Dissertations / Theses on the topic 'DNP derivative'

The discovery of new compounds with antitumoral activity has become one of the most important goals in medicinal chemistry. One interesting group of chemotherapeutic agents used in cancer therapy comprises molecules that interact with DNA. Research in this area has revealed a range of DNA recognizing molecules that act as antitumoral agents, including groove binders, alkylating and intercalator compounds. DNA intercalators are molecules that insert perpendicularly into DNA without forming covalent bonds. The only recognized force that maintain the stability of the DNAintercalators complex, even more than DNA alone, are van der Waals, hydrogen bonding, hydrophobic, and/or charge transfer forces. These molecules have attracted particular attention due to their antitumoral activity. For example, a number of acridine and anthracycline derivatives are excellent DNA intercalators that are now on the market as chemotherapeutic agents. However, the clinical application of these and other compounds of the same class has encountered problems such as multidrug resistance (MDR), and secondary and /or collateral effects. These shortcoming have motivated the search of new compounds to be used either in place of, or in conjunction with, the existing molecules. Along these lines, especially important are the ligands capable of structure- or sequence-selective binding to nucleic acids, since such compounds may purposefully influence the biological functionality of genetic material in vivo. The condensed poly(hetero)aromatic compounds are usually regarded as representative DNA intercalators, especially if they posses electron-deficiency or charged aromatic cores. However, only a few ligands are known that bind to the DNA by the intercalative mode exclusively. A vast number of ligands, which have an intercalating part endowed with a variety of substituents, bind to the DNA by a mixed mode, since the substituents occupy the DNA grooves upon binding. In view of the complexity of the ligand-DNA recognition process, a study with model compounds which posses only one DNAbinding mode is desired. Measurement of the binding constant and biological activity of DNA-intercalator complexes in the 1970’s and QSAR studies in the 1980’s, leads to the conclusion that there should exist a relationship between cytotoxic activity and binding force. Otherwise, cytotoxicity is not only dependent on the ability to interact with DNA, since there are many DNA intercalators that are incapable of working as cytotoxic agents. To be effective, a drug must first overcome many barriers, including metabolic pathways, and cytoplasmatic and nuclear membranes. Once drug is situated in the nucleus, it must be capable of interacting with DNA by intercalating, that is forming a stable complex with a relatively long half-life. Cytotoxicity could be also a consequence of the poisoning of topoisomerases, enzymes that are directly involved in DNA recognition, in the fundamental steps of cellular growth. The spatial arrangement of DNA before, during, and after replication is essential to a high-quality cell division process. In this way, DNA topology is governed by these enzymes. The enzymes can be classified into two main classes: type I, which breaks only one strand of the DNA, although both strands are involved in the interaction with the enzyme, and type II, which breaks both strands of the duplex. They are both a good leads for DNA intercalators, which induce cytotoxicity when they poison the enzymes by stabilizing the ternary, DNA– intercalator–topoisomerase complex in such a way that the enzymatic process cannot continue forward or backward. Finally, once the enzyme–DNA complexes are poisoned by intercalators, the ternary complex is detected by the cell as a damaged portion, which triggers a series of events, which induces cell apoptosis (programmed cell death). In recent years much interest has been focused on molecules that may bind and modify genetic material. Along these lines, there has been increasing attention in the discovery and investigation of compounds that cleave DNA when irradiated with visible or UV light. These molecules are called photonucleases and they exhibit a large potential for therapeutic applications because they are often inert until activated by light and allow control of the reaction both in a spatial and temporal sense. The photonucleases operate by several distinct mechanism. One class of compounds photosensitizes the excitation of reactive intermediates that react with DNA, such as singlet oxygen, or the hydroxyl radical. In a second class, the photonuclease is bound to the nucleic acid before its activation and the DNA damage is thus localized at or near the binding site. These compounds, like any other small DNA-binding molecule, associate by intercalation or fit into the minor groove of the DNA. So, the photosensitized damage of DNA offers a promising tool to destroy DNA on purpose and may have a photobiological effects as they can be applied as phototherapeutics. The photosensitization of cells and tissue using photoactive drugs has been exploited in a variety of phototherapies for the treatment of multiple diseases. In fact in the last 20 years there was the development of dyes for photodynamic therapy, in particular porphyrins and porphyrins-based compounds, or new psoralen derivatives to apply for the well known PUVA therapy. Moreover photosensitization approaches have also been investigated for antimicrobical use, disinfections of blood products, as well as for wound closure in photochemical tissue bonding. Among the compounds investigated along these lines are quinolizinium derivatives such as coralyne and the related molecules. Moreover was recently observed that the tri- and tetra-benzoquinolizinium derivatives and indoloquinolizinium exhibit DNA-binding and, after UV-A irradiation, DNA-photodamaging properties. However, other examples for DNA-binding quinolizinium derivatives with photonuclease activity are still rare. The compounds analyzed in this project are a penta- and hexacyclic derivatives of quinolizinium ion, namely, diazoniapentaphene derivatives, diazoniaanthra[1,2- a]anthracenes, diazoniahexaphene and a partly saturated hydroxyl-substituted diazoniapentaphene. The investigations of these compounds allow to evaluate both the influence of the position of the positive charge and if the extension of ? system may enhance the interaction between DNA base pair. Finally, biological studies are carried out, because their cytotoxic and photocytotoxic activity was never been consider before.

The crystalline compound resulting from thermal cyclization of the Baylis-Hillman product, methyl 3-hydroxy-2-methylene-3-(2-pyridyl)propanoate, has been identified as the indolizine derivative, methyl indolizine-2-carboxylate, and this approach involving the reaction of pyridine-2-carboxaldehydes and acrylate analogues has been established as a general route to 2-substituted indolizines. The ease of cyclization the Baylis-Hillman products to indolizines has been shown to increase by converting the hydroxy group to an acetoxy group, and a range of acetylated Baylis-Hillman products were prepared and cyc1ized to the corresponding 2-substituted indolizines, generally in good overall yield. In the reaction of pyridine-2-carboxaldehyde and methyl vinyl ketone, the intermediate cyclized readily and directly to the corresponding indolizine. One- and two-dimensional ¹H and ¹³C NMR analysis of the 2-substituted indolizine products has permitted complete assignment of all ¹H and ¹³C NMR signals, as well as the measurement of all coupling constants for these compounds. A kinetic and mechanistic study has been conducted on the Baylis-Hillman reaction using ¹H NMR spectroscopy. A range of substrates has been examined and the reaction has been found to be third-order overall. A mechanism involving an addition - elimination sequence is proposed, which fits the kinetic data and accounts for observed substituent effects. Reaction of N,N-dimethylacrylamide with pyridine-2-carboxaldehyde in the presence of the tertiary amine catalyst, DABCO, in chloroform, yielded an unexpected product which has been identified by single crystal X-ray diffraction analysis as 1-(2,2,2-trichloro-1-hydroxyethyl)pyridine. Attempted extension of the general indolizine route to the preparation of chromene systems by reacting salicylaldehyde with methyl acrylate in the presence of DABCO, also led to an unexpected, crystalline material, identified by single crystal X-ray diffraction analysis as the coumarin derivative, 3-[(2-formylphenoxy)methyl]coumarin.A series of chloroquine analogues have been prepared from indolizine-2-carboxylic acid, pyrrolo[I,2-a]quinoline-2-carboxylic acid and imidazo[I,2-a]pyridine-2-carboxylic acid by reaction with suitable amines in the presence of the coupling reagent 1, I' -carbonyldiimidazole. This route has been shown to be a vast improvement on earlier procedures and has provided access to both secondary and tertiary indolizine-2-carboxamides. A range of N,N-dialkylindolizine-2-carboxamides have been prepared by this route, and the influence of substituents on their N-CO rotational energy barriers has been determined using variable temperature ¹H and ¹³C NMR techniques. Intercalation with natural DNA by both chloroquine and the synthesized chloroquine analogues has been examined using UV spectrophotometry, and ¹H and ³¹P NMR spectroscopy. The pyrrolo[I,2-a]quinolines have been shown to be DNA intercalators with binding affinities similar to that of the known antimalarial intercalator, chloroquine. In a preliminary study the synthesis of a short oligonucleotide has been undertaken and changes have been observed in the ¹H and ³¹P NMR spectra of the oligonucleotide on addition of the intercalator, chloroquine.

The Ph.D work was aimed to design, synthesize, and test capabilities of new spermine-porphyrin and BODIPY derivatives, and to study their interactions with various biological targets. The research period was focused on the synthesis of porphyrin derivatives with a different number of spermine pendants, in order to combine the hydrophobic and hydrophilic behaviour in a single molecule with the possibility to modulate the two properties, but meanwhile also to have biocompatible features. In particular, polyamine arms make these compounds quite soluble in aqueous solution and let them to interact with the anionic backbones of different DNA structures, displaying a different protonation degree depending on pH. The results obtained by CD-measurements, UV-Vis, fluorescence and RLS spectroscopy support the good ability of these derivatives (i.e. ZnTCPPSpm4) to act like inducer, probe and stabilizer towards the Z-form of DNA. Moreover, they show a good affinity with G-Quadruplex (i.e. H2TCPPSpm4) opening the way to several supramolecular structures. In addition, these porphyrins are able to self-assembly under hierarchical control, obtaining the desired aggregation state in solution, by choosing the appropriate pH value. The collected results do not allow to propose a definitive mechanism of interactions between porphyrin-porphyrin, and porphyrin-DNA and therefore further work is in progress in order to get a rationale which can help in designing ad hoc molecules to selectively interact with the target biomolecules.

The integrity of DNA in living organisms is permanently threatened by many endogenous and exogenous factors. Damages can lead to cell death and serious diseases and have to be repaired by the organism. The focus of this thesis is the investigation of the ability of high (Bst Pol I, Klenow fragment exo-) and low fidelity polymerases (Pol kappa, Pol eta) to insert and bypass several oxidatively-derived purine-lesions, such as 8-oxopurines (8-oxodA, 8-oxodG) and formamidopyrimidines (FaPydA, FaPydG), but also guanine-derived imidazolone (dIz) and its further degradation products oxazolone and guanidinoformimine. In order to support the biochemical data and to reveal the replication mechanism of a high fidelity polymerase with FaPy-lesions we performed co-crystallization studies with Bst Pol I. After the discovery that oxidative modifications of the base methylcytosine (mC) occur naturally within the genome and play a key role during cellular development we sought to address the question of their mutagenic potential.

