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Orientador: Douglas Soares Galvão / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-09-11T21:00:05Z (GMT). No. of bitstreams: 1 Llanes_ScheilaFurtadoBraga_D.pdf: 1486609 bytes, checksum: 975b9d683860f2c68531081bcd9d855c (MD5) Previous issue date: 2003 / Resumo: Este trabalho envolveu a investigação da atividade biológica de fármacos por meio de descritores quânticos teóricos e a investigação de um sistema molecular de chaveamento ótico. Em uma primeira parte, apresentamos o estudo realizado para um conjunto de 20 taxóides, compostos caracterizados por uma estrutura molecular complexa, e por uma atuação diferenciada no combate ao câncer. Investigamos a estrutura destes compostos através de métodos semiempíricos partindo de seu precursor sintético 10-deacetilbaccatin III. Investigamos a relação entre a atividade experimental dos taxóides e descritores teóricos através de três metodologias de reconhecimento de padrões: a metodologia de índices eletrônicos, a análise de componentes principais e análise hierárquica de clusters. Para estas três investigações distintas pudemos correlacionar a atividade dos taxóides estudados com parâmetros teóricos, em sua maioria eletrônicos. Seguindo o mesmo tipo de análise aplicada para os taxóides, estudamos um conjunto de 41 derivados da benzo[c]quinolizin-3-onas, compostos inibidores das enzimas 5alfa-reductase humana. Aplicando os três métodos acima citados selecionamos descritores relacionados à resposta biológica destes compostos e construímos regras e padrões que os diferenciasse quanto à atividade biológica. Numa investigação especulativa, aplicando as regras e padrões construídos, sugerimos a atividade biológica para alguns compostos ainda não avaliados experimentalmente. Na segunda parte deste trabalho investigamos um sistema orgânico de chaveamento, baseado no comportamento fotocrômico de moléculas quirais. Este é um sistema de grande interesse no desenvolvimento de memórias moleculares que utilizem uma lógica binária. Utilizamos métodos semiempíricos na investigação estrutural e energética do sistema e propusemos uma rota para o funcionamento do sistema, capaz de explicar qualitativa e quantitativamente o seu comportamento / Abstract: This work deals with the structure-activity relationship investigation of pharmacological compounds using theoretical quantum descriptors, and the investigation of an optical molecular switching system. In the first part, we present a study of 20 taxoids, remarkable molecules by their complex molecular structure and dissimilar mode of action as antitumor agent. We investigated the compounds structure with semiempirical methods initiating with their synthetic precursor 10-deacetilbaccatin III. Taxoids experimental activity relationship was investigated with theoretical descriptors applying three pattern recognition methodologies: the electronic indices methodology, principal component analysis and hierarchical cluster analysis. With these three distinct methods we were able to correlate the biological activity of the studied taxoids with theoretical parameters. In a similar approach we studied a set of 41 benzo[c]quinolizin-3-ones derivatives, applied as human 5alpha-reductase inhibitors. Making use of the three methods mentioned above we selected the descriptors related to the compounds biological indices and constructed the rules and patterns able to separate them according to their activity. Considering a speculative approach, we proposed the biological activity to untested compounds, via the obtained rules. In a second part of the work we investigated an organic switching system, based on the photocromic behavior of chiral molecules. This is a system of great interest for molecular memories development based on binary logics. We applied semiempirical methods in the system structural and energetic investigations, and proposed a route able to explain qualitative and quantitatively how the system works / Doutorado / Física / Doutora em Ciências

