Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Neste trabalho foi preparada uma série de onze brometo de 2-[(4-X-benzamido) etil]benzildimetilamônio, compostos I.1-I.11, série I, estruturalmente análogos à procainamida, não descritos na literatura. Os valores do coeficiente de partição, log Papp7.40, destes compostos foram determinados pelo método shake-flask e foram utilizados como parâmetro lipofílico experimental. Os valores de deslocamento químico do grupamento carbonila, δ 13 C=O, foram determinados e foram determinados cm um espectrómetro de ressonância magnética nuclear a 75 MHz. Adicionalmente, outros parâmetros físico-químicos foram retirados da literatura: π, σp, Τ, R e MR4 ou obtidos por cálculo: log Pcalc e πexp. Os valores da concentração inibitória média (IC50) capaz de reduzir a contração máxima a 50% no período de 15 minutos, foram determinados em preparações nervo frênico-músculo diafragma de camundongos. Para verificar a natureza e a contribuição relativa dos parâmetros físico-químicos frente ao bloqueio da transmissão neuromuscular foi feito uma análise de QSAR, obtendo-se equações, usando análise de regressão linear. As análises de QSAR sugerem uma dependência positiva da lipofilicidade para o bloqueio da transmissão neuromuscular expresso por pIC50, segundo o modelo proposto expresso pela equação: (Ver arquivo PDF). / In this work, a set of eleven 2-[(4-X-benzamido)ethyl]benzyl dimethylammonium bromide structurally related to procainamide was synthesized. The apparent partition coefficient values were determined by means of \"shake-flask method and were taken as lipophilic parameters. The carbonyl chemical shifts values were determined in methanol-d4 and taken as electronic parameters. Additionally, physicochemical parameters were either taken from literature: π, σp, Τ, R e MR4 or calculated: log Pcalc and πexp. The median inhibitory concentration values (IC50) able to reduce maximal contraction to 50% at 15 minutes was determined in phrenic nerve-diafragm muscle preparation of mices. In order to verify the nature and relative contribution of the physicochemical parameters to neuromuscular blockage, QSAR equations were derived using regression analysis. The obtained QSAR model, expressed by the equation below, suggest that lipophilicity term plays an important role to neuromuscular blockage. (See files PDF).

Orientador: Clodoaldo Machado. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Exatas e Naturais, Programa de Pós-Graduação em Química.

Die Malaria und andere Infektionskrankheiten sind immer noch die Haupttodesursache in Entwicklungsländern. Durch das jahrzehntelange Versäumnis, neue Wirkstoffe zu entwickeln, und durch die rasante Ausbreitung von Resistenzen gegen herkömmliche Medikamente sind in vielen Regionen der Erde besorgniserregende Zahlen über Neuinfektionen und Todesfälle zu beobachten. Die Suche nach neuen Wirkstoffen ist daher dringend erforderlich und die Hauptaufgabe des Sonderforschungsbereichs 630 an der Universität Würzburg. An diesem interdisziplinären Projekt beteiligt sich unsere Forschungsgruppe vor allem mit der Naturstoffklasse der Naphthylisochinolin-Alkaloide. Neben ihren interessanten strukturellen Eigenschaften haben mehrere Vertreter dieser Sekundärmetabolite vielversprechende Aktivitäten gegen Plasmodien, Leishmanien und Trypanosomen. Dioncophyllin C (24), das bisher wirksamste Naphthylisochinolin gegen P. falciparum, zeigt nicht nur eine exzellente Aktivität in vitro, sondern auch in vivo. In Kooperation mit der Forschergruppe von K. Baumann (Braunschweig) führte man QSAR-Studien durch, um die für die biologische Wirkung entscheidenden Strukturmerkmale zu identifizieren und neue vereinfachte Analoga der Leistruktur 24 vorzuschlagen. Ziel der vorliegenden Arbeit war aufbauend auf Vorarbeiten in unserer Gruppe die Darstellung von strukturell vereinfachten Derivaten des Naturstoffs 24. Die Ergebnisse der biologischen Untersuchungen sollten ausgewertet und somit neue Struktur-Wirkungs-Beziehungen aufgestellt werden. Weiterhin sollten auch Chinolinium-Salze, die man als Analoga der N,C-verknüpften Naphthylisochinoline ansehen kann, synthetisiert werden und innerhalb des SFB 630 und bei unseren Partnern am Schweizerischen Tropen- und Public-Health-Institut auf ihre biologische Aktivität untersucht werden. Man erhoffte sich neben möglichen antiinfektiven Eigenschaften auch Rückschlüsse auf Struktur-Wirkungs-Beziehungen. Zusätzlich sollte die synthetische und analytische chemische Expertise unseren Kooperationspartnern in zwei Projekten außerhalb des SFB 630 zur Verfügung gestellt werden. Dabei handelte es sich einerseits um die Strukturaufklärung