Thymidine dimers 16 and 18, connected by amide or N-methylamide linkages, have been prepared. The dimers were incorporated into normal strands of DNA by solid phase synthesis. Thermal denaturation studies, using complementary single-stranded RNA, indicated that these modifications caused no destabilization of the DNA-RNA duplex.<br>The block synthesis of amide-linked homotetramer 30 is described. The synthesis of the corresponding octamer could not be verified because of lack of solubility. One by one homologation was found to be a suitable method for the preparation of N-methylamide analogues.<br>Poly(ethylene glycol), covalently attached to the 3$ sp prime$ or 5$ sp prime$ end of amide-backbone thymidine homopolymers, was found to greatly increase their solubility. The poly(ethylene glycol) simultaneously served as a soluble solid support for the homologation reactions.$ sp*$ ftn$ sp*$Please refer to the dissertation for diagram.

Guanine-rich telomeric DNA at the end of chromosomes can form a unique DNA tertiary structure - G-quadruplex, which is known to inhibit the binding of telomerase to telomeric regions in cancer cells and thus regulate unrestricted cancer cell growth. Hence, G-quadruplex DNA has recently become a promising target in oncology. The formation of G-quadruplex structures is greatly facilitated by G-quadruplex binding ligands such as Thiazole orange (TO). Compared with other G-quadruplex binding ligands, TO has an intriguing tunable fluorescence property. Upon binding to DNA, the fluorescence of TO can increase up to 1000-fold, making it an attractive probe for studying ligand-DNA interactions. However, the poor binding affinity and minimal binding selectivity towards different DNA conformations greatly limit its applications. My research focuses on developing G-quadruplex binding ligands using TO as a scaffold. In the first part of this work, we investigated the feasibility of increasing the TO binding affinity and selectivity toward G-quadruplex DNA by introducing side chains to the molecule. TO derivatives containing various side chains were successfully synthesized and characterized. Biophysical and biochemical studies with duplex and G-quadruplex DNA showed that tethering side chains to TO is an effective approach to tune its ability of binding to duplex or G-quadruplex DNA. Possible binding modes of the effective derivatives were studied using AutoDock. Their inhibition of telomerase activities was studied using the TRAP assay. The cytotoxicity of these derivatives toward three cancer cell lines was also investigated using the MTS assay. The second part of this work focuses on development of TO-based G-quadruplex DNA binding ligands that can bind to DNA via the dual recognition mode. TO was tethered with pyrene, naphthalene diimide, and anthraquinone respectively to yield three novel conjugates. Further investigation suggested that the conjugate of TO with naphthalene diimide (TO-NF) gave the best G-quadruplex binding affinity. It binds to G-quadruplex DNA via the end stack mode and strongly inhibits the telomerase activity. The cytotoxicity results will also be discussed in this presentation.

Breast cancer is the most commonly occurring cancer in women, with incidence rates approaching 1.38 million cases per year worldwide. Over the last few decades, there have been numerous attempts to develop, synthesise and advance into the clinic novel and selective breast cancer therapies. Research work has shown that bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) exerts potent in vitro anti-cancer activities and strong DNA binding properties. The aim of this thesis was to synthetise novel bisnaphthalimidopropyl derivatives (BNIPs) and investigate their subsequent modes of action within two human metastatic breast cancer cell lines, MDA-MB-231 and SKBR-3. A series of novel BNIPs, bisnaphthalimidopropyl-piperidylpropane (BNIPPiProp), bisnaphthalimidopropyl- ethylenedipiperidine (BNIPPiEth) and (trans(trans))-4,4’-methylenebis-cyclohexylamine (trans,trans-BNIPDaCHM) were synthesised, characterised and studied in comparison to BNIPDaCHM for their DNA binding and anti-cancer activities against MDA-MB-231 and SKBR-3 cells. Thermal denaturation studies have shown that BNIPs can intercalate and stabilize the double helix of Calf Thymus, each BNIP can competitively displace EtBr from DNA in a dose dependent manner and by UV binding studies, high affinity was found for the three novel BNIPs. After 24 hours treatment, all novel BNIPs, exhibited strong cytotoxicity with IC50 values ranging from 1.4 μM to 3.3 μM in MDA-MB-231 cells and 0.2 - 0.7 μM in SKBR-3 cells, confirming the importance of bisnaphthalimidopropyl functionality. BNIPs were also found to increase intracellular ROS levels after 8 hours treatment and induce a significant increase in DNA strand breaks compared to endogenous levels, after 24 hour treatment in both cell lines. After cell synchronisation, cell cycle distribution was studied, revealing that trans,trans-BNIPDaCHM induces sub-G1 cell population arrest in MDA-MB-231 and SKBR-3 cells, after 24 hours treatment. In addition, BNIPs induced apoptotic phosphatidylserine exposure, after 0.5 hours treatment, inhibited Caspase-3 activity and increased autophagy, after 24 hour treatment in MDA-MB-231 and SKBR-3 cells. Moreover, BNIPs inhibited histone deacetylases (HDAC) activity after 24 hours treatment in MDA-MB-231 and SKBR-3 cells and BNIPDaCHM was identified as a potential SIRT2 inhibitor, in SKBR-3 cells. According to Proteome Profiler Arrays, BNIPDaCHM and BNIPPiEth altered the expression of cell stress-related proteins in a cell dependent manner and bioinformatic analysis revealed two novel, putative pathways for BNIP-induced oxidative stress-mediated cell death in MDA-MB-231 and SKBR-3 cells. The above findings indicate that BNIPs represent promising candidates for future breast cancer studies and cancer treatment.

The binding of 9-hydroxyellipticine (9-OHE), Hocchst-33258 (Hoechst) and trans-bis-(4- N-methylpyridiniumyl)diphenylporphyrin (t-112P) to DNA has been studied by means of spectroscopic techniques. The binding modes of t-H2P to calf thymus DNA (ct-DNA), poly[d(G-C)12 and poly[d(A-T)12 were dependent on ionic strength, ligand load on DNA and the base composition at the binding site. At low ionic strength, flow linear dichroism (LD) data suggested t-H2P binds to ct-DNA and poly[d(A-T)12 in an orientation consistent with groove binding and to poly[d(G-C)12 in an orientation consistent with partial intercalation. Interpretation of the spectroscopic data was facilitated by molecular modelling. At high ionic strength, circular dichroism (CD) and resonance light scattering (RLS) measurements indicated the formation of extended t-H2P aggregates on DNA. In addition, the approximate, average transition moment polarisations of t-H2P were assigned from stretched film LD data to facilitate interpretation of the flow LD data. The interaction of Hoechst with poly[d(G-C)12 and poly[d(A-T)12 was studied with CD, LD, RLS and fluorescence spectroscopy. The mode of Hoechst binding to poly[d(G-C)]2 was ligand load independent. The collective spectroscopic data suggested abinding mode in which part of the molecule intercalates and part lies extensively stacked in the poly[d(G-C)12 major groove. Hoechst binding to poly[d(A-T)12 was ligand load dependent. The molecule was observed to bind as dimers or small oligomers at the lower ligand loads studied, with extended aggregates being formed as the concentration of bound Hoechst was increased. LD measurements suggested a binding orientation inconsistent with minor groove binding thus it was concluded that the molecule stacked in the major groove. 9-OHE binding to ct-DNA, poly[d(G-C)12 and poly[d(A-T)12 was studied with LD, RLS and normal absorption spectroscopy. Binding by intercalation was shown to occur with each of the three DNAs. The intercalated mode was shown to be complemented at high ligand load by a stacked binding mode. LD data pertaining to the stacked mode were inconsistent with minor groove binding in which the 9-OHE plane lies at 45' to the helix axis, thus it was concluded that the stacking occurred in the major groove. The stacked binding mode occurred most readily on poly[d(G-C)12. The flow LD data were complemented with film LD measurements from which the approximate, average transition moment polarisations were assigned.

Since cyclothialidine was discovered as the most active DNA gyrase inhibitor in 1994, enormous efforts have been devoted to make it into a commercial medicine by a number of pharmaceutical companies and research groups worldwide. However, no serious breakthrough has been made up to now. An essential problem involved with cyclothialidine is that though it demonstrated the potent inhibition of DNA gyrase, it showed little activity against bacteria. This probably is attributable to its inability to penetrate bacterial cell walls and membranes. We applied the TSAR programme to generate a QSAR equation to the gram-negative organisms. In that equation, LogP is profoundly indicated as the key factor influencing the cyclothialidine activity against bacteria. However, the synthesized new analogues have failed to prove that. In the structure based drug design stage, we designed a group of open chain cyclothialidine derivatives by applying the SPROUT programme and completed the syntheses. Improved activity is found in a few analogues and a 3D pharmacophore of the DNA gyrase B is proposed to lead to synthesis of the new derivatives for development of potent antibiotics.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007.<br>Donald F. Doyle, Committee Member ; Bridgette Anne Barry, Committee Member ; Dr. Gary B. Schuster, Committee Chair ; Nicholas V. Hud, Committee Member ; Roger M. Wartell, Committee Member.

