Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-09-08T19:08:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Karina_Final_CD.pdf: 2797138 bytes, checksum: 5e86db88edfdb58a92aceb27cf908735 (MD5) / Made available in DSpace on 2016-09-08T19:08:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Karina_Final_CD.pdf: 2797138 bytes, checksum: 5e86db88edfdb58a92aceb27cf908735 (MD5) Previous issue date: 2015-10-16 / A partir dos parâmetros eletrônicos obtidos com a Teoria do Funcional Densidade, realizamos um estudo Quantitativo da Relação Estrutura Atividade (QSAR) de derivados de cetonatiossemicarbazonas e de ácidos kójico e benzoico para analisar o potencial de inibição da melanogênese destes compostos. Utilizando técnicas computacionais em conjunção com uma Regressão linear múltipla, obtivemos uma expressão capaz de prever a concentração inibitória (IC50) destes compostos e dos demais aqui propostos. Para a previsão da IC50 foram utilizados os seguintes parâmetros eletrônicos e físico-químicos: afinidade eletrônica (EA), gap de energia (HHL), momento de dipolo (μ) e o logaritmo do coeficiente de partição [octanol/água](LogP). Para as cetonatiossemicarbazonas os descritores eletrônicos que proporcionaram uma boa correlação linear com a IC50 experimental foram: a carga atômica do nitrogênio N2 e a EA. Para as demais moléculas avaliadas nesta pesquisa, além desses parâmetros, foram incluídos o potencial de ionização (IP), a energia do atracamento molecular (G), eletronegatividade absoluta (), dureza (), maciez (S), logaritmo do coeficiente de solubilidade (LogS), volume molar (VM) e o coeficiente de Hansch (). Como resultado da QSAR, os descritores que proporcionaram a melhor correlação linear com a IC50 experimental foram: a HHL e o VM. Na análise QSAR dos derivados dos ácidos kójico e benzoico foi utilizado um conjunto de treinamento formado por dez moléculas e um grupo de teste constituído por duas moléculas para uma validação cruzada tipo boostrap. Os cálculos de G se restringiram a encontrar o valor da energia livre de interação dos derivados dos ácidos kójico e benzoico e da enzima tirosinase por meio da formação do complexo ligante-tirosinase. Os valores das energias de interação obtidos para as moléculas propostas se revelaram promissores, visto que apresentaram valores mais baixos do que o obtido para o complexo ácido kójico-tirosinase. Em todas as análises QSAR, os valores dos parâmetros estatísticos de validação, como coeficiente de correlação, desvio-padrão, teste de Fischer e do nível geral de confiabilidade do modelo, estão dentro do esperado para um bom modelo estatístico. Os modelos obtidos fornecem uma boa previsão das atividades biológicas investigadas neste trabalho apontam para novos compostos candidatos com potencial para inibição da melanogênese. / We have carried ant studies of QSAR using electronic structure derived parameters be the means of Density Functional Theory (DFT) calculations for derivatives of ketonethiosemicarbazones, kojic acid and benzoic acid. The aim was to evaluate the melanogenesis inhibiting potential of these compounds, by using this procedure and performing a Multiple Linear Regression we obtained an expression able to predict the inhibitory concentration (IC50) of the compounds studied here. In predicting the IC50 we used the electronic and physical-chemical parameters of electron affinity (EA), energy gap (HHL), dipole of moment (μ) and the [octanol/water] logarithm of the partition coefficient (LogP). For ketonethiosemicarbazones the electronic parameters that provide a good linear correlation with experimental IC50 are the atomic charge of nitrogen N2 and the EA. For the other molecules analyzed in this study, in addition to these parameters it is included ionization potential (IP), molecular docking of energy (G), absolute electronegativity (), hardness(), softness (S), partition coefficient of molar solubility (LogS), molar volume (VM) and Hansch coefficient (). We found that the parameters leading to the best linear correlation with experimental IC50 are the interaction energy and the molar volume. In the QSAR analysis of derivatives of the benzoic and kojic acids it is used a training group formed by ten molecules and a test group formed of two molecules to realize a bootstrap-type cross validation. The calculations of the molecular docking are restricted to values of free energy of derivatives of the benzoic and kojic acids and to the enzyme tyrosinase forming the tyrosinase-ligand complex. In all QSAR analysis, the statistical validation such as correlation coefficient, standard deviation, Fisher test and the model reliability are in that range expected for a good statistical model. As a conclusion, we show that our model gives a good prediction of the biological activities, which allow us to indicate new compounds with potential in inhibiting melanogenesis.

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / Limonene (LIM) is a monocyclic monoterpene, and one of the main constituents of several essential oils of citric fruits such as orange, tangerine and lemon. Among the essential oils found in citric fruits in general, R-(+)-limonene is the majoritarian component, which can reach concentrations from 90 to 96%. Cyclodextrins (CDs) are cyclic oligosaccharides, most commonly found with six, seven or eight glucose units, known respectively as α-CD, β-CD and γ-CD. They are obtained through the action of the enzyme cyclomaltodextrin glucanotransferase (CGTase) on starch. The CDs are able to form inclusion complexes, altering the physical and chemical properties of the complexed compounds. The objective with this work was to prepare and characterize physical-chemically inclusion complexes of limonene in α-and β-cyclodextrin, studying the properties of the supposed complex formed. Such complex was prepared by means of physical mixture, malaxage and co-evaporation, and was characterized through thermal analysis (differential scanning calorimetry– DSC – and thermogravimetry/derivative thermogravimetry – TG/DTG); gas chromatography coupled to a mass spectrometry -GC/MS; X-ray diffraction – DRX; Fourier infrared transform – FTIR – absorption spectrophotometry; electronic scanning microscopy – ESM;molecular modeling (Docking)and kinetic study of first-phase mass loss. From the results obtained through the analyses, it was possible to point out that both the α-CD and the β-CD formed inclusion complexes with limonene. However, in the DSC curves of the α-CD, which correspond to the methods of MA and CE, it was possible to observe a profile that is different from that observed for pure α-CD and MF. In the DSC curves for β-CD, we observed that the CE presented an endothermic profile that was more significant than were the MF and MA methods. According to the TG/DTGanalyses for α-CD, we can observe that the thermoanalytical profile of the complexes obtained through the MF, MA and CE methods were similar to that of -CD. The TG/DTG curves of limoneneshow mass loss around 100% in the interval of 30-169°C. The TG/DTG curve of α-CD showed two phases of mass loss (between 25 and 120°C) that add up to 10.7% of mass loss followed by decomposition and elimination of carbonaceous material. The TG/DTG curves of β-CD, MF, MA and CE presented a thermoanalytical profile that was similar to the complexes obtained with α-CD. After the evaluation of the mass losses shown on Table 2, we can see figures around 8% for MA and CE in the temperature range between 120 and 270º C, what is an indicator of the higher capacity of limonene complexation. In the results of CG/EM for α-CD, it was possible to observe that MA encapsulated limonene in a ratio of 1:0.49 (limonene: α-CD), but in a ratio that was lower to CE 1:18.11 (limonene: α-CD). Thus, it is shown that CE was the best method, and also that β-CD did not show any complexation in MF and MA. CE showed complexation efficiency of 1:1.44, being considered as the best method for complexation when compared to CE of α-CD. The standard of X-ray diffraction of MF was shown to be quite similar to those found for pure α-CD and β-CD. That is an indicator of low efficiency of complexation. By observing the reflections of MA and CE, we can verify the onset of new peaks and the absence of characteristic peaks of pure α-CD and β-CD. In the analysis of the FTIR spectra corresponding to the inclusion complexes obtained through the methods of MF, MA and CE, it was possible to observe a profile that was quite similar among all the samples. They resemble the spectrum ofpure α-CD and β-CD due to the fact that the complexes have been prepared in a molar ratio of 1:1 (limonene 136.24 g/mol andα-CD 972 g/mol). MEV was performed to evaluate the changes in the crystalline characteristics on the surfaces of pure α-CD and β-CD and after the formation of the inclusion complex, where the results corroborate other findings of the characterization tests. In the molecular modeling (docking), the theoretical results showed more stability in the ligations between α-CD/LIM (-4.49 kcal/mol) than what was observed between β-CD/LIM (-4.04 kcal/mol), once that a lesser expenditure of energy was necessary between α-CD/LIM in relation tothat between β-CD/LIM. The kinetic study of the first phase of decomposition was introduced in this work in order to observe the thermal behavior of the pure α-CD and β-CD, as well as of the complexes obtained in the warming ratios of 2.5-5.0-10 and 15 ºC.min-1, under