Neste trabalho foi realizada a síntese de amidas, bem como de ácidos e ésteres borônicos, derivados dos aminoácidos L-cistina e L-cisteína, através de rota sintética simples, curta e de baixo custo, com o intuito de busca e a identificação de novo(s) inibidor(es) do proteassomo 20S. Esta classe de compostos possui estrutura que permite a inserção de diversos grupos funcionais, o que confere versatilidade e a construção de biblioteca de compostos que contém partes hidrofílicas e hidrofóbicas importantes para posterior avaliação inibitória. Para tanto, empregou-se rota sintética química convencional e rota biocatalisada para a formação da ligação amida. Os compostos derivados de L-cisteína foram obtidos via síntese clássica de peptídeos a qual forneceu os compostos desejados em rendimentos de até 85%. Por outro lado, tentativas de obtenção das amidas via biocatálise não se mostraram efetivas. Já amidas derivadas de L-cistina foram obtidas em rendimentos de até 79%, via síntese tradicional e até 100% de conversão através de rota biocatalítica. A inserção do átomo de boro nas estruturas se deu utilizando-se metodologias sintéticas já bem estabelecidas na literatura. Os ésteres borônicos derivados de L-cisteína foram obtidos em bons rendimentos (até 78%), enquanto que não foi possível obter-se compostos de boro derivados de L-cistina. Por sua vez, os compostos contendo ácido borônico na estrutura foram sintetizados via reação de hidrólise dos respectivos ésteres borônicos, em rendimentos moderados (até 34%). Após a obtenção dos compostos contendo grupamentos organoboro realizou-se avaliação inibitória dos mesmos frente ao proteassomo 20S. Valores de IC50 iguais a 52 µM foram obtidos para composto derivado de L-cisteína contendo grupamento éster borônico, que se mostraram inibidores moderados e reversíveis. Ácidos borônicos se mostraram sem capacidade de inibir o proteassomo 20S. Adicionalmente, realizaram-se estudos de modelagem molecular com a finalidade de elucidar os resultados obtidos experimentalmente. Inicialmente realizaram-se cálculos de modelagem molecular através da realização de docking de alguns compostos e após geração de modelo farmacofórico. De maneira geral observou-se que os inibidores derivados da L-cisteína não ocupam a mesma cavidade que o fármaco bortezomibe, o que pode explicar a diferença na atividade dos compostos frente à inibição do proteassomo 20S. Também se observou que, tendo-se a interação dos inibidores com a enzima, a vizinhança do átomo de boro tem grande influência na capacidade inibitória, uma vez que estes grupamentos determinam qual a região da cavidade do proteassomo 20S será ocupada pelo inibidor. / In our study, amides, boronic acids and esters derivatives from L-cysteine and L-cysteine were synthesized by simple, short and inexpensive synthetic route, in order to search for new inhibitor(s) of the 20S proteasome. This class of compounds has a structure that allows inclusion of various functional groups, giving it versatility and allowing the construction of library compounds containing hydrophilic and hydrophobic moieties, important for further evaluation. To this end, we used conventional chemical synthetic route and biocatalysis for peptide bond formation. The compounds derived from L-cysteine were obtained by classical synthesis of peptides which provided the desired compounds up to 85% yields. On the other hand, attempts to obtain the amides via biocatalysis were not effective. However, amides derived from L-cystine were obtained with up to 79% yields via chemical synthesis and conversion up to 100% using biocatalytic route. The insertion of the boron atom in the structures was possible using synthetic methodologies well established in literature. Boronic esters derived from L-cysteine were obtained in good yields (up to 78%), whereas it was not possible to obtain boron compounds derived from L-cystine. In turn, compounds containing boronic acids in the structure were synthesized by hydrolysis reaction of the respective boronic esters in moderate yields (up to 34%). With organoboron compounds in hand, we turned our attention to inhibitory assessment against the 20S proteasome. IC50 up to 52 µM were obtained when L-cysteine boronic ester derivatives were evaluated. These compounds are moderate and reversible inhibitors. L-cysteine boronic acids derivatives have shown not ability to inhibit the 20S proteasome. Additionally, molecular modeling studies were carried out in order to elucidate the results obtained experimentally. Initially molecular modeling calculations were carried out by performing docking experiments of some compounds. Generation of pharmacophoric model calculations was also executed. In general, it was observed that inhibitors derived from L-cysteine do not occupy the same cavity that drug bortezomib, which may explain the difference in the activity of compounds against the inhibition of 20S proteasome. We also observed that, with the interaction of inhibitors and enzyme, the side chains around boron atom has a great influence on inhibitory capacity, since these groups determine which region of the 20S proteasome cavity is occupied by the inhibitor.

A matriz extracelular e as células musculares lisas das vias aéreas estão intimamente interrelacionadas. Poucos estudos porém, avaliaram a composição dos diferentes componentes da matriz extracelular e seus reguladores na camada do músculo liso brônquico na asma. Utilizando um programa de análise de imagens, a área fracionada do colágeno total e das fibras elásticas foi quantificada no interior do músculo liso brônquico de 35 indivíduos que faleceram devido a um ataque de asma (Asma Fatal), e comparada com 10 casos de indivíduos com asma e que faleceram de outras causas (Asma Não Fatal), e com 22 indivíduos controles sem patologia pulmonar. Expressão dos colágenos I e III, fibronectina, versicam, metaloprotease (MMP)-1, 2, 9 e 12, e inibidores de metaloprotease 1 e 2 foram quantificados no interior do músculo liso brônquico de 22 casos de asma fatal e 10 controles. Nas grandes vias aéreas dos casos de asma fatal, a área fracionada das fibras elásticas foi significativamente maior na camada do músculo liso brônquico quando comparada com os grupos de Asma Não Fatal e Controle. Semelhantemente, fibronectina, MMP-9 e MMP-12 estavam aumentadas no músculo liso das grandes vias aéreas nos casos de asma fatal quando comparadas aos controles. Apenas aumento das fibras elásticas foi observado nas pequenas vias aéreas na Asma Fatal, e somente quando comparadas aos casos de Asma Não Fatal. O conjunto dos resultados mostra que há uma composição alterada dos elementos da matriz extracelular e um ambiente de degradação protéica no músculo liso brônquico de indivíduos que morreram por asma, o qual pode acarretar importantes conseqüências nas funções sintéticas e mecânicas do músculo liso das vias áreas. / There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional area of total collagen and elastic fibres was quantified within the ASM of 35 subjects with Fatal Asthma (FA) and compared with 10 Nonfatal Asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloprotease (MMP)-1, 2, 9 and 12 and tissue inhibitor of metalloprotease-1 and 2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. The results shower that, there is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Orientador: Maria Isabel Nogueira Cano / Resumo: A leishmaniose é uma doença crônica, causada por parasitos flagelados do gênero Leishmania, podendo se apresentar nas formas clínicas, tegumentar (cutânea), mucocutânea e visceral (calazar). A doença é considerada negligenciada pela OMS, pois não existem até o momento métodos eficientes de tratamento e controle para a mesma. Os telômeros desse parasito são um dos potenciais alvos no desenvolvimento de novos fármacos para o combate dessa doença e, para tanto, é necessário o entendimento da biologia desta estrutura. Uma enzima de grande interesse para o estudo dos telômeros é a telomerase que é a responsável pela manutenção e elongação dessas estruturas nos terminais dos cromossomos. A manutenção dos telômeros não é unicamente regulada pelo complexo ribonucleoproteico (RNP) da telomerase, mas também por proteínas que se associam ao complexo e ao DNA telomérico, tornando a ação do complexo mais efetiva e estável. Até o momento, o complexo telomérico de Leishmania amazonensis é o melhor caracterizado dentre os tripanosomatídeos, porém pouco se sabe sobre a biogênese e a composição do complexo RNP telomerase deste parasito. HSP83, ortólogo da HSP90 humana em Leishmania é uma chaperona altamente conservada, dependente de ATP e expressa quando as células são submetidas a diferentes tipos de estresse estando envolvida em transdução de sinal, crescimento, diferenciação celular e sobrevivência. Também é de grande importância para patógenos humanos, em particular aqueles cujo ciclo de v... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leishmaniasis is a chronic disease, caused by flagellated parasites of the genus Leishmania, which could be present in different clinical forms such as, tegumentar (cutaneous), mucocutaneous and visceral (kalazar). WHO classifies leishmaniasis as a neglected disease since there are no efficient methods for disease treatment and control. Parasites telomeres are one of the potential targets for the development of new anti-parasitic drugs to combat this disease and, thus, it is necessary to understand the biology of this structure. Telomerase is the enzyme responsible for maintaining and replicating these structures at the chromosomes termini. However, telomeres maintenance is not only regulated by the telomerase ribonucleoprotein complex (RNP), but also by proteins that associate with the complex and with telomeric DNA, making the action of the complex more effective and stable. To date, the telomeric complex of Leishmania amazonensis is the best characterized among trypanosomatids, although little is known about the biogenesis and composition of the RNP telomerase complex of this parasite. HSP90 is a highly conserved, ATP dependent chaperone and expressed when cells are subjected to different types of stress. It is also involved in signal transduction, growth, cell differentiation and survival of the chaperonin is of great importance for human pathogens, particularly those transmitted by insects to a mammalian host, and which suffer from environmental changes such as temperatu... (Complete abstract click electronic access below) / Mestre

Clostridium difficile (C. difficile) is a gram positive, anaerobic, spore forming bacterium. C. difficile infections are triggered by dysbiosis of the intestinal microbiome linked to age, immune status, and medication; particularly use of antibiotics and proton pump inhibitors (PPI). The spore forming nature of the bacteria gives it the ability to persist in the environment for long periods of time and makes it impervious to many commonly-used hospital cleaning and disinfection products. C. difficile, along with Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE) are some of the leading multi-drug resistant hospital acquired infections in the United States. Environmental contamination and patient susceptibility are hypothesized as major contributors to infection transmission in a healthcare setting. We conducted a cross-sectional pilot study aimed at determining the bioaerosol concentration of C. difficile present in the toilet plume of C. difficile infected patients’ rooms. Patient rooms within the University of Iowa Hospital and Clinics (UIHC) were sampled using a customized bioaerosol air impactor device. Environmental samples were collected before and after flushing the toilet to determine the pre-flush and post-flush levels of aerosolized bacteria. Particle density was collected during both pre and post-flush sampling. Activity levels in the rooms were recorded as a potential confounding variable. A total of 144 environmental samples were collected in 24 rooms. Clostridium difficile was detected in two of the twenty-four rooms (8%). There was a 12% (9/72) positive culture rate pre-flush compared to 23% (19/72) post-flush. Wilcoxon rank sum tests revealed a significant increase in particle concentration at the 5.0µm and 10.0µm size between rooms that produced a bacterial culture compared to rooms that did not (p-values 0.0095 and 0.0082 respectively). There was no significant association between the amount of activity in the room and detectable bioaerosol production (p-value=0.605). Next, we performed a randomized control trial of hospital privacy curtains with antimicrobial properties to determine their ability to resist pathogenic bacterial contamination in an intensive care unit setting. Rooms within the surgical and neurological intensive care unit at UIHC were randomized to receive impregnated curtains, impregnated curtains plus Fuzion hypochlorite spray, or standard control curtains. MRSA, VRE, Pseudomonas spp. and Acinetobacter spp. were the four most frequently cultured pathogenic species. Time to event (contamination) analysis identified a significant difference in time to pathogenic contamination between the control curtains and the impregnated curtains post spray (p-value<0.001). The impregnated curtains post Fuzion spray also grew significantly less colonies of bacteria compared to the control curtains (p-value<0.001). After evaluating environmental risk factors that contribute to Clostridium difficile infection, patient related risk factors for infection were evaluated. Proton pump inhibitors are a class of gastric acid reducers that work by reducing the amount of hydrogen ions produced in the stomach. Recent evidence suggests that prolonged use could negatively affect the intestinal microbiome making it more susceptible to enteric pathogens. A nested case control study was done to determine the association between PPI medication duration and C. difficile infection. Fecal microbiome diversity was analyzed via logistic regression in relation to the development of Clostridium difficile infection. A co-morbidity score was created to adjust for other microbiome altering conditions. PPI duration remained a significant predictor of infection after adjusting for the microbiome influence (p-value=0.0123). Environmental contamination remains a significant risk factor for the transmission of hospital acquired infections including C. difficile. Toilets flushing has been shown to produce pathogenic bioaerosols in the healthcare setting. Hospital privacy curtains have been shown to routinely be contaminated with pathogenic bacteria including other gastrointestinal bacteria that could increase susceptibility to C. difficile infection. PPI medication, which is frequently prescribed in the hospital, has been shown to increase the risk of C. difficile infection, although specific microbiome changes could not be identified.