von Biosyntheseintermediaten mit Hilfe der HPLC-NMR-Kopplung und andererseits um die Darstellung langkettiger Aldehyde für die biologische Untersuchung des Prä-Penetrationsprozesses eines getreideschädigenden Pilzes. / Malaria and other infectious diseases are still the most common cause of death in developing countries. Due to the failure of developing new drugs and the increasing resistance there are alarming numbers of new incidences and death cases in many regions of the world. The search for urgently needed drugs is the main task of the Sonderforschungsbereich 630 (SFB 630) of the University of Würzburg. Our research group participates in this interdisciplinary project with the naphthylisoquinoline alkaloids. Besides their interesting structural properties several of these secondary metabolites also have promising activities against plasmodia, leishmania, and trypanosoma. Dioncophylline C (24), the so far most active naphthylisoquinoline alkaloid against P. falciparum, does not only show activity in vitro but also in vivo. In cooperation with the research group of K. Baumann (Braunschweig) QSAR studies were accomplished in order to find the structural features that are important for the biological effect and to suggest new structurally simplified analogs of the lead structure 24. Based on previous work in our group the main task of this thesis was the synthesis of structurally simplified derivatives of the natural product 24. The results of the biological investigations were to be analyzed and new structure-activity relationships were to be established. Furthermore, quinolinium salts, which can be seen as analogs of the N,C-coupled naphthylisoquinolines, were to be synthesized and tested for their biological properties by our external partners of the Swiss Tropical and Public Health Institute and within the SFB 630. Besides the possible antiinfective activities conclusions on structure-activity relationship studies were of interest. In addition the synthetic and analytical expertise was to be offered to two of our external cooperation partners. The projects were on the one hand the structure elucidation of biosynthetic intermediates using the HPLC-NMR technique and on the other hand the synthesis of very-long chain aldehydes for biological investigations of the pre-penetration process of a cereal-damaging fungus.

<p>This thesis presents a novel bioinformatic methodology denoted the bio-chemometric approach. The methodology is designed for generation of detailed descriptions and predictions of biomolecular interactions. It is based on multivariate analysis of the sensitivity of a biomolecular interaction to multiple minor changes in the experimental conditions. In this work, either the chemical environment where the interaction takes place, or the molecular structure of one of the interacting molecules, was varied. The sensitivity of the interaction to the performed variations was presented as a vector called the sensitivity fingerprint. The bio-chemometric approach was tested on several biomolecular interactions. Useful descriptions of the interactions were obtained by measuring binding kinetics for each interaction in 12-20 different buffers and correlating buffer composition to binding kinetics. The obtained chemical sensitivity fingerprints were reproducible, significantly different and showed a weak correlation to binding site properties for the tested interactions. The results indicate that the fingerprints contained useful information about the binding site. The predictive ability of the bio-chemometric approach was tested on two different biomolecular interactions where one of the binding partners was slightly modified into multiple analogues by amino acid exchanges. In one example, interactions of 18 peptide analogues with an antibody gave data that could be used for accurate prediction of the dissociation rates of novel analogues. Reliable predictions of binding kinetics and affinity were also obtained for single domain camel antibody analogues binding to a protein antigen. By using the three-dimensional structure of camel antibodies and data obtained using the bio-chemometric approach, even the importance of non-exchanged amino acids for the binding could be estimated. The bio-chemometric approach can potentially improve the development of peptides and proteins for therapeutic and diagnostic use. It is suggested to be valid for general use in biochemistry.</p>