nitrogen dynamic atmosphere (100 mL. min-1). / O limoneno (LIM) é um monoterpeno monocíclico, sendo um dos principais constituintes de vários óleos essenciais de frutas cítricas, como laranja, tangerina e limão. Dentre os óleos essenciais encontrados nos cítricos em geral, o R-(+)-limoneno é seu componente majoritário, podendo atingir concentrações de 90 a 96%. As ciclodextrinas (CDs) são oligossacarídeos cíclicos, mais comumente encontradas com seis, sete ou oito unidades de glicose, denominadas respectivamente de α-CD, β-CD e γ-CD. São obtidas pela ação da enzima ciclomaltodextrina glucanotransferase (CGTase) sobre o amido. As CDs são capazes de formar complexos de inclusão, alterando as propriedades físicas e químicas dos compostos complexados. O objetivo com este trabalho foi preparar e caracterizar físico-químicamente complexos de inclusão de limoneno em α e β-ciclodextrina, estudando as propriedades do suposto complexo formado. Tal complexo foi preparado por meio das técnicas de mistura física, malaxagem e co-evaporação, e foi caracterizado por análise térmica (calorimetria exploratória diferencial-DSC e termogravimetria/termogravimetria derivada-TG/DTG); cromatografia gasosa acoplada a espectrometria de massas-CG/EM; difração de raios X-DRX; espectrofotometria de absorção na região do infravermelho com transformada de Fourier-FTIR; microscopia eletrônica de varredura-MEV, modelagem molecular (Docking) e estudo cinético de primeira etapa de perda de massa. Por meio dos resultados obtidos pelas análises foi possível evidenciar que tanto a α-CD quanto a β-CD formaram complexos de inclusão com o limoneno. No entanto, nas curvas DSC da α-CD que correspondem aos métodos de MA e CE foi possível observar um perfil diferente daquele observado para α-CD pura e MF. Nas curvas DSC para β-CD, observou-se que para o método CE apresentou perfil endotérmico mais significativo do que os métodos de MF e MA. De acordo com as análises TG/DTG para α-CD pode-se observar que o perfil termoanalítico dos complexos obtidos pelo método da MF, MA e CE foram semelhantes a -CD. As curvas TG/DTG do limoneno mostra perda de massa em torno de 100% no intervalo de 30-169°C. A curva TG/DTG da α-CD mostrou duas etapas de perdas de massa (entre 25-120°C) somam 10,7% de perda de massa seguidas de decomposição e eliminação do material carbonáceo. Nas curvas TG/DTG da β-CD, MF, MA e CE que apresentaram um perfil termoanalítico similar aos complexos obtidos com a α-CD. Após a avaliação das perdas de massa mostradas na Tabela 2 pode-se verficar valores em torno de 8% para o MA e CE na faixa de temperatura entre 120 e 270º C, o que é um indicativo da maior capacidade de complexação do limoneno. Nos resultados para o CG/EM para α-CD foi possível observar que o MA encapsulou o limoneno em uma razão de 1:0,49 (limoneno: α-CD), mas em uma razão inferior ao CE 1:18,11 (limoneno: α-CD). Mostrando desta forma que a CE foi o melhor método, e para β-CD pode-se observar que não exibiu nenhuma complexação na MF e MA. Já a CE mostrou uma eficiência de complexação igual a 1:1,44, sendo assim considerado o melhor método de complexação quando comparado com o CE da α-CD. No padrão de difração de raios X da MF mostrou ser bastante similar aos encontrados da para a α-CD e β-CD puras. Esse é um indicativo da baixa eficiência de complexação. Ao observar as reflexões da MA e CE, pode-se verificar o surgimento de novos picos e a ausência de picos característicos da α-CD e β-CD puras. Na análise dos espectros no FTIR correspondentes aos complexos de inclusão obtidos pelos métodos da MF, MA e CE foi possível observar um perfil muito semelhante entre todas as amostras. Elas se assemelham a espectro da α-CD e β-CD puras devido aos complexos terem sido preparados em razão molar 1:1 (limoneno 136,24 g/mol e α-CD 972 g/mol). A MEV foi realizada para avaliar as mudanças nas características cristalinas na superfície das α-CD e β-CD puras e após a formação do complexo de inclusão, onde os resultados corroboram com os outros achados dos testes de caracterização. Na modelagem molecular (docking), os resultados teóricos mostraram estabilidade nas ligações entre α-CD/LIM (-4,49 kcal/mol) do que os observados entre β-CD/LIM (-4,04 kcal/mol), pois foi necessário menor gasto de energia entre α-CD/LIM do que entre β-CD/LIM. O estudo cinético da primeira etapa de decomposição foi introduzido neste trabalho para observar o comportamento térmico das α-CD e β-CD puras e dos complexos obtidos nas razões de aquecimento de 2,5-5,0-10 e 15 ºC.min-1, sob atmosfera dinâmica de nitrogênio (100 mL. min-1).

As estruturas e propriedades biológicas das amidas piplartina e a piperina, isoladas respectivamente de Piper tuberculatum e P. nigrum, inspiraram a síntese de 89 derivados e 7 esters estruturalmente relacionadas. As preparações envolveram metodologias tradicionais e os compostos purificados tiveram suas estruturas caracterizadas por análises espectroscópicas e espectrométricas. Os estudos de fragmentação por IE e IES indicaram a clivagem preferencial da ligação N-CO no caso das cinamamidas, dienamidas e cinamimidas. Estudos computacionais envolvendo afinidade protônica e energias de ligação confirmaram a fragmentação preferencial da ligação amídica para as amidas. A citotoxicidade de 89 substâncias foi avaliada contra três células leucêmicas (K562, Nalm6 e Raji) e a partir dos valores de IC50 foram realizados estudos de relação estrutura-atividade (SAR). As linhagens K562 e a Nalm6 foram a mais resistente e vulnerável, respectivamente, e as amidas piplartina (1a), N-Ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4,5-trimetoxifenil)propanamida (1n), e (E)-N,N-dibutil-3-(3,4-dimetoxifenil)acrilamida (13h) foram as mais ativas com IC50 de 0,34 µM; 0,84 µM e 1,88 µM contra K562 e (E)-N-ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4-dimetoxifenil)acrylamida (13i) com IC50 de 0,98 µM contra Nalm6. A avaliação de atividade leishmanicida de 18 substâncias não se mostrou promissora. As abordagens qualitativas e quantitativas foram feitas baseadas nos descritores moleculares gerados pelo programa VolSurf+. A partir de métodos quimiométricos tais com PLS, algoritmo genético, árvores de decisão foi possível gerar modelos para correlacionar às propriedades moleculares com a atividade biológica. As propriedades de absorção, distribuição, metabolismo e excreção e os equilíbrios entres as regiões hidrofílicas e hidrofóbicas foram importantes para atividade citotóxica. O estudo de ancoragem molecular mostrou que as amidas (E)-N,N-dibutil-3-(3,4,5-trimetoxifenil)acrilamida (1l), 1n, (E)-3-(4-clorofenil)-N-ciclohexil-N-(ciclohexilcarbamoil)acrilamida (5a), 13h e 13i podem atuar como inibidores das histonas desacetilases particularmente HDAC4 e HDAC8. / The structures and biological properties of the amides piplartine and piperine isolated from Piper tuberculatum and P. nigrum respectively, inspired the synthesis of derivatives 89 and 7 esters structurally related. Their preparations were achieved using classical procedures and the purified amides were submitted to spectroscopic and spectrometric characterization. The study of fragmentation process by EI and ESI suggested the preferential cleavage of the N-CO bond of cinnamamides, dienamides and cinnamimides. The cytotoxicity of 89 compounds was evaluated against three leukemic cells (K562, Nalm6 and Raji) and based on IC50 values the structure-activity relationship (SAR) was performed. While the K562 and Nalm6 cells were the more resistant and more sensitive, respectively, the amides piplartine (1a), N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4,5-trimethoxyphenyl)propanamide (1n) and (E)-N,N-dibutyl-3-(3,4-dimethoxyphenyl)acrylamide (13h) were in general the most active with IC50 of 0.34 µM, 0.84 µM and 1.88 µM against K562 and (E)-N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4-dimethoxyphenyl)acrylamide (13i) with IC50 of 0.98 µM against Nalm6. The evaluation of leishmanicidal activity of 18 substances was also performed but was not promising. Qualitative and quantitative approaches were made based on molecular descriptors generated by VolSurf+ program. The chemometric methods such as PLS, genetic algorithm, decision trees generated models to correlate molecular properties with the biological activity. The absorption, distribution, metabolism and excretion properties and a balance between hydrophilic and hydrophobic moieties of the amides were important for an optimized activity. The molecular docking revealed that amides such as (E)-N,N-dibutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (1l), 1n, (E)-3-(4-chlorophenyl)-N-cyclohexil-N-(cyclohexylcarbamoyl)acrylamide (5a), 13h and 13i have potential to act as possible inhibitors of histone deacetylase proteins particularly HDAC4 and HDAC8.

A enzima diidroorotato desidrogenase (DHODH) catalisa a conversão de diidroorotato em orotato, a quarta e única reação redox da via metabólica da síntese de novo de nucleotídeos de pirimidina. DHODH tem sido explorada como alvo validado para terapias contra doenças proliferativas e parasitárias e, em particular, tem sido considerada um alvo atraente para o planejamento de fármacos com ação contra tripanossomatídeos, como parasitos dos gêneros Trypanosoma e Leishmania, que conjuntamente são responsáveis por doenças e mortes que acometem milhões de pessoas em todo o mundo. Neste trabalho, através da combinação de técnicas de DNA recombinante, termofluor, cristalografia de raios-X e ensaios de inibição in vitro e in silico, foi possível identificar sítios alvos na estrutura da DHODH para o desenvolvimento de ligantes, identificar inibidores potentes e seletivos contra as DHODHs de Leishmania major e Trypanosoma cruzi e, caracterizar seus mecanismos de inibição. Finalmente, o efeito leishmanicida observado em nossos ensaios anti-promastigota e os baixos níveis de citotoxicidade observados em células de mamíferos sugerem que alguns dos compostos identificados durante o desenvolvimento deste projeto como potentes inibidores da enzima DHODH poderão ser utilizados como protótipos para o desenvolvimento de fármacos com ação leishmanicida e tripanocida. Combinados, nossos resultados forneceram uma nova e importante contribuição para a compreensão do mecanismo de ação das enzimas DHODH da classe 1A e para o desenho de fármacos baseado nas estruturas das enzimas diidroorotato desidrogenase de Leishmania major e Trypanosoma cruzi. Além disso, a alta identidade sequencial e estrutural observada entre as enzimas de tripanossomatídeos sugerem que uma única estratégia para o desenho de inibidores baseado em estrutura poderá ser usada para explorar a enzima DHODH como alvo terapêutico para várias doenças negligenciadas tropicais como Leishmaniose, Doença do sono e Doença de Chagas / Dihydroorotate dehydrogenase (DHODH) catalyses the conversion of dihydroorote to orotate, the fourth step and only redox reaction in the de novo pyrimidine biosynthetic pathway. DHODH has been exploited as a validated target for therapy against proliferative and parasitic diseases, and in particular, has been considered to be an attractive target for drug development against trypanosomatids, such as parasites from the genera Leishmania and Trypanosoma that collectively cause disease and death in millions of humans. In this work, by combining recombinant DNA technology, thermofluor, X-ray crystallography and in vitro and in silico inhibition assays, we have been able to identify target sites for ligand design, identify potent and selective inhibitors against trypanosomatid DHODHs and fully characterize their mechanism of inibition. Finally, the anti-leishmanial effect observed in our anti-promastigote assays and the low citotoxicity levels observed against mammaliam cells strongly suggest that some of the compounds identified during the development of this project as potent DHODH inhibitors can be used as prototytes for the development of anti-leishmania and anti-trypanosoma drugs. Altogether, our findings provide a new and important contribution to the understanding of the mechanism of action of class 1A DHODHs and for the structure-assisted design of inhibitors against trypanosomatid DHODHs. Furthermore, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to exploit DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping Sickness and Chagas\' Disease.

As metaloproteinases de matriz (MMP) são enzimas superexpressas em quase todos os tumores humanos, sendo que os subtipos MMP-2 e MMP-9 têm sido associados ao potencial metastático e prognóstico desfavorável em neoplasias malignas como, por exemplo, melanoma metastático e glioma. Compostos capazes de inibir a atividade destas enzimas podem representar potenciais agentes terapêuticos. O composto 4-nerolidilcatecol (4-NC), isolado de plantas do gênero Pothomorphe, apresentou resultados promissores para o tratamento do melanoma e glioma e foi capaz de atuar em várias etapas bioquímicas importantes envolvidas na progressão dessas patologias, inclusive inibindo MMP-2 e MMP-9. No entanto, o mecanismo de ação do 4-NC não está completamente elucidado. O presente estudo envolveu a aplicação de métodos de modelagem molecular e de formalismos do planejamento de novas moléculas auxiliado por computador, CAMD (Computer-Aided Molecular Design) a fim de explorar a interação entre esta molécula e as enzimas MMP-2 e MMP-9, além de planejar novos inibidores para estes alvos. Análise exploratória de dados, que compreende a análise de agrupamentos hierárquicos e de componentes principais. foi desenvolvida para um conjunto de hidroxamatos (N=64) descritos como inibidores de MMP-2 e MMP-9, a fim de identificar as propriedades moleculares que mais influenciavam o processo de discriminação dos compostos. As propriedades termodinâmicas, eletrônicas e estéricas foram importantes para descrever os compostos mais ativos no conjunto de dados da MMP-2. Para a MMP-9, o coeficiente de distribuição (ClogD) em pH 1,5 foi relevante no processo de discriminação do conjunto. A presença de substituintes volumosos na porção R3 parece ser crucial para o conjunto de inibidores investigados. Esta região está envolvida em interações moleculares com a cavidade S1 de ambas as enzimas, mas há um limite de volume a ser considerado para estes substituintes. O formalismo QSAR-4D independente do receptor (IR) foi aplicado ao mesmo conjunto de dados e permitiu estabelecer o mapeamento do farmacóforo, além de explorar diferentes alinhamentos para a obtenção da hipótese de conformação bioativa prevista pelo melhor modelo de QSAR. OS modelos QSAR apresentaram boa capacidade de previsão, auxiliaram na proposição de novos inibidores e estimaram a atividade do 4-NC. Com o melhor modelo QSAR para MMP-9 (N=64), a atividade prevista para o 4-NC foi classificada na faixa dos inibidores com atividade moderada. Entretanto, o melhor modelo QSAR obtido para MMP-2 (N=38) não foi capaz de prever, de forma adequada, a atividade de compostos com arcabouço químico diferente daqueles utilizados na construção dos modelos. Estudos de ancoramento molecular foram desenvolvidos para investigar a orientação do 4-NC no sitio catalítico das duas enzimas e as interações que poderiam ser estabelecidas nestes complexos. Duas conformações favoráveis foram encontradas. Simulações computacionais de dinâmica molecular foram desenvolvidas com os complexos mais promissores selecionados nos estudos de ancoramento, a fim de obter informações mais detalhadas e de maior confiabilidade. sobre