Imidazolium trans-[tetrachloro (dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, is an experimental metastasis inhibitor whose specific mechanism of activation and action remains to be elucidated. In the nucleophilic and reducing physiological environment; it is anticipated that the most relevant and available reductants upon introduction of NAMI-A as a therapeutic agent will be the biologically-relevant free thiols. The kinetics and mechanisms of interaction of NAMI-A with biologically-active thiols cysteamine, glutathione, cysteine and a popular chemoprotectant, 2-mercaptoethane sulfonate (MESNA) have been studied spectrophotometrically under physiologically-relevant conditions. The reactions are characterized by initial reduction of NAMI-A with simultaneous formation of dimeric thiol and subsequent ligand exchange with water to various degrees as evidenced by Electospray Ionization Mass Spectrometry. Stoichiometry of reactions shows that one molecule of NAMI-A reacted with one mole of thiol to form corresponding disulfide cystamine, dimeric MESNA, oxidized glutathione and cystine. Observed rate constants, ko, for the reaction of NAMI-A with cysteamine, MESNA, GSH and cysteine were deduced to be 6.85 + 0.3 x 10-1, 9.4 + 0.5 x 10-2 , 7.42 + 0.4 x 10-3 and 3.63 + 0.3 x 10-2 s-1 respectively. Activation parameters determined from Arrhenius plots are indicative of formation of associative intermediates prior to formation of products. A negative correlation was obtained from the Brønsted plot derived from observed rate constants and the pKa of the different thiols demonstrating significant contribution of thiolate species towards the rate. In conclusion, interactions of NAMI-A with biologically-active thiols are kinetically and thermodynamically favored and should play significant roles in in vivo metabolism of NAMI-A.

Myeloid Derived Suppressor Cells (MDSC) represent a significant hurdle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous studies have reported on the myelo-depletive effects of certain chemotherapies. Using guadecitabine, a next-generation DNA methyltransferase inhibitor (DNMTi), we observed significantly reduced tumor burden in the 4T1 murine mammary carcinoma model. Guadecitabine treatment prevents excessive tumor-induced myeloid proliferation and systemic accumulation, and skews remaining MDSCs toward a beneficial antigen-presenting phenotype. Together, this alters the splenic environment to improve T cell activation and interferon-gamma (IFNg) production. Additionally, guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. Based on these findings, the immune-modulatory effects of guadecitabine can help rescue the anti-tumor immune response and could contribute to the overall effectiveness of current cancer immunotherapies. Allergies and asthma are common ailments that are on the rise around the world. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. In studies using ADAM17-deficient mast cells, percent degranulation and cytokines released by IgE-mediated activation were significantly reduced. Interestingly, ionomycin-activation was unchanged, suggesting ADAM17 may be involved in IgE-mediated mast cell activation upstream of calcium release. Additionally, ADAM17MC-/- mice showed protection from IgE-, but not histamine-, mediated passive systemic anaphylaxis (PSA). The underlying mechanism behind the reduced degranulation occurs through signaling deficiencies downstream of Lyn phosphorylation. Together, the data suggest that ADAM17 is required for proper mast cell signaling through its interaction with the Src family kinase, Lyn.