Paclitaxel (PTX) is one of the leading drugs against breast and ovarian cancer. Due to its low solubility, treatment of the patients with this drug requires a very well-suited combination with a soluble pharmaceutical excipient to increase the bioavailability and reduce the strong side ef-fects. One efficient way to achieve this in the future could be the incorporation of PTX into pol-ymeric micelles composed of poly(2-oxazoline) based triblock copolymers (POL) which ena-bles PTX loadings of up to 50 wt.%. However, structural information at an atomic level and thus the knowledge of interaction sites within these promising but complex PTX-POL formula-tions were not yet available. Such results could support the future development of improved excipients for PTX and suitable excipients for other pharmaceutical drugs. Therefore, a solid-state MAS NMR investigation of these amorphous formulations with different POL-PTX com-positions was performed in this thesis as this gives insights of the local structure at an atomic level in its solid state. NMR in solution showed very broad 13C signals of PTX for this system due to the reduced mobility of the incorporated drug which exclude this as an analytical meth-od. In a first study, crystalline PTX was structurally characterized by solid-state NMR as no com-plete 13C spectrum assignment and no 1H NMR data existed for the solid state. In addition, the asymmetric unit of the PTX crystal structure consists of two molecules (Z'=2) that can only be investigated in its solid state. As crystalline PTX in total has about 100 different 13C and 1H chemical shifts with very small differences due to Z’=2, and furthermore, its unit cell consisting of more than 900 atoms, accompanying GIPAW (CASTEP) calculations were required for NMR signal assignments. These calculations were performed using the first three available purely hydrous and anhydrous PTX structures, which were determined by XRD and published by Vel-la-Zarb et al. in 2013. Within this thesis, is was discovered that two investigated batches of commercially available PTX from the same supplier both contained an identical and so far un-known PTX phase that was elucidated by PXRD as well as solid-state NMR data. One of the two batches consists of an additional phase that was shown to be very similar to a known hy-drated phase published in 2013.[1] By heating the batch with the mixture of the two phases un-der vacuum, it is transformed completely to the new dry phase occurring in both PTX batches. Since the drying conditions to obtain anhydrous PTX in-situ on the PXRD setup described by Vella-Zarb et. al.[1] were much softer than ours, we identify our dry phase as a relaxed version of their published anhydrate structure. The PXRD data of the new anhydrate phase was trans-ferred into a new structural model, which currently undergoes geometry optimization. Based on solid-state NMR data at MAS spinning frequencies up to 100 kHz, a 13C and a partial 1H signal assignment for the new anhydrous structure were achieved. These results provided sufficient structural information for further investigations of the micellar POL-PTX system. In a second study, the applicability and benefit of two-dimensional solid-state 14N-1H HMQC MAS NMR spectra for the characterization of amorphous POL-PTX formulations was investi-gated. The mentioned technique has never been applied to a system of similar complexity be-fore and was chosen because around 84% of the small-molecule drugs contain at least one nitrogen atom. In addition, the number of nitrogen atoms in both POL and PTX is much smaller than the number of carbons or hydrogens, which significantly reduces the spectral complexity. 14N has a natural abundance of 99.6% but leads to quadrupolar broadening due to its nuclear spin quantum number I = 1. While this is usually undesirable due to broadening in the resulting 1D 14N NMR spectra, this effect is explicitly used in the 2D 14N-1H HMQC MAS experiment. The indirect 14N measurement can avoid the broadening while maintaining the advantage of the high natural abundance and making use of the much more dispersed signals due to the additional quadrupolar shifts as compared to 15N. This measurement method could be successfully applied to the complex amorphous POL-PTX mixtures. With increasing PTX loading of the formulations, additional peaks arise as spatial proximities of the amide nitrogens of POL to NH or OH groups of PTX. In addition, the 14N quadrupolar shift of these amide nitrogens decreases with increasing PTX content indicating a more symmetric nitrogen environment. The latter can be explained by a transformation of the trigonal planar coordination of the tertiary amide nitrogen atoms in pure POL towards a more tetrahedral environment upon PTX loading induced by the formation of hydrogen bonds with NH/OH groups of PTX. In the third and last project, the results of the two abovementioned studies were used and ex-tended by solid state 13C and two-dimensional 1H-13C as well as 1H-1H MAS NMR data with the aim to derive a structural model of the POL-PTX formulations at an atomic level. The knowledge of the NMR signal assignments for crystalline PTX was transferred to amorphous PTX (present in the micelles of the formulations). The 