suas interações intermoleculares. O 4-NC tende a se orientar no sítio de forma a acomodar sua cadeia lateral no bolso S1 adjacente ao sítio catalítico em ambas as enzimas. Ensaios de zimografia também foram realizados com o objetivo de elucidar possíveis contribuições da cadeia lateral e do núcleo catecólico do 4-NC na atividade inibitória frente às enzimas em estudo. O núcleo catecólico parece ser o responsável por sua atividade, pois o composto 1,2dimetoxibenzeno, que possui as hidroxilas bloqueadas por grupos metil, não foi capaz de exercer atividade inibitória significante frente à MMP-2 e MMP-9. Estudos de voltametria reforçaram a hipótese de que o 4-NC tem a capacidade de quelar os íons zinco presentes no tampão de incubação. / Matrix metalloproteinases (MMP) enzymes are overexpressed in almost all human tumors, and MMP-2 and MMP-9 subtypes have been associated with metastatic potential and poor prognosis in malignant tumors, such as metastatic melanoma and glioma. Compounds capable of inhibiting the activity of theses enzymes would be considered as potential therapeutic agents. The 4-nerolidylcatechol compound (4-NC), isolated from plants of genus Pothomorphe, has showed promising results in the treatment of melanoma and glioma, and was able to act in several important biochemical steps involved in the progression of these diseases, as well as inhibiting MMP-2 and MMP-9. However, the 4-NC mechanism of action is not completely understood. This study has involved the application of molecular modeling methods and formalisms of computer-aided molecular design (CAMD) in order to explore the interaction between 4-NC and MMP-2/MMP-9, and to design new inhibitors for these targets. Exploratory data analysis, which comprises hierarchical cluster analysis and principal components analysis, was performed to a set of hydroxamates (N=64). previously reported as MMP-2 and MMP-9 inhibitors, in order lo identify the molecular properties that is most critical for the discrimination process regarding the investigated compounds. The thermodynamic, electronic, and steric properties were: quite important to describe the highly active compounds in the data set of MMP-2, whereas the apparent partition coefficient (ClogD) at pH 1.5 was the property more relevant for MMP-9 data set. The presence of bulky substituents on the R3 moiety seems to be crucial for this set of inhibitors due to the molecular interaction with the S1 subsite of both enzymes. However, there is a limit regarding the substituents volume in this region. Receptor independent (RI) 4D-QSAR analysis was applied lo the same data set and it was possible to establish the pharmacophore mapping, besides to explore different alignments in order to generate the hypothesized bioactive conformation through the best QSAR model. The QSAR models have presented good predictability, assisted in proposing new inhibitors, and estimated the activity of 4-NC. Regarding the best QSAR model for MMP-9 (N=64), the 4-NC predicted activity was classified in the range of the moderate active inhibitors. The best QSAR model obtained for MMP-2 (N=38), however was not able to properly predict the activity for compounds with different chemical scaffold from those used to build up the QSAR model. Molecular docking studies have been developed to investigate the 4-NC binding mode into the catalytic site of the two enzymes and the interactions that could be established in those complexes. The results have shown two favorable conformers regarding the MMP inhibition. Molecular dynamics computational simulation were combined to molecular docking studies in order to obtain more detailed and reliable information regarding the intermolecular interactions of each complex. The 4-NC molecule tends to accommodate the side chain in the S1 pocket adjacent to the catalytic site in both enzymes. Experimental zymography assays were also performed to elucidate the possible contribution of the side chain and the catechol core in the 4-NC inhibitory activity against the MMP-2 and MMP-9 enzymes. The catechol core seems to be responsible for its activity, since the 1,2 dimethoxybenzene compound, which has the hydroxyl blocked by a methyl group, was not able to exert any significant inhibition on enzymes. Voltametric assays confirmed the hypothesis that 4-NC chelates zinc ions present in the incubation buffer.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used classes of drugs. They target the cyclooxygenases (COX) 1 and 2 to reduce the physiological responses of pain, fever, and inflammation. Due to their role in inducing angiogenesis, COX proteins have also been identified as targets in cancer therapies. In this thesis, I describe computational protocols of molecular docking, molecular dynamics simulations and free energy calculations. These methods were used in this thesis to determine structure-activity relationships of a diverse set of NSAIDs in binding to their target proteins COX-1 and 2. Binding affinities were calculated and used to predict the binding modes. Based on combinations of molecular dynamics simulations and free energy calculations, binding mechanisms of sub-classes of NSAIDs were also proposed. Two stable conformations of COX were probed to understand how they affect inhibitor affinities. Finally, a brief discussion on selectivity towards either COX isoform is discussed. These results will be useful in future de novo design and testing of third-generation NSAIDs.