Peptides are emerging as promising therapeutics due to their inhibitory affinity towards protein-protein interactions (PPI). However, peptides have been limited mainly by their poor bio-stability and lack of cell permeability. Efforts to generate drug-like peptides have led to the development of macrocyclic peptides, which exhibit improved stability. Yet, most macrocyclic peptides still require the assistance of a cell penetrating peptide (CPP) for cellular entry. High throughput technologies have been exceptional tools for the discovery of peptides to interrupt PPIs. This work details the recent advancements we have made to improve our high throughput technique, mRNA display, to yield more therapeutically relevant peptides to inhibit PPIs. Our advancements are focused on cell permeability, protease stability, and secondary structure for enhanced affinity. Here we develop and optimize a cyclic CPP that can be included in future mRNA display libraries. We also tested the ability of our CPP to deliver an impermeable peptide cargo into cells. We rationally designed and tested linear and cyclic peptides to improve affinity to the BRCA1 protein. We used computational work to complement our experimental results for our CPPs and BRCA1 inhibitors. We examined peptides that arose from a library containing a mix of linear, monocyclic, and bicyclic peptides constructed using orthogonal cyclization chemistries. We rationally designed cyclic peptides and tested their affinity against Hsp70. We proposed a novel selection strategy to find optimal CPP motifs.

The use of the versatile universal adhesive systems aims to improve adhesion to dentin and simplify the bonding procedure. The association between functional monomer as 10-methacryloyloxydecyl-dihydrogen phosphate (10-MDP) and proteolytic inhibitors seems to be a promising strategy to improve the longevity of hybrid layer. Therefore, this study aimed to evaluate the performance of Adper Single Bond Universal (SU) combined with proteolytic inhibitors, especially chlorhexidine (CHX), in different dentin substrates overtime. In article 1, the interaction between CHX and E-64 with SU (in etch-and-rinse mode) was investigated in sound, artificial carious and eroded dentin over 18 months aging. It was found that carious substrate was the most affected and none of the inhibitors tested were able to maintain stability over 18 months. Furthermore, it was observed that CHX negatively impacted regardless of the substrate, leading to the hypothesis of possible competition between CHX and 10-MDP, since both involve calcium ions in their mechanism of action. For a better comprehension, article 2 purposed the evaluation of proteolytic activity and bonding to dentin tests, focusing on the performance of SU in both modes (etch-and-rinse and self-etching) compared to a conventional MDP-free 2- step adhesive system (Adper Single Bond 2), associated with CHX over 6 months aging. It was observed that proteolytic activity was evidenced when all dentin bonding systems (DBS) was used. SU in self-etching mode showed the highest values of microtensile bond strength. CHX was able to reduce proteolytic activity, regardless of DBS even in 6 months aging. Moreover, CHX did not affect negatively mechanical properties. In conclusion, CHX is capable of reduce proteolytic activity, however it did not provide long lasting up to 18 months. / A utilização dos versáteis sistemas adesivos universais tem por objetivo melhorar à adesão à dentina e simplificar o procedimento adesivo. A associação entre monômeros funcionais como o 10-Metacriloiloxidecil dihidrogênio fosfato (10-MDP) e inibidores proteolíticos tende a ser uma estratégia promissora para melhorar a longevidade da camada hibrida. Desta forma, este trabalho tem por objetivo avaliar o desempenho do Adper Single Bond Universal (SU) combinado com inibidores proteolíticos, especialmente a clorexidina (CHX), em diferentes substratos dentinários ao longo do tempo. No artigo 1, a interação entre CHX e E-64 com SU (modo convencional) foi investigada em dentina sadia, artificialmente cariada e erodida em 18 meses de envelhecimento. Foi encontrado que o substrato cariado foi o mais afetado e nenhum dos inibidores testados foram capazes de manter a estabilidade ao longo de 18 meses. Além do mais, observou-se que a CHX impactou de forma negativa independente do substrato avaliado, levando a hipótese de uma possível competição entre ela e o 10-MDP, visto que ambos envolvem íons Ca em seus mecanismos de ação. Para uma melhor compreensão, no artigo 2 foi proposto testes para avaliação de atividade proteolítica e resistência de união à dentina, com foco no desempenho do SU nos dois modos (convencional e autocondicionantes) comparado a um convencional de 2 passos livre de MDP (Adper Single Bond 2), associados com CHX em 6 meses de envelhecimento. Foi observado que a atividade proteolítica foi evidente em todos os sistemas adesivos (SA). O SU no modo autocondicionantes apresentou os maiores valores de resistência de união. A CHX foi capaz de reduzir a atividade proteolítica, independente dos SA mesmo em 6 meses de envelhecimento. Além disso, a CHX não afetou negativamente as propriedades mecânicas. A CHX é capaz de reduzir a atividade proteolítica, no entanto não perdura até 18 meses.