13C solid-state NMR signals were evalu-ated concerning changes in chemical shifts and full widths of half maximum (FWHM) for the different PTX loadings. In this way, the required information about possible interaction sites at an atomic level becomes available. Due to the complexity of these systems, such proximities often cannot be assigned to special atoms, but more to groups of atoms, as the individual de-velopments of line widths and line shifts are mutually dependent. An advantageous aspect for this analysis was that pure POL already forms unloaded micelles. The evaluation of the data showed that the terminal phenyl groups of PTX seem to be most involved in the interaction by the establishment of the micelle for lowest drug loading and that they are likely to react to the change in the amount of PTX molecules as well. For the incorporation of PTX in the micelles, the following model could be obtained: For lowest drug loading, PTX is mainly located in the inner part of the micelles. Upon further increasing of the loading, it progressively extends to-ward the micellar shell. This could be well shown by the increasing interactions of the hydro-phobic butyl chain of POL and PTX, proceeding in the direction of the polymer backbone with rising drug load. Furthermore, due to the size of PTX and the hydrodynamic radius of the mi-celles, even at the lowest loading, the PTX molecules partially reach the core-shell interface of the micelle. Upon increasing the drug loading, the surface coverage with PTX clusters increas-es based on the obtained model approach. The latter result is supported by DLS and SANS data of this system. The abovementioned results of the 14N-1H HMQC MAS investigation of the POL-PTX formulations support the outlined model. As an outlook, the currently running geometry optimization and subsequently scheduled calcu-lation of the chemical shieldings of the newly obtained anhydrous PTX crystal structure can further improve the solid-state NMR characterization through determination of further spatial proximities among protons using the existing 2D 1H(DQ)-1H(SQ) solid-state MAS NMR spec-trum at 100 kHz rotor spinning frequency. The 2D 14N-1H HMQC MAS NMR experiments were shown to have great potential as a technique for the analysis of other disordered and amor-phous drug delivery systems as well. The results of this thesis should be subsequently applied to other micellar systems with varying pharmaceutical excipients or active ingredients with the goal of systematically achieving higher drug loadings (e.g., for the investigated PTX, the similar drug docetaxel or even different natural products). Additionally, it is planned to transfer the knowledge to another complex polymer system containing poly(amino acids) which offers hy-drogen bonding donor sites for additional intermolecular interactions. Currently, the POL-PTX system is investigated by further SANS studies that may provide another puzzle piece to the model as complementary measurement method in the future. In addition, the use of MD simu-lations might be considered in the future. This would allow a computerized linking of the differ-ent pieces of information with the aim to determine the most likely model. / Paclitaxel (PTX) ist eines der führenden Medikamente gegen Brust-und Eierstockkrebs. Aufgrund seiner geringen Löslichkeit erfordert die Behandlung der Patienten mit diesem Medikament eine sehr gut geeignete Kombination mit einem löslichen pharmazeutischenHilfsstoff, um die Bioverfügbarkeit zu erhöhen und die starken Nebenwirkungen zu reduzieren. Ein effizienter Weg, dies in Zukunft zu erreichen, könnte der Einbau von PTX in polymere Mizellen sein, die aus Poly(2-oxazolin)-basierten Triblock-Copolymeren (POL) bestehen und PTX-Beladungen von bis zu 50 Gew.-% ermöglichen. Strukturelle Informationen auf atomarer Ebene und damit die Kenntnis von Wechselwirkungeninnerhalb dieser vielversprechenden, aber komplexen PTX-POL-Formulierungen waren jedoch bisher nichtverfügbar. Solche Ergebnisse könnten die zukünftige Entwicklung von verbesserten Hilfsstoffen für PTX und von geeigneten Hilfsstoffen für andere pharmazeutische Wirkstoffe unterstützen. Aus diesem Grund wurdenin der vorliegenden DissertationFestkörper-NMR-Untersuchungen andiesenamorphen Formulierungen mit unterschiedlichen POL-PTX Zusammensetzungen durchgeführt, weil damit Einblickein die lokale Struktur auf atomarer Ebene im festen Zustand erhalten werden können. Aufgrund der verringerten Mobilität des eingebrachten Wirkstoffs in diesem System ergeben NMR-Messungen in Lösung sehr breite 13C-PTX-Signale, was diese Technikals Analysemethode ausschließt. ...