Neoplastic diseases have become one of the most important causes of death in the world. In USA, cancer is the second cause of death after the cardiovascular diseases. Therefore, the research, the discovery and the development of new compounds with antitumoral activity have become one of the most important goals in medicinal chemistry, also trying to make a selective toxicity towards the diseased or cancer cells, thus not involving the healthy cells. Many therapeutic approaches are available for the treatment of cancer in clinical use: surgery, radiotherapy are used for localized cancer; chemotherapy, hormone-therapy and immunotherapy are considered useful, as systemic treatments, for leukemia and metastatic tumours. In the chemotherapy a high number of molecules interacts with nucleic acids like groove binders, alkylating and intercalator compounds. The molecules that belong to the latter class, interact with DNA by intercalation in the base pairs through van der Waals interactions, hydrogen bonds, hydrophobic and/or charge transfer forces. Therefore, these molecules have attracted, during their development, particular attention as chemotherapeutic agents in medicinal chemistry because the consequences of DNA intercalation by exogenous molecules lead to a significant modification of the DNA structure and may result in a hindered or suppressed function of the nucleic acid in physiological processes. But the clinic application of these compounds has shown some problems such as multidrug resistance (MDR), and secondary and/or collateral effects. These shortcomings have motivated the search of new compounds to be used either in place of, or in conjunction with, the existing molecules. Condensed poly(hetero)aromatic compounds are usually regarded as representative DNA intercalators, especially if they contain electron-deficient or charged aromatic cores in the structure. Measurement of the binding constant and biological activity of DNA-intercalator complexes and QSAR studies lead to the conclusion that there should exist a relationship between cytotoxic activity and binding force. Otherwise, cytotoxicity is not only dependent on the ability to interact with DNA, since there are many DNA intercalators that are incapable of working as cytotoxic agents: to be effective, a drug must first overcome many barriers, including metabolic pathways, cytoplasmatic and nuclear membranes. Cytotoxicity could be also a consequence of the poisoning of topoisomerases, enzymes that are directly involved in DNA recognition and that regulate DNA topology. They induce cytotoxicity when they act as poisons towards the enzymes by stabilizing the ternary DNA-intercalator-topoisomerase complex in such a way that the enzymatic process cannot continue forward or backward. This complex is detected by the cell as a damaged portion, which triggers a series of events such as cell apoptosis. Some compounds, called photonucleases, which induce DNA damage after UV-VIS-irradiation, have become interesting; while the association of cationic dyes to DNA is a reversible process, the DNA damage, which frequently occurs on irradiation of ligand-DNA complexes, is often irreversible. The latter DNA damage may lead to cell death or mutation, and must be avoided in healthy systems. However, this photoinduced DNA-damage may be applied in photochemotherapy to remove unwanted cells. Among the compounds investigated along these lines, the quinolizinium derivatives, such as coralyne and the related molecules, have attracted particular attention. They are arenes containing quaternary bridgehead nitrogen atom and have been shown to bind to DNA and may be employed as a central unit in DNA-targeting drugs. During the studies of the influence of the substituition pattern of quinolizinum derivatives on their intercalation with DNA, it has been shown that the chemical structure of the tetracyclic naphtho[1,2-b]quinolizinium bromide 2 has interesting properties with respect to the binding to nucleic acids. In particular, these intercalators may exhibit a stronger interaction with nucleic acids as compared with the tricyclic benzo[b]quinolizinium 1: the additional benzene moiety extends the surface of the planar chromophore and increases the  stacking between the dye and the DNA bases, resulting in higher binding constants. Other important aspects are represented by the photobiological properties: it was shown that an efficient DNA-strand cleavage is photoinduced by the naphtho[1,2-b]quinolizinium bromide 2. The compounds synthesized and analyzed in this project were 3-aryl-substituted-naphtho[1,2-b]quinolizinium derivatives; then studies about the DNA-binding properties and cytotoxic activity were carried out. The investigation of these compound allows to evaluate the effects of the extension of  system, by the introduction of fourth aromatic ring, and the effects of the substituent in position 3. This position was chosen for structural analogy with some tricyclic benzo[b]quinolizinium 1, with better biological activity with respect to the not-substituted compound. After these, experiments in comparison to the naphtho[1,2-b]quinolizinum bromide 2, without substituents in position 3, to investigate preliminary molecular target (topoisomerase I and II), to attempt a structure-relactionship-activity and finally photobiological tests will be carried out.<br>Le neoplasie risultano essere una delle più importanti cause di morte nel mondo: negli Stati Uniti il cancro rappresenta la seconda causa di morte dopo le malattie cardiovascolari. Quindi la ricerca, la scoperta e lo sviluppo di nuovi composti a potenziale attività antitumorale è considerato uno dei più importanti obiettivi in campo della chimica farmaceutica, cercando anche di distinguere in termini di citotossicità le cellule sane da quelle cancerose e malate. Ad oggi, molti approcci terapeutici sono disponibili per il trattamento del cancro in ambito clinico: chirurgia, radioterapia sono usate nel trattamento di tumori localizzati; chemioterapia, terapie ormonali, immunoterapia si sono invece rivelate utili nella cura di leucemia e tumori metastatici. Nell’approccio chemioterapico un alto numero di molecole interagisce con gli acidi nucleici come groove binders, agenti alchilanti e intercalanti. Le molecole che appartengono a quest’ultima classe interagiscono con il DNA intercalandosi appunto fra le coppie di basi attraverso interazioni di Van der Waals, legami idrogeno, legami idrofobici e/o interazioni di carica. Molte di queste molecole hanno suscitato particolare interesse durante il loro sviluppo come potenziali agenti chemioterapici perché è noto che una conseguenza dell’intercalazione da parte di molecole esogene nella doppia elica è proprio una modifica strutturale e chimico-fisica della struttura che vede come risultato un’alterata o arrestata funzione del DNA nei processi fisiologici. Ma l’applicazione clinica di questi composti ha mostrato problemi in termini di insorgenza di resistenze (MDR), effetti secondari o collaterali. Questi inconvenienti hanno spinto alla ricerca di nuovi composti che potessero sostituire o migliorare i composti già esistenti. I poli(etero)cicli aromatici condensati sono solitamente considerati buoni agenti intercalanti, specialmente se nel core aromatico presentano cariche positive. Gli studi sulla valutazione quantitativa della costante di binding e sull’attività biologica dei complessi DNA-agenti intercalanti e di QSAR hanno portano alla conclusione che potrebbe esistere una relazione tra attività citotossica e forza di binding. Comunque la citotossicità non è soltanto dipendente dall’abilità a interagire con il DNA, dal momento che alcuni intercalanti si sono rivelati anche non citotossici: infatti per poter esercitare il suo effetto, un farmaco deve prima passare barriere, vie metaboliche, membrane citoplasmatiche e nucleari. La citotossicità può anche essere conseguenza dell’azione di veleno contro le topoisomerasi, enzimi direttamente coinvolti in processi che coinvolgono gli acidi nucleici in fondamentali steps della crescita cellulare e regolano la topologia del DNA. Gli intercalanti del DNA possono esplicare un’azione citotossica attraverso stabilizzazione del complesso DNA-intercalatore-topoisomerasi in modo che il ciclo biochimico venga bloccato. Questo complesso con il DNA danneggiato poi può essere riconosciuto dalla cellula che attiva una serie di vie biochimiche che portano all’apoptosi. Si sono rivelati anche interessanti quei composti, chiamati fotonucleasi, che inducono danno al DNA dopo irradiazione con luce UV-VIS; mentre l’associazione di cationi organici al DNA è un processo reversibile, il danno al DNA generato dopo irradiazione del complesso ligando-DNA è spesso irreversibile e questo può portare a morte cellulare, mutazioni che dovrebbero essere evitati in sistemi sani. Comunque, un fotodanno al DNA potrebbe essere applicato in fotochemioterapia al fine di rimuovere cellule malate. Tra i composti studiati in tutto questo contesto, i derivati del chinolizinio, come la coralina e molecole correlate, hanno suscitato particolare interesse. Essi constano di anelli aromatici condensati con una carica positiva sull’azoto quaternario e hanno mostrato di legare il DNA. Durante gli studi sulle modifiche chimiche del chinolizinio in termini di legame al DNA, è stato mostrato che la struttura chimica tetraciclica del nafto[1,2-b]chinolizinio bromuro 2 presenta interessanti proprietà di binding agli acidi nucleici: in particolare viene osservata una forte interazione se comparata al derivato triciclico benzo[b]chinolizinio 1, dovuta all’addizione del quarto anello aromatico che estende la superficie planare del cromoforo ed aumenta le interazioni  tra il composto e le basi del DNA. Altro aspetto importante è rappresentato dalle attività fotobiologiche: è stato mostrato che esiste un efficiente taglio del DNA fotoindotto appunto dal nafto[1,2-b]chinolizinio bromuro 2. In questo progetto di ricerca sono stati analizzati e sintetizzati derivati 3-aril del nafto[1,2-b]chinolizinio; e successivamente sono stati effettuati studi per determinare la tipologia di binding al DNA e l’attività citotossica. Particolare attenzione è stata riposta sulla valutazione dell’effetto dell’introduzione del quarto anello aromatico e della sostituzione in posizione 3. Quest’ultima posizione è stata scelta in analogia a alcuni derivati del triciclico benzo[b]chinolizinio 1 con migliore attività biologica rispetto alla molecola di partenza. Successivamente prendendo come riferimento il nafto[1,2-b]chinolizinio bromuro 2, senza quindi sostituenti in posizione 3, si sono condotti studi preliminari sull’identificazione, oltre agli acidi nucleici, di un probabile target molecolare (topoisomerasi I e II); si è cercato di ipotizzare una relazione struttura-attività e infine di valutare l’attività fotobiologica.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.<br>Vita. Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>Linear polyethylenimine (PEI) is the "gold standard" of polycationic gene delivery vectors. However, little focus has been placed on enhancing or understanding the specificity of PEImediated gene delivery. Herein we evaluated the effect of chemical modification on the specificity of PEI-mediated nucleic acid delivery. We found that low molecular weight PEI (2 kDa) does not mediate efficient gene expression while high molecular weight (> 87 kDa) leads to toxicity. However, linear PEI of 25 kDa is an efficient gene delivery vector for both DNA and siRNA. Therefore, this PEI was chemically modified to explore the relationship between structure and specificity. First, PEI was covalently attached to a monoclonal anti-angiotensin I-converting enzyme (ACE) antibody (PEI-9B9) and evaluated for its ability to target PEI-9B9 polyplexes following intravenous delivery in a rat. Although mAb 9B9 retains affinity for its substrate ACE, PEI-9B9 does not enhance delivery to its intended target, the lung. Clearance of PEI-9B9 from circulation likely occurs before antibody binding to the surface expressed antigen. Next, we evaluated the ability of hydrophobic modification to modulate specificity of PEIbased gene delivery. Linear PEI was alkylated with variable length hydrocarbon chains at varying percent modification and evaluated for effective and specific gene delivery following intravenous delivery in mice. Modest alkylation (11% modification with ethyl chains to produce N-ethyl-PEI) enhances gene delivery in the lung 26-fold while quadrupling the ratio of gene product expressed in the lung relative to other organs. Interestingly, specificity profiles of the various alkyl chain derivatives vary among the organs examined. Additionally, a topical approach to gene delivery was investigated. Small branched PEI was cross-linked to gold to create PEI-gold nanoparticles (PEI-GNPs). These polycations were complexed with DNA and delivered topically to scratched rabbit cornea. PEI-GNPs effectively transfected corneal endothelium and evoked expression of the plasmid DNA without causing significant immunogenicity or toxicity. Finally, the effect of radiation on biologics was evaluated using a rigorously controlled experimental design with extreme conditions to unequivocally determine if radiofrequency radiation (RFR) has a non-thermal effect on biologics. Neither enzymes nor living cells (both bacterial and mammalian) were affect non-thermally by RFR.<br>by Jennifer A. Fortune.<br>Ph.D.

Quinocarcin binds to d(ATGCAT)₂ with a preferred direction of 3' and the R configuration at C4 of the drug. A mode of action involving ring opening of the oxazolidine ring to form an iminium ion which can then alkylate the N2 of guanine has been reinforced by the current computer modeling study. The absolute configuration for quinocarcin should be reversed based on the fact that the optical isomer of the structure arbitrarily assigned in the literature forms a much better binding complex to DNA. Anthramycin binds to the 2-amino group of guanine but its mechanism of action proceeds through a neutral imine. The 3' direction is again favored but for this molecule, the preferred configuration is S. This computer modeling study provided a basis for a 2D NMR study which confirmed that anthramycin forms a 3'S adduct when it binds to d(ATGCAT)₂. Bisantrene and R9 are synthetic anthracene derivatives with antitumor activity. Use of UV spectroscopy provided insight into the ability of these compounds to intercalate between the base pairs of double helical DNA. Standard Scatchard plot analysis proved useless in determining the binding parameters. A McGhee-von Hippel equation was able to describe a portion of the data but a smoothing spline function was able to describe the data completely. Naphthyridinomycin studies indicate that it too prefers a covalent adduct in which the direction is 3' and the configuration is R at C7. When the noncovalent drug binds to d(ATGCAT)₂ it may bind with either the C3a face or the C7 face closest to N2 of guanine. Iminium ion mechanisms have been proposed for the binding of naphthyridinomycin to N2 of guanine in the minor groove of DNA and the computer modeling presents evidence to support such mechanisms. Saframycin A binds much better to d(GATGCATC)₂ as a hydroquinone species but the quinone can still bind in the same site. The 3' direction is clearly preferred with the R configuration at C7. The hydrogen bonding network of the hydroquinone is conserved in the noncovalent, iminium ion, and covalent 3'R models after 32 ps of dynamics. Iminium ion mechanisms have been proposed for the binding of saframycin A to N2 of guanine in the minor groove of DNA and the computer modeling presents evidence to support such mechanisms.

The synthesis and characterisation of novel bisnaphthalimidopropyl polyamine (BNIPP) derivatives, has gained pace over the last couple of years, as they have enhanced aqueous solubility, without loss of biological activity, in contrast to parent bisnaphthalimide derivatives. Recent work has shown that bisnaphthalimidopropyl spermidine (BNIPSpd) bis-intercalates to DNA, induces oxidative DNA damage, depletes polyamine levels and causes cell death, by apoptosis, in human colon cancer CaCO-2 and HT-29 cells. The aim of this thesis was to synthesise new BNIPP derivatives to highlight the important structural features required for biological activity, particularly, bisnaphthalimidopropyl functionality, and investigate their subsequent modes of action in breast cancer MDA-MB-231 and breast epithelial MCF-10A cells. Initially, work focused on determining the DNA binding affinities and biological activity of BNIPP derivatives. All BNIPP derivatives, except bisphthalimidopropyl diaminodecane (BPHPDadec) and mononaphthalimidopropylamine (NPA) (Δ Tm values of 15.8 and 10.2 °C, respectively, C50 values of > 10 μM, IC50 values of > 40 μM), exhibited strong DNA binding affinities and cytotoxic properties in both cell lines. Results indicate that BNIPP derivatives interact with DNA by bis-intercalation suggesting, therefore, that BNIPP derivatives target DNA. For the first time, an investigation into the mechanism of cellular entry, via the polyamine transport (PAT) system, was studied. However, none of the BNIPP derivatives utilised the MGBG-specific PAT system, suggesting that BNIPP derivatives utilise other modes of cellular entry. Two BNIPP derivatives, BNIPSpd and BNIPDaCHM, were further investigated, and results show that these derivatives significantly induced a dose dependent increase in DNA strand breaks from ≥ 0.1 μM, after 4 hours. BNIPSpd and BNIPDaCHM (at non toxic concentrations) also inhibited the repair of oxidative (H2O2) and methylative (MMS)-induced DNA strand breaks. Based on phosphatidylserine exposure and membrane integrity analyses, early apoptotic cell death was determined as a mode of cell death utilised by both BNIPSpd and BNIPDaCHM (5 μM), after only 0.5 hours treatment in MDA-MB-231 cells. Interestingly, BNIPDaCHM was identified, using HDAC assay kits, as a potent and selective SIRT2 enzyme inhibitor, thus, identifying, a novel structural backbone for the selective inhibition of HDAC enzymes.

The major target of ionising radiation has been determined as cellular DNA. Damage to DNA, as detected at 77K under conditions of direct damage by ESR, is localised on the bases thymine and guanine. This damage leads to single and double strand breaks, precursors of cell death and mutagenesis. In an attempt to intercept the damage at the bases, before formation of strand breaks, the use of polycations as potential drug delivery systems to DNA has been examined. Magnetic resonance techniques have been used to establish that polyamines used are present almost completely as polyammonium cations at pH 7 and to probe the interactions of a number of polycations with DNA. Sodium-NMR was used to investigate the affinity of polyamines, poly- aminothiols and transition-metal complexes for DNA, via sodium ion displacement from the DNA region. It was found that small metal complexes displace a greater number of sodium ions than polyamines of similar charge. Application of the counterion condensation theory led to a model of the counterions existing within a cylinder around the DNA of approximate radius 20A. The mode of interaction of polyammonium cations was studied using proton magnetic resonance. Linewidths, related to the transverse relaxation rate, give information on the motion of compounds close to DNA. Comparison of linewidths in the presence and absence of DNA revealed no significant broadening. This was interpreted as indicative of a loose, electrostatic interaction, not significantly hindering motion of the cations close to DNA, suggesting rapid motion of polyammonium ions along the DNA. The radioprotection of DNA by various transition-metal complexes was studied using ESR. Certain compounds exhibited protection via electron transfer, resulting in a decreased radical yield.

4′,6-Diamidine-2-phenylindole (DAPI) is a common fluorochrome that is able to bind to deoxyribonucleic acid (DNA) with distinct, sequence-dependent enhancement of fluorescence. This work presents the synthesis of a new multifunctional compound that includes the fluorescent dye as a ⁹⁹ᵐTechnetium (⁹⁹ᵐTc) carrier. A new technique for the bioconjugation of DAPI with 6-hydrazinonicotinic acid (HYNIC) through an amide linkage was developed. The radiolabelling was performed with HYNIC as a chelator and N-IJ2-hydroxy-1,1-bisIJhydroxymethyl)ethyl)glycine (tricine) as a coligand. Furthermore, experimental evidence showed that ⁹⁹ᵐTc complexes with DAPI as DNA-binding moieties are detectable in living Fischer rat thyroid follicular cell line 5 (FRTL5) and their nuclei. The investigations indicated further that the new HYNIC-DAPI derivative is able to interact with double-stranded DNA. This establishes the possibility of locating ⁹⁹ᵐTc in close proximity to biological structures of living cells, of which especially the genetic information-carrying cell compartments are at the centre of interest. In this context, further investigations are related to the radiotoxic effects of DNA-bound ⁹⁹ᵐTc-HYNIC-DAPI derivatives and dosimetric calculations.

Nucleic acids play important roles in living systems by storing and transferring genetic information and directing protein synthesis. Recently, it was found that nucleic acids can catalyze chemical and biochemical reactions similar to protein enzymes. In addition, they can also serve as drug targets for the treatment of deadly diseases such as AIDS and cancers. As a result, the 3D structure study of nucleic acids and proteinnucleic acids complexes by X-ray crystallography has become one of the most active research areas. However, the two intrinsic bottlenecks of macromolecule X-ray crystallography, including crystallization and phase determination, have significantly limited its application in study and discovery of the new structures and folds, as well as in exploration of the biological mechanisms. So far, the selenium derivatization (Se-Met) of proteins and multiple anomalous dispersion (MAD) or single anomalous dispersion (SAD) technology have revolutionized the protein crystallography field by providing a rational solution to solve the phase determination problem. Similarly, it’s important and urgent to develop a corresponding methodology for nucleic acid X-ray crystallography. The work presented here includes two general research directions: the selenium derivatized nucleic acids (SeNA) and tellurium derivatized nucleic acids (TeNA): 1) The SeNA strategy by site-specifically replacing oxygen with selenium at the 2’ and 4 positions of thymidine and uridine has been developed. We found that the selenium derivatization at both sites are relatively stable and doesn’t cause significant structure perturbations by comparing with their corresponding native counterparts. In addition to the phase determination, the 2’-Se modification can also facilitate crystal growth of many oligonucleotides. Moreover, we have observed colorful DNAs and RNAs with the 4-Se modification for the first time. 2) The TeNA strategy by covalently incorporating tellurium functionalities into different positions of nucleic acids, particularly at the 2’ and 5 position of thymidine, has been developed. We have demonstrated the compatibility of the tellurium modification and solid-phase synthesis, as well as the potential application of the tellurium modifications in anti-viral drug synthesis and DNA-damage investigation.

Nucleoside 5-triphosphates are the building blocks to synthesis of nucleic acids. Nucleic acids (RNA and DNA) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. Nucleoside 5- triphosphates have many important therapeutic and diagnostic applications. To understand how these triphosphates are utilized in living systems, numerous synthetic mimics have been prepared and used as active metabolites of certain drugs and molecular probes. Over the years, nucleic acids have been modified at the nucleobase, sugar moiety and phosphate backbone with the aim of understanding their structures and functions. We have site-specifically replaced selected oxygen atoms of nucleosides and nucleotides with selenium atom in order to enzymatically synthesize selenium-derivatized DNAs for obtaining insights into the DNA flexibility, duplex recognition and stability. Although triphosphates have important biological and medicinal significance, they are however, very difficult to synthesize and isolate in high purity and yield. There are many approaches to the synthesis of nucleoside 5-triphosphates, but there is no general strategy that allows simple and direct synthesis of nucleoside triphosphates. To face the challenges, we have developed a new approach in the absence of protecting groups to quickly and efficiently synthesized native deoxynucleoside 5-triphosphates and deoxynucleoside 5-(α- P-seleno)- P-seleno)triphosphates. Syntheses of the triphosphates containing selenium-derivatized nucleobases were also successfully accomplished. After replacing the oxygen atoms at the 4-position of thymidine and uridine, and the 6-position of guanosine, we observed most strikingly, a large bathrochromic shift of over 100 nm, relative to their native counterparts of UV absorbance of 260 nm. Consequently, the synthesized selenium base modified triphosphates are yellow. We also synthesized 2-selenothymidine and 5-methylseleno thymidine 5-triphosphates. We conducted stability study on the colored 4-selenothymidine and used the 5- triphosphate analog (4-SeTTP) as substrate for polymerase recognition. The Klenow polymerase incorporated the 4-SeTTP with efficiency equal to that of the native counterpart. Finally, 4-SeTTP was used to demonstrate UVdamage resistance of selenium-derivatized DNAs and plasmid.

Thesis (Ph. D.)--Georgia State University, 2006.<br>Title from title screen. William H. Nelson, committee chair; Thomas L. Netzel , A.G. Unil Perera, Brian D. Thoms, Gary Hastings, committee members. Electronic text (243 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Aug. 16, 2007. Includes bibliographical references.

The primary aim of this study was to investigate the potential of an aroA mutant of Pasteurella multocida B:2 (vaccine strain JRMT12) as a candidate for DNA vaccine delivery (bactofection). First, the invasive property of the vaccine strain was assessed for its interaction with different mammalian cell lines. Next, a eukaryotic expression plasmid that could be maintained in Pasteurella was modified to contain a prokaryotic reporter gene to help in determining the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of the mammalian cells. This plasmid was further developed to function with a dual prokaryotic and eukaryotic reporter system in order to demonstrate expression of the plasmid DNA in the mammalian cells. During interaction of strain JRMT12 with mammalian cell lines, the ability of the bacterium to adhere, invade and survive intracellularly was monitored and assessed. Three mammalian cell lines were used: a mouse macrophage-like cell line, J774.2; a bovine-lymphoma cell line, BL-3; and an embryonic bovine lung cell line, EBL. The JRMT12 strain was compared with strains of the wild-type P. multocida B:2 (85020), bovine P. multocida A:3, Mannheimia haemolytica A:1 and Escherichia coli XL-1 BLUE. Both P. multocida B:2 strains were capable of adhering to and invading J774.2, BL-3 and EBL cells. All of the Pasteurella and Mannheimia strains tested were able to adhere to EBL cells but only B:2 strains were taken up intracellularly in significant numbers. The vaccine strain, JRMT12 was found to survive intracellularly in EBL cells for at least 7 h although a steady decline in the number of viable intracellular bacteria was noted with time. In an invasion inhibition assay, the use of the microfilament formation inhibitor cytochalasin D suggested that the entry into mammalian cells was by an actin-dependent process. Cell viability assessment by trypan blue staining indicated that none of the bacterial strains was toxic for the mammalian cells. Upon entry into the mammalian cells the JRMT12 strain resided in a vacuolar compartment, as demonstrated by transmission electron microscopy. However, P. multocida A:3 and M. haemolytica A:1 were only found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. Further morphological assessment by TEM showed that only a low percentage of mammalian cells appeared to contain one or more JRMT12, suggesting that only certain cells in the population were capable of being invaded by, or taking up, the bacteria. Attempts were made to construct a Pasteurella eukaryotic expression plasmid using a gene sequence from the Pasteurella shuttle plasmid pAKA16, developed previously in this laboratory, and the commercial eukaryotic expression plasmid pCMV-sCRIPT, but these were only partially successful. The origin of replication gene (oriP) in the Pasteurella shuttle plasmid was isolated and sequenced. Analysis of oriP showed sequence similarity with the known origins of replication in other Pasteurella plasmids. The E. coli plasmid origin of replication (oriE) was removed from pCMV-sCRIPT and the oriP gene was ligated into the oriE-free pCMV-sCRIPT but attempts to transform the resulting plasmid into P. multocida B:2 were not successful. An alternative approach to plasmid development was made using another commercial eukaryotic expression vector, pEGFP-N1. This plasmid has the same properties as pCMV-sCRIPT but has an additional, fluorescent reporter gene under the control of a eukaryotic promoter. It was found to be able to replicate in P. multocida B:2 but positive transformants were only recovered after prolonged incubation after electroporation. The plasmid was stably maintained in strain JRMT12 for at least 14 days with or without antibiotic selection. It was also successfully transfected into EBL cells, as shown by expression of green fluorescent protein (GFP) in individual cells. The P. multocida vaccine strain JRMT12 was also able to deliver the plasmid into EBL cells, although the number of EBL cells expressing GFP after bacterial delivery was lower than by direct transfection of the plasmid. Next, plasmid pMK-Express, a Pasteurellaceae prokaryotic expression vector with a gfp reporter gene, was used. When this was electroporated into the vaccine strain, the strain was shown to express GFP maximally as measured by fluorimetry, during the early exponential phase of bacterial growth. The DsRed.M1 gene coding for red fluorescent protein (RFP) from plasmid pDsRed-Monomer was then used to replace the gfp gene in pMK-Express to make the construct pMK-RED. After electroporation of pMK-RED into the JRMT12, RFP expression was detected maximally during the early exponential phase of bacterial growth. The same strain expressing RFP could also be detected in the intracellular compartment of the EBL cells by fluorescence microscopy at 3 h post-invasion. Finally, plasmid pSRG, our so-called “traffic light” plasmid with a dual reporter system was constructed. This was made from plasmid pEGFP-N1 (with its existing eukaryotic expression system for GFP expression) and the sodRED fragment (with a Pasteurella promoter controlling the DsRED.M1 gene for RFP expression) isolated from plasmid pMK-RED. This plasmid was stable in strain JRMT12 with or without antibiotic selection for 14 passages. RFP expression from JRMT12 was detected maximally during the early exponential phase of bacterial growth. Transfection of pSRG into EBL cells gave individual cells expressing GFP. Invasion assays with EBL cells and P. multocida B:2 JRMT12 pSRG+ showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously. At 5 h post-invasion, some of the EBL cells were still harbouring RFP-expressing bacteria and at the same time expressing GFP themselves. Concurrently, some Pasteurella free-EBL cells were shown to express GFP. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine. An apparent immunosuppressive effect of P. multocida B:2 on the proliferative response to concanavalin A (ConA) of peripheral blood mononuclear cells (PBMC) had been reported by Ataei (2007). The PBMC had been taken from calves infected with P. multocida B:2 or from normal calves and treated in vitro with extracts of P. multocida B:2. In the present study, in vitro assays with PBMC from normal calves were undertaken in an attempt to confirm these findings. A cell-free extract (CFE) of the vaccine strain JRMT12 was found to suppress the subsequent proliferation of PBMC in response to ConA in a dose-dependent manner. However, the results were not consistently reproducible and the same effect could not be demonstrated with CFE from the wild-type strain 85020.