Lactacystin is a microbial metabolite isolated by Omura that exhibits neurotrophic activity in neuroblastoma cell lines. Lactacystin and especially its β-lactone analog are the first examples of non-polypeptide small molecules capable of specifically inhibiting the 20S proteasome. Various asymmetric total syntheses of lactacystin and its analogs have been reported. The total synthesis of clasto -lactacystin β-lactone is achieved using L-serine methyl ester as the starting material and the sole source of stereochemical induction. The success of this synthesis hinges on two featured transformations. The first key step involves formation of the γ -lactam core via rhodium (II) catalyzed intramolecular C-H insertion of the α-diazo-α-(phenylsulfonyl)acetamide intermediate. The methodology for this transformation has been developed and applied to the synthesis of highly functionalized stereogenic γ-lactams from natural α-amino acids. Three control elements that govern γ-lactam formation are described. This step is highlighted by the xvi simultaneous creation of two stereogenic centers of the γ-lactam core. The second key step involves the late stage aldol coupling for quaternary carbon formation and installation of the hydroxyisobutyl group. In all previously reported syntheses, this is the very first aspect which is addressed. The stereochemical outcome of this step is directed by the chiral environment of the enolate itself. Various attempts to achieve selectivity are explored and reported. Completion of the synthesis of clasto-lactacystin β-lactone requires 17 steps with an overall yield of 10%. Some general attempts for optimizing the synthetic scheme are discussed as well as the future direction of this research.

Im Mittelpunkt dieser Arbeit stand die Entwicklung neuer Wirkstoffe gegen Tuberkulose, einer schwerwiegenden bakteriellen Infektionskrankheit, die am häufigsten die Lunge befällt. Die Entwicklung neuer Arzneistoffe gegen diese Erkrankung ist immens wichtig, da nach Angaben der WHO weltweit jährlich über 1 Million Menschen an den Folgen der Tuberkulose sterben, derzeit kein effizienter Impfstoff zur Verfügung steht und sich die Therapiemöglichkeiten auf wenige Arzneistoffe beschränken. Zudem steigt weltweit das Auftreten von arzneistoff- und totalresistenten Tuberkuloseformen. Tuberkulose wird vorwiegend durch das Mycobacterium tuberculosis erregt. Eine Besonderheit des M. tuberculosis stellt die mykobakterielle Zellwand dar, da diese durch einen hohen Anteil an Fettsäuren besonders wachsartig und dick ist. Die mykobakterielle Fettsäuresynthese unterscheidet sich signifikant von der Synthese eukaryotischer Fettsäuren. Daher besteht die Möglichkeit, Inhibitoren der mykobakteriellen Fettsäuresynthese als effektive und selektive neue Antituberkulotika zu entwickeln. Zielstruktur dieser Arbeit ist KasA (β-Ketoacylsynthase), ein Enzym der mykobakteriellen Fettsäuresynthese II, das die Kondensation zwischen der wachsenden Fettsäurekette und Malonyl-ACP katalysiert. Ein literaturbekannter KasA-Inhibitor ist Thiolactomycin, ein Thiolacton-Derivat mit einer schwachen inhibitorischen Aktivität (IC50: 242 µM; Kd 226 µM), für den eine KasA-Komplexstruktur verfügbar ist. Ziel der Arbeit war es, mittels computergestützten Wirkstoffdesigns neue Leitstrukturen für KasA-Inhibitoren zu entwickeln und davon abgeleitet Substanzbibliotheken kleiner Moleküle zu synthetisieren. Zur Bestimmung der In-vitro-Aktivitäten sollte KasA exprimiert und ein Assay etabliert werden. Theoretische und experimentelle Affinitäten sollten anschließend analysiert und bewertet werden. Zur Identifizierung neuer potenzieller KasA-Inhibitoren wurde mit Hilfe des Thiolactomycin-Bindemodus