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

Central Nervous System (CNS) injury may lead to irreversible damage to cognitive and motor abilities when injured. This is due to the inability of axons to regenerate. This thesis focuses on two methods of promoting axonal regeneration: microtubule stabilization and upregulation of the intrinsic growth capacity of the neuron via the mechanistic target of rapamycin (mTOR) pathway. Both have shown promising results in potentially being a therapeutic treatment for CNS trauma. This research seeks to (1) test a combinatorial method of axonal regeneration utilizing both methods simultaneously and (2) compare microtubule stabilization and upregulation of the mTOR pathway as neuronal regeneration methods. Aim 1 serves to test the combinatorial treatment of Taxol, a microtubule stabilizer, and cRheb transfection, which upregulates the mTOR pathway, on neuronal cell cultures. Cells were cultured in either a growth-promoting substrate or a mix of growth-promoting and growth-inhibitory substrates. The results of this study revealed combinatorial treatment of 2DIV Taxol application with cRheb transfection as a promising treatment that yielded significantly greater axonal outgrowth than either treatment alone. Aim 2 serves to compare the two established methods of axonal regeneration in the scientific community. Based off of a meta-analysis, results of this aim indicate upregulation of mTOR is more effective at promoting axonal regeneration than microtubule stabilization.

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Taxol™, isolated from the bark of Taxus brevifolia in the late 1960s, and the semisynthetic analog Taxotere™ have proven clinical importance for the treatment of ovarian and breast cancer. Taxol™ exerts its biological effect by binding to polymerized tubulin and stabilizing the resulting microtubules. Studies aimed at understanding the biologically active conformation of taxol and its binding environment on β-tubulin are described. This knowledge is important because it could lead to the design of structurally less complicated drugs with better efficacy and better bioavailability. Moreover, the information can be extended to other natural products that possess microtubule-stabilizing properties similar to Taxol™. In this work, the synthesis of a triply labeled taxol analog is described as well as REDOR studies of this compound complexed to tubulin are in progress. Macrocyclic analogs of taxol have been prepared and their biological activities were evaluated. Chemical modeling of these analogs and their activities agrees with the hypothesis that Taxol™ adopts T-shaped conformation. Difficulties were encountered with the key ring-closing metathesis strategy, suggesting that a more flexible and efficient macrocyclization method will be needed to synthesize additional macrocyclic analogs. / Ph. D.

Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anticancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditions chemo- and radio-therapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities.While various telomerase inhibition strategies cause cancer cells to undergo apoptosis car senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient.We blocked telomerase by targeting hTR and hTERT, individually and collectively utilizing synthetic short interfering RNA (siRNA), short hairpin RNA (siRNA), and a dominant negative form of hTERT (DN-hTERT) in MCF-7 breast cancer cells. We analyzed the efficiency of telomerase inhibition for each strategy alone and then treated the cells with two mainstay chemotherapeutic agents, Adriamycin (AdR) and Taxol. The most effective telomerase inhibition strategies were synthetic siRNA and DN-hTERT, individually. After treatment with various concentrations of AdR or Taxol, breast cancer cells with inhibited telomerase grew significantly slower and exhibited widespread senescence or apoptosis within a much shorter time period and at a dose that is insufficient to trigger cytostasis. In addition, we provide evidence that cells in which telomerase was inhibited were more sensitive to anti-cancer agents, whether the drug inhibited topoisomerase II resulting in DNA damage (AdR) or blocked mitosis via protracted microtubule stabilization (Taxol). Collectively, our data indicate that alone, anti-telomerase inhibition strategies differ in their efficacy. However, when used in the adjuvant setting with diverse acting chemotherapeutic agents, there is a potent synergy resulting in chemotherapeutic sensitization characterized in part by widespread senescence and/or apoptosis.

Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis. This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen-independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells.