridil. La resolució de la seva estructura per difracció de Raig-X ha permès determinar l'existència d'una interacció intramolecular Pt-Pt de 3.474 Å. / Cisplatin, PtCl2(NH3)2, has been one of the most used drugs for the cancer chemotherapy. However, due to its high toxicity and important side effects, new complexes have been synthesized in order to reduce these disadvantages.In that way, the work presented in this thesis is the synthesis and characterization of 13 Pt(II) complexes in order to study their antitumor activity. These complexes present some common traits: cis geometry, two labile chloride ligands, and one diamine chelating ligand derived from d,l-2,3-diaminopropionic acid (Hdap) and d,l-2,4-diaminobutyric acid (Hdab). These ligands have been derived using new synthetic strategies with different functional groups as ester, aminoacid and peptide type: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], being Metala= methyl ester of L-alanine, phe= L-phenylalanine and Mettrp= methyl ester of L-triptophan. These ligands have been used for preparing the following Pt(II) complexes: PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).Using different techniques and biologic assays (circular dichroism, agarose gel electrophoresis, atomic force microscopy, flow cytometry, cell proliferation assays) the antitumor activity of these complexes has been demonstrated.With circular dichroism it has been demonstrated that free ligands do not covalently bind Calf Thymus-DNA and do not modify its secondary structure. In constrast, the corresponding Pt(II) complexes are able to interact with DNA and modify its secondary structure. Complexes PtCl2(Hdap), PtCl2(Hdab) and PtCl2(dab-phe) behave in the same direction as cisplatin, forming cis-bifunctional intrastrand adducts that probably open the double helix. The other complexes, PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2), interact with DNA generating a BC transformation with increasing winding of the double helix by rotation of the bases. The influence of the complex structure in the effect on the secondary structure of DNA has been proved. Firstly, a difference in the behaviour exists depending on the ring size, so complexes with (dab) ligand cause bigger modifications. This kind of behaviour is also seen between complexes with ester groups at the C-terminus and complexes with carboxylic group. Thus, there is a greater effect in the secondary structure of the DNA in those complexes that have the (dab) ligand and a protonated carboxylate group.The interaction between the platinum complexes and 5'-GMP has been studied by 1H-NMR, following the variation of H8 signals from 5'-GMP. It has been demonstrated that all complexes covalently interact with 5'-GMP through the N7 of the base, as cisplatin do.The effect generated by free ligands and their Pt(II) complexes in the tertiary structure of pBR322 DNA has been determined by agarose gel electrophoresis and atomic force microscopy (AFM). Generally, the free ligands studied generate an increase of DNA aggregation and a slight coiling of the double helix, due to their possibility of forming hydrogen bonds with the DNA bases through different functional groups of their structure. The Pt(II) complexes generate an important coiling and aggregation of DNA, probably due to the covalent binding Pt-DNA and hydrogen bond interactions through their ligands.The cytotoxic activity of the Pt(II) complexes has been evaluated through cell proliferation assays in A431, HeLa and HL-60 cell lines using different incubation times (24h, 48h, 72h). The water-soluble complexes, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), show antitumor activity in a dose- and time- dependent manner, gradually decreasing the cell survival percentage when the complex concentration increase, in the same direction as carboplatin. All these complexes present the highest activity at long incubation times and high complex concentration. The alanine complexes, PtCl2(dap-ala) and PtCl2(dab-ala) are the most active and those of phenylalanine are the less active, probably due to the ligand size, which can make its transport through cell membrane more difficult.The water-insoluble complexes, PtCl2(Etdap) and PtCl2(Etdab), apparently do not show antitumor activity due to the presence of 12% DMSO necessary por solving these complexes. In these conditions they do not cause a decrease in cell survival. The DMSO could interact with platinum, displacing the labile ligands of the complex and not allowing it to interact with DNA. Complex PtCl2(dap-ASTTTNYT-NH2) is also insoluble in water, but can be dissolved in 2% DMSO solution. Cell proliferation assays show that the free peptide, ASTTTNYT-NH2, and the platinum complex are both active. The activity of the complex is higher than the free peptide, probably because platinum can covalently interact with DNA and increase the cytotoxic effect. This result suggest that the ligand can act as a carrier ligand and make the interaction metal-DNA be easier.Flow cytometry assays have demonstrated that cell death generated by the complexes synthesized is apoptosis. Agarose gel electrophoresis of extracted DNA show a classical 200-base pair integer oligonucleosome ladder.A new trinuclear Pt(II) complex, {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2} being Me2Bpy= 4,4'-dimethyl-2,2'-dipyridyl, has been synthesized and characterized. The X-ray diffraction of its structure shows the existence of an intramolecular Pt-Pt interaction of 3.474 Å.