This thesis presents a novel bioinformatic methodology denoted the bio-chemometric approach. The methodology is designed for generation of detailed descriptions and predictions of biomolecular interactions. It is based on multivariate analysis of the sensitivity of a biomolecular interaction to multiple minor changes in the experimental conditions. In this work, either the chemical environment where the interaction takes place, or the molecular structure of one of the interacting molecules, was varied. The sensitivity of the interaction to the performed variations was presented as a vector called the sensitivity fingerprint. The bio-chemometric approach was tested on several biomolecular interactions. Useful descriptions of the interactions were obtained by measuring binding kinetics for each interaction in 12-20 different buffers and correlating buffer composition to binding kinetics. The obtained chemical sensitivity fingerprints were reproducible, significantly different and showed a weak correlation to binding site properties for the tested interactions. The results indicate that the fingerprints contained useful information about the binding site. The predictive ability of the bio-chemometric approach was tested on two different biomolecular interactions where one of the binding partners was slightly modified into multiple analogues by amino acid exchanges. In one example, interactions of 18 peptide analogues with an antibody gave data that could be used for accurate prediction of the dissociation rates of novel analogues. Reliable predictions of binding kinetics and affinity were also obtained for single domain camel antibody analogues binding to a protein antigen. By using the three-dimensional structure of camel antibodies and data obtained using the bio-chemometric approach, even the importance of non-exchanged amino acids for the binding could be estimated. The bio-chemometric approach can potentially improve the development of peptides and proteins for therapeutic and diagnostic use. It is suggested to be valid for general use in biochemistry.

Over a billion individuals worldwide suffer from neglected diseases. This equates to approximately one-sixth of the human population. These infections are often endemic in remote tropical regions of impoverished populations where vectors can flourish and infected individuals cannot be effectively treated due to a lack of hospitals, medical equipment, drugs, and trained personnel. The few drugs that have been approved for the treatments of such illnesses are not widely used because they are riddled with inadequate implications of cost, safety, drug availability, administration, and resistance. Hence, there exists an eminent need for the design and development of improved new therapeutics. Influential world-renowned scientists in the Consortium for Parasitic Drug Development (CPDD) have preformed extensive biological testing for compounds active against parasites that cause neglected diseases. These data were acquired through several collaborations and found applicable to computational studies that examine quantitative structure-activity relationships through the development of predictive models and explore structural relationships through docking. Both of these in silico tools can contribute to an understanding of compound structural importance for specific targets. The compilation of manuscripts presented in this dissertation focus on three neglected diseases: trypanosomiasis, Chagas disease, and leishmaniasis. These diseases are caused by kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., respectively. Statistically significant predictive devices were developed for the inhibition of the: (1) T. brucei P2 nucleoside transporter, (2) T. cruzi parasite at two temperatures, and (3) two species of Leishmania. From these studies compound structural importance was assessed for the targeting of each parasitic system. Since these three parasites are all from the Order Kinetoplastida and the kinetoplast DNA has been determined a viable target, compound interactions with DNA were explored to gain insight into binding modes of known and novel compounds.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès En aquesta tesis es pretén aplicar metodologies computacionals (generació de farmacòfors i docking proteïna lligand) en l'àmbit de la nutigenòmica (ciència que pretén entendre, a nivell molecular, com els nutrients afecten la salut). S'aplicaran metodologies in silico per entendre a nivell molecular com productes naturals com els compostos fenòlics presents en la nostra dieta, poden modular la funció d'una diana comportant un efect en la salut. Aquest efecte es creu que podria ser degut a la seva interacció directa amb proteïnes de vies de senyalització molecular o bé a la modificació indirecta de l'expressió gènica. Donat que el coneixement de l'estructura del complex