ein Pharmakophor-Modell erstellt. In diesem wurden die essentielle Wasserstoffbrücke zwischen den Histidinen und dem Carbonyl-Sauerstoff des Thiolactonrings, zwei hydrophobe Bereiche und ein verbindendes Strukturelement definiert und das Volumen des Pharmakophors begrenzt. Das Screening der Datenbank erfolgte mit GOLD4.0 und GOLDscore. Zur Identifizierung der 16 aussichtsreichsten Verbindungen wurden Rescorings mit ChemScore und sfc_score290m durchgeführt, sowie verschiedene physikochemische Deskriptoren und der errechnete Bindungsmodus einbezogen. Ausgewählte Verbindungen des Screenings wurden synthetisiert. Weitere Variationen wurden durch Einführung von Substituenten und Bromierung und Nitrierung der Grundgerüste erhalten. Zur biologischen Testung dieser Verbindungen konnte KasA in M. smegmatis exprimiert werden. Die Reinigung des Proteins erfolgte mittels Affinitäts- und Größenausschlusschromatographie. Affinitätswerte an KasA konnten mit einem Fluoreszenzassays bestimmt werden, da in jedem KasA-Monomer vier Tryptophane zur intrinsischen Fluoreszenz beitragen. Die Bindung eines Inhibitors in die TLM-Bindetasche führte zum Quenching der Fluoreszenz von KasA und konnte unter Berücksichtigung von Verdünnungs- und inneren Filtereffekten zur Berechnung der Dissoziationskonstante Kd herangezogen werden. Die In-vitro-Untersuchungen der Inhibitoren von KasA zeigten im Vergleich zu TLM eine Verbesserung der Affinität bis zu einem Faktor von 11, die beste Verbindung war das Nitroisatin-Derivat 2l (22.1 µM). Einen Hinweis auf Hemmung des Wachstums von Mykobakterien war für die Verbindungen 2e (5-Nitro-1-phenethyl-2,3-indolindion) und 3a (5,7-Dibrom-1-(4-chlorbenzyl)indolin-2,3-dion) ersichtlich. Die übrigen Verbindungen zeigten keine Aktivität, was dadurch bedingt sein kann, dass sie Substanzen nicht lipophil genug sind (clogP-Werte zwischen 1 und 3), um die mykobakterielle Zellwand zu durchdringen. Analog dem Docking im Rahmen des virtuellen Screenings wurde ein Docking mit GOLD4.0 und GOLDscore für die Substanzbibliothek durchgeführt. Verglichen mit den In-vitro-Affinitäten konnte eine gute Übereinstimmung in der Differenzierung der Substanzklassen gefunden werden. Da kleine Moleküle mit großer biologischer Aktivität zu bevorzugen sind, wurde die „ligand efficiency“, die inhibitorische Potenz unabhängig vom Molekulargewicht, für die Verbindungen berechnet. Für die Substanzbibliothek wurde eine gute Korrelation von „ligand efficiency“ und GOLDscore pro Schweratom erzielt (R2=0.65), beste Substanzgruppen waren monoalkylierte Uracil- und Isatin-Derivate. Der beste Wert wurde für das Isatin-Derivat 1a erzielt. Mit den erarbeiteten theoretischen und experimentellen Ergebnissen und den etablierten Methoden bietet diese Arbeit eine wichtige Grundlage, um erste „hits“ von KasA-Inhibitoren zu neuen Leitstrukturen für Wirkstoffe gegen Mycobakterium tuberculosis zu entwickeln. / This work focused on the development of new antibiotics against tuberculosis, a severe bacterial infection mainly affecting the lung. Currently, according to the WHO more than 1 million people annually die from tuberculosis. Furthermore, the therapy is limited to inefficient vaccines and a small number of antibiotics, and complicated by multi- or even totally resistant mycobacterial strains occurring worldwide. Thus, new active compounds against tuberculosis are urgently needed. Tuberculosis is mainly caused by Mycobacterium tuberculosis, which is characterized by a unique thick and waxy cell wall containing a high percentage of mycolic acids. Due to the fact that the biosynthesis of mycolic acids is not carried out in eukaryotes, it is a reasonable strategy to design inhibitors of the FAS