The thesis entitled “Design and Synthesis of Benzimidazole Based Templates in Duplex and Quadruplex DNA Recognition and in Topoisomerase Inhibition” deals with the design and synthesis of several benzimidazole based molecules and their interaction with duplex and quadruplex DNA structures. It also elucidates the inhibition effect of the compounds on the activity of topoisomerase I enzyme of parasitic pathogen Leishmania donovani. The work has been divided into five chapters. Chapter 1: An Introduction to DNA and its Interaction with Small molecules. The first chapter provides an introduction to the double helical structure of DNA and the central dogma that suggests the flow of genetic information from DNA to RNA to protein. This chapter also presents an overview on the various types of small molecules that interact with duplex and quadruplex structures of DNA or interfere with the activity of DNA targeted enzymes like topoisomerase. This chapter describes the importance of such molecules as chemotherapeutic agents. Chapter 2 deals with three isomeric, symmetrical bisbenzimidazole derivatives bearing pyridine on the two termini. The syntheses, duplex DNA binding and computational structure analyses of the molecules have been divided into two sections. Chapter 2A: Novel Symmetrical Pyridine Derivatized Bisbenzimidazoles: Synthesis and Unique Metal Ion Mediated Tunable DNA Minor Groove Binding. The first chapter deals with the synthesis and double stranded (ds) DNA binding characteristics of the three bisbenzimidazole derivatives. Despite being positional isomers, their relative binding affinities towards ds-DNA varied considerably. Fluorescence, circular dichroism and temperature dependent UV-absorption spectroscopy have been employed to characterize ligand-DNA binding interaction. All spectroscopic studies revealed the strong A-T selective DNA binding affinities of the p- and m-pyridine derivatized molecules (p-pyben and m-pyben respectively) and indicated dramatically weak binding interaction of the ortho derivative (o-pyben) to ds-DNA. Additionally, unique transition metal ion mediated tunable DNA binding shown by o-pyben has been described in this chapter. While the ds-DNA binding characteristics of p- and m-pyben remained unaffected in presence of metal ions, that of o-pyben could be reversibly ‘switched off’ in the presence of divalent transition metal ions like Co2+, Ni2+, and Cu2+. Addition of EDTA reversed the effects and DNA binding was again observed. This interesting observation provides valuable insight into the DNA recognition property of these isomeric bisbenzimidazole derivatives. Figure 1. Molecular structures of pyridine derivatized symmetrical bisbenzimidazoles. Chapter 2B: Differential Binding of Positional Isomers of Symmetric Bisbenzimidazoles on DNA Minor-Groove: A Computational study. To explain the weak DNA binding affinity of o-pyben, compared to p- or m-pyben, detailed ab initio/DFT computational analyses of the inherent structural features of the three isomers were performed both in the gas-phase and in water. The study revealed the presence of intramolecular hydrogen bond existing in the opyben, between the benzimidazole proton (H3) and the pyridine nitrogen (N1). Additionally, potential energy scans for rotation about the bonds connecting the pyridine-benzimidazole and benzimidazole-benzimidazole fragments were performed. This revealed surprising conformational rigidity existing in the o- isomer that resisted any out-of-plane twisting of the pyridine-benzimidazole fragment. The presence of intramolecular H-bonding was further confirmed by experimental determination of pKa of the three isomers. The molecules being bisbenzimidazole derivatives bound to the minor groove of ds-DNA, the benzimidazole protons forming hydrogen bonded interactions with the DNA bases. However in the o- derivative, the intramolecular hydrogen bonding made the crucial benzimidazole protons unavailable for DNA binding thereby leading to its poor interaction with DNA. Chapter 3. Novel Series of Anthra[1,2-d]imidazole-6,11-dione Derivatives: Synthesis, DNA Binding and Inhibition of Topoisomerase I of Leishmania donovani This chapter describes the synthesis of nine imidazole fused anthraquinone derivatives and their interaction with double-stranded DNA, investigated by UV-visible absorption spectroscopy and viscometric titrations. Figure 2. Molecular structures of the imidazole fused anthraquinone derivatives. All the molecules showed intercalative mode of binding to double stranded DNA, though their relative binding affinities were different. Next their inhibitory effects on the catalytic activity of topoisomerase I enzyme of Leismania donovani were investigated. L. donovani is the causative agent for human visceral leishmaniasis; a fatal disease affecting liver and spleen. Five out of the nine derivatives tested, proved to be extremely efficient inhibitors of the enzyme. Of them, three showed greater inhibition potency than camptothecin, a well-established topoisomerase I inhibitor and the precursor for several clinically useful anti-tumor drugs. The molecules were shown to inhibit by the stabilization of enzyme-DNA cleavable complex, and the inhibition efficiency was found to be highly dependent on the pKa of the side-chain nitrogen. These results provide useful insights towards developing more potent inhibitors