lligand-receptor és bàsic per entendre el mecanisme d'acció d'aquests lligands s'aplica la metodologia docking per predir l'estructura tridimensional del complex. En aquest sentit, un dels programes de docking és AutoGrid/AutoDock (un dels més citats). No obstant, l'automatització d'AutoGrid/AutoDock no és trivial tan per (a) la cerca virtual en una llibreria de lligands contra un grup de possibles receptors, (b) l'ús de flexibilitat, i (c) realitzar un docking a cegues utilitzant tota la superfície del receptor. Per aquest motiu, es dissenya una interfície gràfica de fàcil ús per utilitzar AutoGrid/AutoDock. Blind Docking Tester (BDT) és una aplicació gràfica que s'executa sobre quatre programes escrits en Fortran i que controla les condicions de les execucions d'AutoGrid i AutoDock. BDT pot ser utilitzat per equips d'investigadors en el camp de la química i de ciències de la vida interessats en dur a terme aquest tipus d'experiments però que no tenen suficient habilitats en programació. En la modulació del metabolisme de la glucosa, treballs in vivio i in vitro en el nostre grup de recerca s'han atribuït els efectes beneficiosos de l'extracte de pinyol de raïm en induir captació de glucosa (punt crític pel manteniment de l'homeostasis de la glucosa). No obstant alguns compostos fenòlics no tenen efecte en la captació de la glucosa, d'altres l'inhibeixen reversiblement. En alguns casos aquesta inhibició és el resultat de la competició dels compostos fenòlics amb ATP pel lloc d'unió de l'ATP de la subunitat catalítica de la fosfatidil inositol 3-kinasa (PI3K). Estudis recents amb inhibidors específics d'isoforma han identificat la p110&#945; (la subunitat catalítica de PI3K&#945;) com la isoforma crucial per la captació de glucosa estimulada per insulina en algunes línies cel·lulars. Els programes computacionals han estat aplicats per tal de correlacionar l'activitat biològica dels compostos fenòlics amb informació estructural per obtenir una relació quantitativa estructura-activitat (3D-QSAR) i obtenir informació dels requeriments estructura-lligand per augmentar l'afinitat i/o selectivitat amb la diana (proteïna). Tot hi haver-se demostrat que l'adició d'extractes de compostos fenòlics en l'aliment pot tenir en general un benefici per la salut, s'ha de tenir en compte que l'estudi 3D-QSAR (construït a partir d'inhibidors sintètics de p110&#945;) prediu que algunes d'aquestes molècules poden agreujar la resistència a la insulina en individus susceptibles dificultant la capatació de glucosa en múscul i teixit adipós i, per tant, produir un efecte secundari indesitjat. Resultats en el nostre grup de recerca han demostrat que compostos fenòlics presents en extractes de llavor de raïm incrementen l'activitat del receptor "farnesoid x receptor" (FXR) de manera dosi depenent quan el lligand natural de FXR (CDCA) és present. Les metodologies in silico, docking i 3D-QSAR, han estat aplicades juntament amb dades biològiques d'agonistes no esteroidals de FXR que s'uneixen a un lloc d'unió proper però diferent al lligand esteroidal 6CDCA. Els resultats han mostrat que els compostos fenòlics no són capaços d'activar FXR per ells mateixos però poden afegir noves interaccions que estabilitzarien la conformació activa de FXR en presència del lligand natural CDCA. Els compostos fenòlics podrien induir canvis conformacionals específics que augmentarien l'activitat de FXR. In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès This thesis was written with the aim of applying computational methods that have already been developed for molecular design and simulation (i.e. pharmacophore generation and protein-ligand docking) to nutrigenomics. So, in silico tools that are routinely used by the pharmaceutical industry to develop drugs have been used to understand, at the molecular level, how natural products such as phenolic compounds (i.e. molecules that are commonly found in fruits and vegetables) can improve health and prevent diseases. Therefore, we first focused on predicting the structure of protein-ligand complexes. The docking algorithms can use the individual structures from receptor and ligand to predict (1) whether they can form a complex and (2) if so, the structure of the resulting complex. This prediction can be made, for instance, with AutoGrid/AutoDock, the most cited docking software in the literature. The automation of AutoGrid/AutoDock is not trivial for tasks such as (1) the virtual screening of a library of ligands against a set of possible receptors; (2) the use of receptor flexibility and (3) making a blind-docking experiment with the whole receptor surface. Therefore, in order to circumvent