Topoisomerase I (TopoI) is an essential DNA-targeting enzyme which alters the supercoiling of DNA through a concerted process of breaking and rejoining of a DNA strand, thereby controlling the DNA topology required for replication and transcription. TopoI mediates relaxation of supercoiled DNA by creating a transient single-strand break in the DNA duplex that originates from a transesterification reaction involving a nucleophilic attack by the active-site tyrosine (Tyr723) hydroxyl group on a DNA phosphodiester bond situated at the site of cleavage. The resulting 3'-phosphotyrosine enzyme-DNA complex (“covalent binary complex” or “cleavable complex”) is then reversed, after DNA relaxation through strand passage, when the released 5'-OH of the nicked strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. These events result in the relaxation of the DNA structure, which is required during transcription or replication. TopoI is a specific target for the pentacyclic alkaloid Camptothecin (CPT), which was first isolated in 1966 from extracts of Camptotheca Acuminata, and its derivatives, known as TopoI poisons. These molecules block DNA religation, thus converting TopoI into a DNA-damaging agent. In the presence of a TopoI poison a ternary complex between DNA, an intercalator and topoisomerase is formed. Such a ternary complex is more stable than the DNA-TopoI associate, which may lead to an enhanced lifetime of the initially cleaved DNA. As a consequence of the misalignment of the free 5-hydroxyl group and the scissile tyrosine-DNA bond due to the presence of the drug, the religation of the broken strand cannot take place, i.e. the strand break persists. This activates a complex sequence of intacellular responses, that ultimately lead to cell death by apoptosis. Therefore, molecules which form such stabilized ternary complexes with DNA and TopoI exhibit a high potential as DNA-targeting anticancer drugs. Camptothecin was early shown to be clinically problematic because, in addition to its negligible water solubility, its active “ring-closed” ?-hydroxylactone form is rapidly converted under physiological conditions to the open carboxylate form, which is inactive and readily binds to human serum albumin, making it inaccessible for cellular uptake. To date, only two semisynthetic analogs of Camptothecin (Topotecan and Irinotecan) have been approved by FDA for the clinical treatment of the ovarian, small cell-lung and colon cancers. Solving crystal structures of Top1 in complex both with Camptothecin and Topotecan and with structurally different molecules (indolocarbazoles and indoloquinolines) has significantly increased the amount of structural information about the interaction between the Top1-DNA binary complex and the poison molecule. This encouraged the application of structure-based drug design to investigate and rationalize the activity of Top1 poisons and to rationally design new potential anticancer drugs. Thus, we considered the possibility to find a new pharmacophore exploiting all the available crystal structures to find the set of structural features which are in common to all of the five TopoI poisons. In order to obtain new derivatives easier to be synthesized while maintaining the pharmacophoric features required for the formation of a stable TopoI-DNA-poison ternary complex, the synthesis of simplified CPT derivatives was proposed. It is possible to suppose that a pentacyclic scaffold is not necessarily required for a molecule to effectively bind the TopoI-DNA binary complex, providing that it maintains an aromatic planar system for the intercalation and appropriate functional groups to interact with specific residues of TopoI. On the basis of this assumption, several scaffolds were designed and docking studies with different search algorithms were performed to predict the ability of simplified analogs to form stable ternary complex with TopoI and DNA. Moreover, we performed a molecular docking analysis on a series of new 5-substituted CPT derivatives to achieve more insight into the interactions of the new compounds with the binary TopoI-DNA complex. The introduction of substituents in position 5 of the CPT scaffold results in derivatives with reduced cytotoxic potency compared with the reference drugs. In general, the presence of a small lipophilic substituent is well tolerated while the modification with hydrophilic groups results in reduced affinity for the binding site. The presence of bulky groups is detrimental for cytotoxic potency because of the loss of important stabilizing interactions. Computational results indicated as a general feature that the 5-?-epimer is better tolerated in the binding site compared to the 5-?-epimer, resulting in higher biological activity. Recently, a new series of 16a-thio-CPT derivatives has been synthesized. Biological assays for this class of compounds revealed that 16a-thio-CPT derivatives have higher anticancer activity compared to their oxo-analogs, both in vitro and in vivo, and are potent TopoI inhibitors. In order to find a rational explanation to their remarkable activity, a computational analysis was performed, both for predicting elements of their pharmacokinetic behavior and for describing their binding mode through a docking analysis. Docking results did not reveal striking differences between thio- and oxo-derivatives in terms of binding mode and docking score. On the contrary, the predicted values for features such as lipophilicity, water solubility and cell permeability indicated that these properties may be responsible for a significantly different pharmacokinetic profile for the two classes of compounds. For the docking analysis of 5-substituted and 16a-thio CPT derivatives we used the quantum mechanics (QM)-polarized ligand docking (QPLD) protocol (Schrödinger software suite) with an aim to improve accuracy in docking calculations. Traditional docking methods employ an approximated physical chemistry-based representation of protein-ligand interactions, obtaining charges from a molecular mechanics force-field. The QPLD protocol uses an ab initio methodology to calculate ligand charges within the protein environment, thus taking into account the charge polarization induced by the protein environment. The work on 5-substituted and 16a-thio CPT derivative was sponsored by the pharmaceutical company Indena S.p.A., Milano, Italy. The work was carried out in collaboration with the group of Professor Arturo Battaglia (Centro Nazionale Richerche Bologna, Italy) for the synthesis of the compounds, and with the group of Professor Franco Zunino (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy), that was concerned with biological assays.<br>La Topoisomerasi I eucariotica (TopoI) è un enzima essenziale che modifica il grado di superavvolgimento del DNA mediante un processo di rottura e successiva ricongiunzione di uno dei due filamenti del DNA, regolandone lo stato topologico richiesto nei processi di replicazione e di trascrizione. Il rilassamento del DNA superavvolto indotto dalla TopoI avviene attraverso la rottura transitoria di un filamento per mezzo di una reazione di transesterificazione. Quest'ultima coinvolge l'attacco nucleo lo da parte del gruppo ossidrilico di un residuo di tirosina (Tyr723) del sito attivo dell'enzima ad un legame fosfodiesterico del DNA a livello del sito di taglio, con formazione di un complesso covalente 3'-fosfotirosina TopoI-DNA (noto anche come cleavable complex). La rotazione del filamento tagliato intorno a quello intatto rende possibile il rilassamento della molecola di DNA. Il complesso binario covalente subisce in seguito un attacco nucleofilo a livello della fosfotirosina da parte dell'estremità libera 5'-OH del lamento rotto, che permette la riformazione del legame fosfodiesterico. La TopoI è il target specifico dell'alcaloide pentaciclico Camptotecina (CPT), isolato per la prima volta nel 1966 dalla corteccia di Camptotheca Acuminata, e dei suoi derivati, noti come veleni di TopoI. Queste molecole bloccano il processo di ricongiunzione del filamento di DNA tagliato, convertendo la TopoI in un agente dannoso per il DNA stesso. Queste molecole si intercalano tra le basi del DNA a livello del sito di taglio dando luogo alla formazione di un complesso ternario con la TopoI e il DNA. La maggiore stabilità del complesso ternario rispetto al complesso binario TopoI-DNA risulta in un aumento del tempo di vita del DNA tagliato. Poiché la presenza del veleno induce il disallineamento dell'estremità libera 5'-OH del filamento rotto con il legame fosfotirosinico, il processo di ricongiunzione non può avvenire. Questo porta all'attivazione di una serie complessa di segnali intracellulari, il cui risultato ultimo è la morte della cellula per apoptosi. Di conseguenza, le molecole in grado di formare complessi ternari stabili hanno attività antitumorale. L'utilizzo clinico della CPT è ostacolato sia dalla sua scarsissima idrosolubilità, sia perchè la sua forma attiva lattonica “chiusa” è rapidamente convertita, in condizioni fisiologiche, nella forma carbossilica “aperta”, che è inattiva e si lega in elevata percentuale all'albumina serica. Attualmente solo due derivati semisintetici della CPT (Topotecan ed Irinotecan) sono utilizzati nella pratica clinica per il trattamento di cancro ovarico, polmonare e colo-rettale. La recente risoluzione, mediante cristallografia a raggi X, della struttura tridimensionale di TopoI in complesso sia con la Camptotecina e il Topotecan che con derivati strutturalmente diversi (indolocarbazolici ed isochinolinici), ha fornito chiarimenti sulle caratteristiche dell'interazione enzima-inibitore. Tali informazioni costituiscono elementi molto utili nello studio dei veleni di TopoI con un approccio di tipo computazionale il cui scopo è comprendere in che modo queste molecole interagiscono con il loro target e progettare nuovi nuovi derivati come potenziali farmaci ad attività antitumorale. È stato quindi possibile individuare un nuovo farmacoforo sfruttando le cinque strutture cristallogra che disponibili, al fine di rintracciare un set di caratteristiche strutturali comuni a tutti i veleni di TopoI. Con lo scopo di disporre di nuovi derivati più semplici da ottenere dal punto di vista della sintesi ma che mantengano tutte le caratteristiche farmacoforiche richieste per la formazione di complessi ternari stabili con TopoI e DNA, è stata proposta la sintesi di analoghi “semplificati” della CPT. Si può infatti ipotizzare che una molecola non debba necessariamente possedere uno scaffold pentaciclico per legarsi al complesso binario TopoI-DNA, purchè mantenga un sistema planare aromatico per intercalare il DNA e opportuni gruppi funzionali in grado di interagire con specifici aminoacidi dell'enzima. Sulla base di questa assunzione, diversi possibili scaffold sono stati disegnati e sottoposti a studi di docking con diversi protocolli di ricerca per predire la loro capacità di formare stabili complessi ternari con TopoI e DNA. È stato inoltre condotto uno studio di docking molecolare per una serie di nuovi derivati della CPT sostituiti in posizione 5 (5-derivati) per descrivere la loro modalità di interazione con il complesso TopoI-DNA. In generale, l'introduzione di sostituenti in posizione 5 ha come risultato una diminuzione dell'attività citotossica rispetto alla CPT. La presenza di sostituenti lipofili di piccole dimensioni sembra essere tollerata all'interno del sito di binding, mentre l'introduzione di gruppi idro lici dà luogo ad una diminuzione dell'affinità. L'introduzione di gruppi stericamente ingombranti provoca la perdita di importanti interazioni al'interno del sito di legame. I risultati computazionali indicano come caratteristica generale che l'epimero ? dei 5-derivati ha un'affinità maggiore per il sito di binding rispetto all'epimero ?. Recentemente sono stati sintetizzati una serie di 16a-tio-CPT analoghi. I saggi biologici condotti su questi composti hanno rivelato che i tio-derivati sono potenti inibitori della TopoI ed hanno attività antitumorale maggiore rispetto ai loro oxo-analoghi, sia in vitro che in vivo. Al fine di razionalizzare la maggiore attività dei tio-analoghi rispetto ai derivati tradizionali, è stato eseguito uno studio computazionale volto sia a predire proprietá chimico-fisiche rilevanti per la descrizione del possibile profilo farmacocinetico delle nuove molecole, sia a descriverne la modalità di legame nel sito del complesso binario TopoI-DNA tramite studi di docking. I risultati di docking non hanno messo in evidenza differenze di rilievo tra i tio- e gli oxo-derivati in termini di modalità di binding e di docking score. Al contrario, i valori predetti per proprietà come la lipofilicità, la solubilità in acqua e la permeabilità cellulare indicano che il profilo farmacocinetico delle due classi di composti potrebbe essere significativamente diverso. Per le simulazioni di docking condotte sui 5-CPT-derivati e sui tio-CPT-derivati è stato utilizzato il protocollo quantum mechanics (QM)-polarized ligand docking (QPLD) (implementato nella suite Schrödinger) con lo scopo di aumentare l'accuratezza dei calcoli di docking. I metodi tradizionali utilizzati per il docking molecolare utilizzano una rappresentazione chimico-fisica approssimata delle interazioni proteina-ligando, dove le cariche atomiche sono calcolate in base al campo di forza. Il protocollo QPLD utilizza invece una metodologia ab initio per il calcolo delle cariche del ligando all'interno della cavità della proteina, tenendo in considerazione la polarizzazione di carica indotta dagli aminoacidi del sito di legame sul ligando. Il lavoro relativo ai 5-CPT-derivati e ai tio-CPT-derivati à stato supportato dall'azienda farmaceutica Indena S.p.A., Milano, Italia. Il lavoro è stato condotto in collaborazione con il gruppo del Professor Arturo Battaglia (Centro Nazionale Richerche Bologna) per la sintesi dei composti e con il gruppo del Professor Franco Zunino (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano) per i saggi biologici.