Proteins and the formation of large protein complexes are essential parts of living organisms. Proteins are present in all aspects of life processes, performing a multitude of various functions ranging from being structural components of cells, to facilitating the passage of certain molecules between various regions of cells. The 'protein docking problem' refers to the computational method of predicting the appropriate matching pair of a protein (receptor) with respect to another protein (ligand), when attempting to bind to one another to form a stable complex. Research shows that matching the three-dimensional (3D) geometric structures of candidate proteins plays a key role in determining a so-called docking pair, which is one of the key aspects of the Computer Aided Drug Design process. However, the active sites which are responsible for binding do not always present a rigid-body shape matching problem. Rather, they may undergo sufficient deformation when docking occurs, which complicates the problem of finding a match. To address this issue, we present an isometry-invariant and topologically robust partial shape matching method for finding complementary protein binding sites, which we call the ProtoDock algorithm. The ProtoDock algorithm comes in two variations. The first version performs a partial shape complementarity matching by initially segmenting the underlying protein object mesh into smaller portions using a spectral mesh segmentation approach. The Heat Kernel Signature (HKS), the underlying basis of our shape descriptor, is subsequently computed for the obtained segments. A final descriptor vector is constructed from the Heat Kernel Signatures and used as the basis for the segment matching. The three different descriptor methods employed are, the accepted Bag of Features (BoF) technique, and our two novel approaches, Closest Medoid Set (CMS) and Medoid Set Average (MSA). The second variation of our ProtoDock algorithm aims to perform the partial matching by utilizing the pointwise HKS descriptors. The use of the pointwise HKS is mainly motivated by the suggestion that, at adequate times, the Heat Kernel Signature of a point on a surface sufficiently describes its neighbourhood. Hence, the HKS of a point may serve as the representative descriptor of its given region of which it forms a part. We propose three (3) sampling methods---Uniform, Random, and Segment-based Random sampling---for selecting these points for the partial matching. Random and Segment-based Random sampling both prove superior to the Uniform sampling method. Our experimental results, run against the Protein-Protein Benchmark 4.0, demonstrate the viability of our approach, in that, it successfully returns known binding segments for known pairing proteins. Furthermore, our ProtoDock-1 algorithm still still yields good results for low resolution protein meshes. This results in even faster processing and matching times with sufficiently reduced computational requirements when obtaining the HKS.

Effective maintenance planning is essential and important in any organisation that is responsible for procuring and managing complex assets. In the marine shipping industry maintenance planning is very significant due to its complexity and the obligations on shipping organisations to comply with certain regulations and requirements. Moreover, improper planning can reduce the ship's availability, which may in turn, be reflected in the revenue of the company. Another issue that requires attention in this field is the cost of maintenance, since improper or inadequate planning could result in breakdowns that could increase the cost of maintenance.This research aims to identify the key factors that affect ship maintenance planning and to provide a framework that can help the decision maker to identify and choose optimum decisions regarding ship maintenance. The research is divided into four stages in order to achieve its objectives and to address the research problem.The first stage is the review of the literature to identify the need for maintenance and to select the key factors that affect maintenance planning. The findings indicate that: maintenance scheduling, selection of maintenance strategy, ship construction, crew compensation, and shipyard selection are the most important factors.The second stage is to evaluate maintenance performance measurements for the marine shipping industry by conducting case study and interviews with professionals involved in the mercantile industry. Semi-structured interviews were conducted with six senior staff experts from three different organisations. The results show that: dry docking scheduling, maintenance costs and budgets, customer satisfaction, employees' satisfaction, classification requirements, and the ship's maintenance requirements are the main factors that have great influence on maintenance planning.The third stage is to develop new methodology to measure the maintenance performance in the marine shipping organisation which is the ship maintenance performance measurement (SMPM) framework. The developed method was validated to assist managers in making the right decisions in ship maintenance planning. The framework was developed based on ten thematic criteria that can be used as indicators for potential organisation growth, i.e., maintenance strategy; dry docking scheduling; budget and costs; the ship's equipment; customer satisfaction; employees; health, safety and environment; learning and growth; classification requirements; and the ship's operation and demands requirements. Interviews were conducted with key personnel from the Kuwait Oil Tanker Company (KOTC) to validate the framework.The fourth stage demonstrates that an optimised schedule for the dry docking of ships for routine maintenance has been constructed. This is accomplished on the basis of one measured criterion, dry docking scheduling, by using an integer programming model to maximise the ship's availability within the company fleet. The model is defined by three constraints: the maintenance window, maintenance completion, and the ship's limit. The model was validated using data from KOTC, and the results depict an optimum solution for maintenance scheduling, maximising the ship's availability to 100% and not less than 92%.

This thesis contains three main sections. In the first section, we examine methodologies to discriminate Type II protein kinase inhibitors from the Type I inhibitors. We have studied the structure of 55 Type II kinase inhibitors and have notice specific descriptive geometric features. Using this information we have developed a pharmacophore and a shape based screening approach. We have found that these methods did not effectively discriminate between the two inhibitor types used independently, but when combined in a consecutive way – pharmacophore search first, then shape based screening, we have found a method that successfully filtered out all Type I molecules. The effect of protonation states and using different conformer generators were studied as well. This method was then tested on a freely available database of decoy molecules and again shown to be discriminative. In the second section of the thesis, we implement and assess swarm-based docking methods. We implement a repulsive particle swarm optimization (RPSO) based conformational search approach into Autodock 3.05. The performance of this approach with different parameters was then tested on a set of 51 protein ligand complexes. The effect of using different factoring for the cognitive, social and repulsive terms and the importance of the inertia weight were explored. We found that the RPSO method gives similar performance to the particle swarm optimization method. Compared to the genetic algorithm approach used in Autodock 3.05, our RPSO method gives better results in terms of finding lower energy conformations. In the final, third section we have implemented a Monte Carlo (MC) based conformer searching approach into Gaussian03. This enables high level quantum mechanics/molecular mechanics (QM/MM) potentials to be used in docking molecules in a protein active site. This program was tested on two Zn2+ ion-containing complexes, carbonic anhydrase II and cytidine deaminase. The effects of different QM region definitions were explored in both systems. A consecutive and a parallel docking approach were used to study the volume of the active site explored by the MC search algorithm. In case of the carbonic anhydrase II complex, we have used 1,2-difluorobenzene as a ligand to explore the favourable interactions within the binding site. With the cytidine deaminase complex, we have evaluated the ability of the approach to discriminate the native pose from other higher energy conformations during the exploration of the active site of the protein. We find from our initial calculations, that our program is able to perform a conformational search in both cases, and the effect of QM region definition is noticeable, especially in the description of the hydrophobic interactions within the carbonic anhydrase II system. Our approach is also able to find poses of the cytidine deaminase ligand within 1 Å of the native pose.

Métodos computacionais são usados para gerar estruturas de complexo proteína-ligante e estimar suas afinidades. Esse trabalho investigou como as diferentes representações da flexibilidade proteica afetam as poses obtidas por ancoragem molecular e as afinidades atribuídas a essas poses. Os mutantes L99A e L99A/M102Q da lisozima T4 foram escolhidos como sistemas modelo. Um descritor para predição de afinidades baseado na aproximação de energia de interação linear (LIE) foi parametrizado especificamente para ligantes da lisozima e foi usado para estimar as afinidades. A proteína foi representada como um grupo de estruturas cristalográficas ou de estruturas de trajetória de dinâmica molecular. O campo de força OPLS-AA para modelar a proteína e os ligantes e a aproximação de Born generalizada para modelar o solvente foram empregados. O descritor de afinidades parametrizado resultou em desvios médios entre afinidades experimentais e calculadas de 1,8 kcal/mol para um conjunto de testes. O descritor teve desempenho satisfatório na separação entre poses cristalográficas e poses falso-positivo e na identificação de poses falso-positivo. Experimentos de agrupamento de complexos realizados com o objetivo de reduzir o custo computacional para estimar afinidades apresentaram resultados insatisfatórios. As melhores aproximações da teoria do ligante implícito propostas aqui para estimar afinidades consideram conjuntos de estruturas de receptor com o mesmo peso. Configurações de ligante também apresentam o mesmo peso ou são dominadas por uma única configuração. A representação da flexibilidade requer um tratamento estatístico adequado para estimativa de afinidades. Aqui, a associação entre LIE e a teoria do ligante implícito mostrou-se frutífera. / Computational methods are used to generate protein-ligand complex structures and estimate their binding affinities. This work investigated how different representations of protein flexibility affect poses obtained by molecular docking and the affinities attributed to these poses. T4 lysozyme mutants L99A and L99A/M102Q were chosen as model systems. A descriptor for prediction of affinities based on linear interaction energy (LIE) approximation was parametrized specifically to lysozyme ligands and was used to estimate affinities. The protein was represented as a group of crystal structures or as structures from a molecular dynamics trajectory. OPLS-AA force field was used to model protein and ligands and the Generalized Born approximation was used to model solvent. The parametrized affinity descriptor resulted in average deviations between experimental and calculated affinities of 1.8 kcal/mol for a test set. Descriptor performance was satisfactory in the separation between crystal poses and false-positive ones and in the identification of false-positive poses. Clustering of complexes was tried out to reduce computational cost to estimate affinities, but results were poor. The best approximations to the implicit ligand theory proposed here in order to estimate affinities consider groups of receptor structures with the same weight. Ligand configurations also have the same weight or are dominated by only one configuration. The representation of protein flexibility requires an adequate statistical treatment when used to estimate affinities. Here, the linking between LIE and the implicit ligand theory proved itself useful.