II system as effective and selective antibiotics against mycobacteria. The enzyme of interest in our work is the β-keto-acyl ACP synthase (KasA), an elongating enzyme in the FAS II system of Mycobacterium tuberculosis which catalyses the condensation between the mycolic acid and malonyl-ACP. Recently, a crystal structure of KasA in complex with Thiolactomycin, a weak thiolactone-type inhibitor (IC50: 242 µM; Kd 226 µM), was solved. Aim of this work was to identify new potential lead structures for KasA-inhibitors by virtual screening. A library of small molecules was synthesized and tested for ability to inhibit KasA, therefore KasA was expressed. In silico and in vitro affinities were analyzed and compared. To identify new lead structures for potential KasA inhibitors, a pharmacophore model based on TLM was developed. This contained the essential H-bond between the carbonyl-oxygen of TLM with the histidines, two hydrophobic features and a linker feature between them. Additionally, volume constraints were applied to limit the size of molecules matching the pharmacophore model. Screening of a database of commercially available compounds was performed with GOLD4.0 and GOLDscore. 16 Promising structures were identified by implementation of rescorings with ChemScore and sfc_score290m, by calculation of physicochemical descriptors and by visual inspection of the predicted binding mode. Selected substances of the virtual screening were synthesized. Based on these substances the core fragments were varied by bromination and nitration. Via subsequent introduction of substituents a small library of compounds was created. For biological testings KasA was expressed in M. smegmatis. Purification of the protein was achieved by affinity and size exclusion chromatography. Dissociation constants were determined by a fluorescence assay: In each KasA monomer four tryptophanes cause intrinsic fluorescence, thus binding of inhibitors led to quenching of the fluorescence. Therefore, dissociation constants of ligands were calculated considering the dilution and inner filter effects. The in vitro studies of the KasA inhibitors showed, in comparison to TLM, a 11fold improvement of the affinity. The best inhibitor was the nitroisatine derivative 2l (22.1 µM). 2e (5-Nitro-1-phenethyl-2,3-indolindione) and 3a (5,7-Dibromo-1-(4-chlorbenzyl)indolin-2,3-dione) were able to inhibit the growth of mycobacteria. No other substances showed any antimycobacterial activity, which might be due to their low lipophilicity (clogP varies from 1 to 3), hence which hinders an efficient penetration through the highly lipophilic mycobacterial cell wall. In the future, the precise cause of this fact has to be determined to counteract with systematic structural modifications. The compound library was docked into KasA by using GOLD4.0 and GOLDscore with analogous settings as in the virtual screening. The analysis of the results showed an agreement between in vitro and in silico outcomes for the substance classes. As small molecules of high activity are preferred in drug development, ligand efficiencies of the inhibitors were calculated which describe inhibitory potency independent of molecular weight. A good correlation between ligand efficiency and GOLDscore per heavy atom was observed. Best ligand efficiencies were obtained by the classes of monoalkylated uracile- and isatine-derivatives, the best substance was the isatine-derivative 1a. Due to the established methods combined with computer-based and experimental results, this work provides an important foundation for the future development of first “hits” of KasA-inhibitors to new lead structures of new drugs against mycobacterium tuberculosis.