Thesis (Ph. D.)--Georgia State University, 2008.<br>Title from file title page. Thomas L. Netzel, Dabney W. Dixon, committee co-chairs; David Boykin, Jerry Smith, committee members. Electronic text (250 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Nov. 20, 2008. Includes bibliographical references.

This thesis investigates how pairwise combinatorial gene and stimulus perturbation experiments are conducted and interpreted. In particular, I investigate gene perturbation in the form of knockout, which can be achieved in a pairwise manner by SGA or CRISPR/Cas9 methods. In the present literature, I distinguish two approaches to interpretation: the calculation of stimulus and gene interactions, and the identification of equality among phenotypes measured for distinct perturbation conditions. I describe how each approach has been applied to derive hypotheses about gene regulatory networks. I identify conflicts and uncertainties in the assumptions allowing these derivations, and explore theoretically and experimentally approaches to improve the interpretation of genetic interaction data. I apply the approaches to a well-studied gene regulatory branch of the DNA damage checkpoint (DDC) pathway of Saccharomyces cerevisiae, and confirm the known order of genes within this pathway. I also describe observations that seem inconsistent with this pathway structure. I explore this inconsistency experimentally and discover that high concentrations of the DNA alkylating drug methyl methanesulfonate cause a cell division arrest program distinct from a G1 or G2/M checkpoint or from DNA damage adaptation, that resembles an endocycle.

Nucleic acids are macromolecules in cells for storing and transferring genetic information. Moreover, nucleic acids, especially RNAs, can fold into well-defined 3D structures and catalyze biochemical reactions. As ubiquitous biological molecules in all living systems, nucleic acids are important drug targets, and they can also be used in diagnostics and therapeutics. Structural information of nucleic acids provides the foundation for DNA and RNA function studies. X-ray crystallography has been a useful tool for structural studies of bio-macromolecules at atomic level. There are two major problems in macromolecular crystal structure determination: phasing and crystallization. Although selenium derivatization is routinely used for solving novel protein structures through the MAD phasing technique, the phase problem is still a critical issue in nucleic acid crystallography. The covalent selenium-derivatization of nucleic acids has been proven to be a useful strategy for solving the phase problem in nucleic acid X-ray crystallography. Besides the facilitation of nucleic acid crystallography, there is also a wide range of other applications for selenium-derivatized nucleic acids (SeNA). The investigation presented in this dissertation mainly focuses on the following research subjects (1) Synthesis and characterization of selenium-derivatized nucleic acids for X-ray crystallography, especially phosphoroselenoate RNAs. They are generated and used for crystallization. (2) Application of selenium-derivatized RNA for RNA interference. Phosphoroselenoate RNAs are tested for RNAi activities. (3) Synthesis and characterization of the uridine 5’-triphosphate modified with selenium at position 4. (4) Facile synthesis and antitumor activities of selenium modified deoxyribonucleosides. MeSe-thymidine nucleosides have shown antitumor activity in cell assays.

Anthraquinone (AQ) derivatives with relatively high reduction potentials have been synthesized to afford good candidates for electron transfer studies in DNA. Electron withdrawing groups on the anthraquinone ring gave derivatives with less negative reduction potentials. The anthraquinone imide (AQI) derivatives had reduction potentials less negative than AQ derivatives. The AQI ring system was subject to base-induced hydrolysis. Water-soluble sulfonated tetraarylporphyrins have been studied in a wide variety of contexts. Herein, we report the first synthesis of a pentasulfonated porphyrin bearing an internal cyclic sulfone ring. Treatment of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) with fuming H2SO4 gave a structure consistent with initial sulfonation followed by dehydration to give a sulfone bridge between an ortho-position of one of the phenyl groups and a β-pyrrole position on the porphine ring (TPPS4Sc). The structure was established by ESI-MS and 1HNMR. The Soret absorption is red shifted by about 32 nm compared to that of TPPS4. Streptococcus pyogenes obtains iron by taking up heme from the environment during infection. One of the heme uptake pathways is the Sia or Hts pathway. The initial protein in this pathway is Shr, which has two heme-binding NEAT domains, NEAT1 nearer the N-terminus, and NEAT2 nearer the C-terminus. We report biophysical characteristics of these two NEAT domains. To assess stability of this domain towards heme release, denaturation studies of the Fe(II) and Fe(III) forms were performed. For each domain, both the Fe(II) and the Fe(III) forms behave similarly in thermal denaturation and guanidinium denaturation. Overall, NEAT2 is more stable than NEAT1. Spectral signatures, sequence alignment and homology modeling for both domains suggest that one of the axial ligands is methionine. NEAT2 autoreduces as the pH increases and autooxidizes as the pH decreases. Heme uptake from the host environment is the only iron acquisition pathway in S. pyogenes; inhibition of this pathway might be an approach to infection control. Compounds that might inhibit the heme uptake pathway were selected via virtual screening.

The aim of the present work lies in the analylis of two biological important enzymes: Human TopoisomeraseI and Protein Kinase A. Human Topoisomerase I (Topo I) is one of the enzyme involved into the control of the replication of the cell through the management of the topology of DNA, in particular the DNA supercoiling. Top1 relaxs supercoiled DNA operating a break in only one strand of the DNA duplex; it plays a fundamental step in the replicative process of the cell. Due to its crucial role, great efforts were made to obtain inhibitors of this enzime in order to obtain potential anticancer drug. Camptothecin (CPT), a natural pentacyclic alkaloid and its derivatives are one of the more representative class of inhibitors of Topo I. These molecules are able to intercalate inside the covalent binary complex Top1-DNA forming a reversible ternary complex Top1-DNA-CPT more stable respect to the previous one. This enhanced stability lead to a dimished possibility to religate the open strand and to the begin of the apoptotic process, when the replicative fork approaches the complex. Several efforts were made to better understand the mechanism of action of the camptothecin. We thus decided to use a spectroscopic technique to study this system, the electron paramagnetic resonance (EPR), by which is possible to register the behaviour of a paramagnetic probe placed inside the system under investigation, in particular its different mobility. Several camptothecin derivatives bringing the nitroxide moiety were synthetized followed by the synthsis of spin-labeled derivatives of a DNA oligonucleotide strand containing an high affinity cleavage site fo the topoisomerase I enzyme. Biological tests of these molecules were also performed to evaluate the ability of them to interact with the enzyme. The Protein Kinase A (PKA) is a four-membered structure with two regulatory (R) and two catalytic (C) subunits which control a wide number of biological processes of the cell, such as lipid and sugar metabolism, gene expression, cell replication and apoptosis. It controls all these processes through the phosphorilation of other substrates, such as proteins, which in turn activates other substrates until the biological response is produced (such kind of domino reaction are called enzymatic “cascade”). The activity of the PKA is controlled by the cyclic adenosine monophosphate (cAMP), a second messenger synthesized from ATP in response of an exreacellular stimulus. Failure to keep PKA under control can have disastrous consequences, including diseases such as cancer. In fact, a study conducted in the University of Padua showed an abnormal concentration of one of the regulatory subunit (RII alpha) in gliomas affected cells. Thus, to further investigate this aspect, cAMP based probe was realized using rhodamine as fluorescent moiety.<br>Lo scopo del presente lavoro consta nell'analisi di due importanti enzimi: la Topoisomerasi I umana e la Protein Chinasi A. Le topoisomerasi sono un gruppo di enzimi deputati al controllo del superavvolgimento del DNA durante la fase di replicazione cellulare. La topoisomerasi I (sottoclasse B) espressa nell‟uomo e‟ in grado di rilassare il DNA attraverso il taglio di un singolo filamento ed il successivo scorrimento del filamento intatto senza consumo di ATP. Per il suo ruolo chiave nel controllo della replicazione cellulare la topoisomerasi e‟ stata presa in considerazione come possibile bersaglio per la cura di patologie implicanti una rapida (ed incontrollata) replicazione, quali le neoplasie. Tra gli inibitori più importanti di questo enzima, spicca sicuramente la camptotecina. Questa molecola svolge la propria azione stabilizzando il complesso binario DNA-enzima attraverso la formazione di un complesso ternario DNA-enzima-camptotecina. Il danno subentra quando avviene la collisione tra la forcella replicativa e il complesso ternario: il mancato riconoscimento tra i due sistemi sfocia nella successiva attivazione del programma di apoptosi. Uno degli aspetti più interessanti di questa molecola riguarda il suo meccanismo di azione. Per ottenere ulteriori informazioni abbiamo utilizzato la spettroscopia di risonanza di spin elettronico (ESR od EPR), dove viene eccitato e successivamente registrato lo spin dell‟elettrone. Sono stati dunque sintetizzati diversi derivati della camptotecina recanti una sonda paramagnetica (in questo caso, un nitrossido) e derivati nitrossido-marcati di una sequenza oligonucleotidica di DNA con un sito ad alta affinità per la topoisomerasi I. Vengono anche mostrati i saggi biologici realizzati per valutare la loro attività nei confronti dell’enzima. Le protein chinasi cAMP dipendenti (PKA) sono enzimi dipendenti dal secondo messaggero, il cAMP (3‟-5‟-adenosina monofosfato ciclico). Questi enzimi si presentano in forma di tetrameri composti de due subunità regolatorie (R) e due catalitiche (C); il cAMP lega le subunità regolatorie delle PKA e converte quest‟ultima nella conformazione attiva grazie alla quale è in grado di trasferire gruppi fosfato da una molecola donatrice quale l‟ATP a specifici substrati. Questo processo, definito fosforilazione, è alla base di numerosi processi metabolici cellulari, quali la trascrizione, il metabolismo, il ciclo cellulare, la differenziazione, la crescita, lo sviluppo e l‟apoptosi. Un’ incontrollata regolazione della PKA può portare all’ insorgenza di patologie gravi, tra cui anche il cancro. Uno studio recente condotto dall’Università di Padova ha infatti mostrato una anomala distribuzione di una della subunità regolatorie della PKA in cellule cerebrali affette da glioma. Per investigare più dettagliatamente questo aspetto, è stata realizzata una sonda basata sulla struttura AMP ciclica recante un fluoroforo facilmente misurabile quale la rodamina.

Malgré l’intérêt et la diversité des propriétés pharmacologiques des 2,5-dicétopipérazines (DKP), les voies de biosynthèse de ces molécules d’origine microbienne sont très peu connues. L’objectif de mes travaux de thèse a été i) de documenter de nouvelles voies de biosynthèse de DKP qui se caractérisent par la présence d’une synthase de cyclodipeptides (CDPS) travaillant souvent de concert avec une ou plusieurs enzymes de modification des cyclodipeptides et ii) d’explorer la diversité chimique codée par ces voies. Dans un premier temps, je me suis intéressée aux CDPS. Après la sélection par bioinformatique de candidats dans les bases de données génomiques, j’ai pu identifier 51 nouvelles CDPS actives et montrer que ces enzymes peuvent incorporer 17 des 20 acides aminés naturels. Par ailleurs, ce travail a permis de mieux caractériser la famille des CDPS, de définir l’existence de plusieurs sous-familles aux signatures fonctionnelles spécifiques et d’établir les premiers éléments d’un code de spécificité pour la synthèse de cyclodipeptides. Dans un second temps, je me suis attachée à caractériser les enzymes de modification associées aux nouvelles CDPS et, en particulier, les dioxygénases dépendant du Fe(II) et du 2-oxoglutarate (OG) qui sont très représentées dans ces voies. J’ai ainsi pu détecter une activité in vivo pour 11 OG et poursuivre la caractérisation in vitro pour l’une de ces OG, ce qui a permis de caractériser les DKP qu’elle synthétise et d’ainsi montrer la complexité des modifications chimiques introduites. L’ensemble de ces travaux a donc permis d’identifier et de caractériser de nouvelles voies de biosynthèse qui donnent accès à une diversité accrue de DKP<br>Despite the interest and diversity of the pharmacological properties of 2,5-diketopiperazines (DKPs), the biosynthetic pathways of these microbial molecules are poorly documented. The aim of my doctoral work was i) to identify new DKP biosynthetic pathways that are characterized by the presence of a cyclodipeptide synthase (CDPS) often associated with one or more cyclodipeptide-tailoring enzymes and ii) to explore the chemical diversity encoded by these pathways. First of all, my study focused on CDPSs. After the bioinformatics-based selection of candidates, 51 novel CDPS were characterized, revealing the incorporation of 17 of the 20 proteinogenic amino acids. Moreover, this work has allowed a better characterization of the CDPS family, by showing the existence of several subfamilies with specific functional signatures and laying the foundations of a specificity conferring code for the synthesis of cyclodipeptides. Second, I characterized the tailoring enzymes associated with the newly identified CDPSs and, in particular, the Fe(II) and oxoglutarate dependent dioxygenases (OGs) that are highly represented in these pathways. I detected the in vivo activity for 11 OGs and characterized the in vitro activity for one of them, showing the complexity of the chemical modifications introduced into the cyclodipeptide. This work has led to identify and characterize novel biosynthetic pathways that provide access to a greater diversity of DKPs

Die Verpackung und Spaltung konkatemerer DNA ist ein essentieller Prozeß bei der Reifung von Virionen. Die an diesem Prozess maßgeblich beteiligten Proteine werden als Terminase bezeichnet. Die Inhibition der HCMV Terminase bietet einen attraktiven alternativen Ansatzpunkt für die antivirale Therapie. Zur Inhibition der Terminase Aktivität wurden die neuen Benzimidazol D-Ribonukleosid Derivate BTCRB und Cl4RB auf ihre antivirale Wirkung analysiert. Die neuen Benzimidazol D-Ribonukleosid Derivate BTCRB und Cl4RB zeigten sowohl gegen den HCMV Laborstamm AD169 als auch gegen klinische HCMV-Isolaten eine Wirkung. Weiterhin wurde die Wirksamkeit der Substanzen auf anderen Herpesviren getestet. Interessanterweise zeigten BTCRB und Cl4RB sowohl einen Effekt gegen Varicella-Zoster-Virus (VZV) als auch Ratten-Cytomegalovirus (RCMV), wohingegen der Effekt gegen Herpes-simplex-Virus Typ-1 (HSV-1) und Maus-Cytomegalovirus (MCMV) gering war. Infolgedessen eignen sich beide Substanzen BTCRB und Cl4RB als attraktive alternative Inhibitoren für die weitere Entwicklung einer antiviralen Therapie.<br>DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminase. The development for an inhibitor of HCMV terminase would be of great value, because it would act subsequent to DNA synthesis and block the first steps in viral maturation. Therefore we characterized two inhibitors targeting the HCMV terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetly-ß-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetly-ß-D-ribofuranosyl) benzimidazole (Cl4RB). By using viral plaque formation, viral yield, viral growth kinetics and electron microscopy, we demonstrated that two compounds BTCRB und Cl4RB are highly active against HCMV AD 169 and HCMV clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly BTCRB was active all tested herpesviruses. The best effects were observed on VZV-and RCMV-infected cells. Therefore new compounds might be promising attractive compounds for antiviral therapy in the future.

Cette thèse traite de la reconstruction d'hypersurfaces au sein de champs de normales en dimension quelconque et trouve des applications dans l’analyse des empreintes digitales (lignes dermiques), des images satellites météorologiques (lieux de turbulence) et astrophysiques (bras de galaxies) ainsi que dans l’analyse stratigraphique des images sismiques (horizons). Les méthodes développées s’appuient sur la minimisation d’une équation aux dérivées partielles non linéaire reliant une hypersurface au pendage déduit d’un champ de normales. Elles prennent en compte des contraintes diverses telles que des points de passages, des frontières, des bornes et des discontinuités. La contribution principale de la thèse réside dans l’introduction d’un changement d’espace du pendage qui permet de reconstruire aussi bien des hypersurfaces exprimées sous des formes implicites dans les repères de définition des champs de normales que des horizons sismiques de manière rapide et interactive. Deux schémas de reconstruction d’horizons sismiques unidimensionnels présentant une discontinuité d’amplitude et de lieu inconnus sont également proposés<br>This thesis deals with the reconstruction of hypersurfaces from a finite-dimensional normal vector field. Application scopes can be found in the analysis of fingerprints (epidermal ridges), meteorological images (eddies and cyclones), astrophysical images (galaxy arms) and in the stratigraphic analysis of seismic images (horizons). The hypersurfaces are obtained by solving a non-linear partial derivative equation relied on the local dip deduced from a normal vector field. Several constraints such as boundaries, bounds, points belonging to the hypersurface or discontinuities can be considered.The major contribution of this thesis consists in a local dip transformation which allows to reconstruct implicit hypersurfaces as well as seismic horizons by a fast and interactive method. Two schemes dedicated to the reconstruction of discontinuous one-dimensional seismic horizons are also proposed when the discontinuity location and jump are unknown

Notre travail s'inscrit dans le cadre de l'etude par resonance magnetique nucleaire (rmn) d'une des lesions majeures de l'adn : le site abasique. Celui-ci resulte de la perte d'une base nucleique conduisant a la formation du 2-desoxyribose ou de sa forme oxyde, la 2-desoxyribonolactone. La 2-desoxyribonolactone fait l'objet de nombreux travaux mais reste une lesion mal connue, tant d'un point de vue chimique, biologique que structural. Nous avons determine par rmn et modelisation moleculaire, la premiere structure d'un oligonucleotide contenant cette lesion. Par comparaison avec la structure du duplex de reference non modifie et caracterise de la meme facon, nous avons mis en evidence les deformations specifiques induites par la 2-desoxyribonolactone. Une deuxieme partie est consacree a la determination par rmn du mode d'interaction de composes qui reconnaissent specifiquement le site 2-desoxyribose. L'interet majeur de ces molecules est qu'elles pourraient inhiber le systeme de reparation de l'adn dans la cellule. L'etude a ete realisee sur un oligonucleotide contenant un residu tetrahydrofurane, analogue stable du 2-desoxyribose. Nous avons montre qu'une molecule de type base-chaine-intercalant qui potentialise in vitro et in vivo l'effet cytotoxique d'un agent alkylant anticancereux (le bcnu), se complexe de facon specifique avec l'adn. La base de la drogue s'insere notamment dans la loge abasique et forme des liaisons hydrogene de type watson-crick avec la thymine qui fait face a lesion. Par ailleurs, l'etude de l'interaction entre un macrocycle de type bisacridine et ce meme oligonucleotide a montre que la molecule traverse l'adn, une acridine s'intercalant dans la loge abasique, l'autre a une paire de base cote 3, et les chaines polyaminees se positionnent chacune dans un sillon. Une telle molecule constitue ainsi un bon module de reconnaissance du site 2-desoxyribose et pourrait servir de modele a la conception de molecules inhibitrices du systeme de reparation. Ce travail apporte des informations nouvelles sur les lesions abasiques de l'adn et devrait, contribuer a une meilleure comprehension des phenomenes biologiques s'y rapportant ainsi qu'au developpement d'autres molecules a activite anticancereuses.