of the parasitic enzyme. As the compounds are synthetically facile, chemically stable and possess long shelf life, they should be attractive candidates for design of novel family of topoisomerase I inhibitor. Indeed the nature of amine based side chain and its pKa would hold the key in such design. Chapter 4 deals with a series of symmetrical bisbenzimidazole derivatives in which the benzimidazole units have been connected via different aromatic linkers. The syntheses, duplex DNA interaction, topoisomerase inhibition and quadruplex DNA stabilization shown by these four molecules have been divided into two sections. Chapter 4A. Synthesis, Duplex DNA Binding and Topoisomerase I Inhibition by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. This chapter describes the synthesis of four symmetrical bisbenzimidazole derivatives bearing aromatic linkers, phenyl, naphthyl or anthryl between the benzimidazole rings. Next their interaction with duplex DNA was investigated using fluorescence and temperature dependent UV absorption spectroscopy and viscometric titration techniques. Addition of DNA caused fluorescence enhancement of the molecules implying their interaction with duplex DNA. All the four molecules on binding to double helical DNA induced thermal stabilization of the latter. Viscometric titration of calf thymus DNA with the four compounds revealed a partial-intercalative mode of binding for the anthracene derivatized molecule 4. Next, their inhibitory effects on the catalytic activity of topoisomerase I enzyme were studied. The anthracene derivatized compound (4) showed high inhibition of the enzyme catalyzed relaxation of supercoiled plasmid DNA. Naphthalene derivatized compound (3) exhibited weak inhibition whereas the derivatives bearing 1,4- and 1,3-disubstitued benzene (1 and 2 respectively) units showed no inhibition. Figure 3. Molecular structures of the symmetrical bisbenzimidazole derivatives. Chapter 4B. Quadruplex DNA Stabilization by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. The ability of the aforementioned molecules to stabilize G-quadruplex structures was investigated next. DNA quadruplex secondary structures are potential molecular targets for new generation chemotherapeutic drugs; hence there is an impetus in developing quadruplex targeting molecules. The Tetrahymena thermophilia telomeric sequence 5´-(T2G4)4-3´ was selected for the studies as it exhibits interesting structural polymorphism depending on whether quadruplex formation occurs in presence of Na+ or K+. Circular dichroism and fluorescence anisotropy techniques were used to study the interaction of these newly synthesized molecules with quadruplex DNA. Also thermal stabilization of quadruplex structure induced by the molecules was determined by temperature dependent UV absorption studies. The compounds 1, 3 and 4 stabilized Na+ induced quadruplex without causing any structural alterations of the latter. However, the m-phenyl linker bearing molecule 2, above a certain [ligand]/[DNA] concentration ratio, caused uniquestructural alteration of the Na+ induced quadruplex such that the CD-signature of the latter resembled that of a K+ induced quadruplex structure. This result was corroborated by quadruplex thermal melting data and fluorescence anisotropy. Interestingly this ligand was also able to induce secondary structure formation in randomly oriented ss-DNA, akin to K+ induced quadruplex structure, even in the absence of Na+ or K+. Chapter 5. Synthesis and DNA Binding of Novel Biscationic Dimers of Bisbenzimidazole Systems. This chapter describes the design, synthesis and ds-DNA binding properties of four dicationic dimers of bisbenzimidazoles. Targeting long base pair sequences in double helical DNA is a key issue in chemical biology and connecting different DNA binding modules by appropriate linkers is an attractive strategy for achieving the same. The precursor monomer unit was a bisbenzimidazole derivative and an analogue of Hoechst 33258. Two such moieties were connected via bisoxyethylenic or 6- or 3-methylenic or piperazinyl units to achieve linker of varying length, rigidity and hydrophilicity. To study the interaction of the dimers with duplex DNA, fluorescence and circular dichroism spectroscopy were used. Two of the dimers, (bbim-2ox-bbim and bbim-6met-bbim) bearing long flexible spacers, were able to target 13-AT base pairs long oligonucleotide sequences in a 1:1 binding mode with an affinity 8-10 times better than the precursor monomer or Hoechst 33258. Also thermal denaturation experiments showed high duplex stabilization induced by the same two dimers. All studies indicated a bidentate mode of binding where both the arms of the dimers participated in DNA binding. The molecules bearing the short and rigid linkers (bbim-3met-bbim and bbimpiper- bbim) on the other hand showed low binding affinity towards duplex DNA, as indicated by fluorescence, circular dichroism and thermal melting studies. The short linkers probably did not favor simultaneous binding of both the monomeric arms of the dimers to DNA minor groove. The work reported in this chapter indicates the strong influence of the length and nature of linker in determining drug/DNA binding affinity. Figure 4. Molecular structures of dicationic dimeric bisbenzimidazole derivatives.(Refer PDF File)

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.