these limitations, we have designed BDT (i.e. blind-docking tester; http://www.quimica.urv.cat/~pujadas/BDT), an easy-to-use graphic interface for using AutoGrid/AutoDock. BDT is a Tcl/Tk graphic front-end application that runs on top of four Fortran programs and which controls the conditions of the AutoGrid and AutoDock runs. As far as the modulation of the glucose metabolism is concerned, several in vivo and in vitro results obtained by our group have shown that grape seed procyanidin extracts (GSPE) stimulate glucose uptake in 3T3-L1 adipocytes and thus help to maintain their glucose homeostasis. In contrast, it is also well known that although some phenolic compounds do not affect glucose uptake, others reversibly inhibit it in several cell lines. Moreover, for at least some of these phenolic compounds, this inhibition is the result of their competition with ATP for the ATP-binding site in p110&#945; (i.e. the &#945; isoform of the catalytic subunit of phosphoinositide 3-kinase or PI3K&#945;). Furthermore, recent studies with isoform-specific inhibitors have identified p110&#945; as the crucial isoform for insulin-stimulated glucose-uptake in some cell lines. Therefore, although it has been proved that the addition of phenolic compound extracts to food can have an overall benefit on health, it should be taken into account that some of these molecules may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in muscle and adipose tissues and, therefore, produce an undesirable side effect. In this context, we have applied computational approaches (i.e. protein-ligand docking and 3D-QSAR) to predict the IC50 (i.e. the concentration that reduces the p110&#945; activity to 50%). Our results agree with previous experimental results and predict that some compounds are potential inhibitors of this enzyme. Recent results in our research group have demonstrated that the phenolic compounds in GSPE increase the activity of the farnesoid X receptor (i.e. FXR) in a dose-dependent way when the natural ligand of FXR (i.e. CDCA) is also present. The phenolic compounds might induce specific conformational changes that increase FXR activity and then contribute to cardioprotection through mechanisms that are independent of their intrinsic antioxidant capacities but that involve direct interaction with FXR to modulate gene expression. Taking into account this hypothesis a 3D-QSAR analysis was made in an attempt to understand how phenolic compounds activate FXR. So, our results explain why phenolic compounds cannot activate FXR by themselves and how they can add new interactions to stabilize the active conformation of FXR when its natural ligand (i.e. CDCA) is present. Therefore, we proposed a mechanism of FXR activation by dietary phenolic compounds in which they may enhance bile acid-bound FXR activity.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants known for their photoinduced toxicity. There are two mechanisms through which this may occur: photosensitization and photomodification. Photosensitization generally leads to the production of singlet oxygen, a reactive oxygen species (ROS), which is highly damaging to biological molecules. Photomodification of PAHs, usually via oxygenation, results in the formation of new compounds (oxyPAHs), and can occur under environmentally relevant levels of actinic radiation. <br /><br /> PAHs and oxyPAHs readily adsorb to the organic phase of particulate matter in the environment such as sediments. It is logical to conclude that sediment transport will also facilitate the transport of these contaminants, and it has been shown that in the course of transport, degradative processes evoke a change in the profile of the PAHs present. Sediment samples taken along a transect from Hamilton Harbour were fractionated, and analyzed using a 2D HPLC method. All sediments contained intact and modified PAHs, although a marked change was noted in the profile of compounds present in the samples, which differ in distance from shore. Fractions of sediment extract were tested for toxicity using a bacterial respiration assay. Toxicity was observed in fractions containing modified PAHs, and was similar to that of intact PAH-containing fractions. <br /><br /> Subsequently, the toxicities of 16 intact PAHs were assessed to <i>Daphnia magna</i> under two ultraviolet radiation (UV) conditions. The toxicity of intact PAHs generally increased in the presence of full spectrum simulated solar radiation (SSR), relative to visible light plus UVA only. To expand the existing data on the effects of PAH photoproducts to animals, fourteen oxyPAHs were also assayed with <i>D. magna</i>, most of which were highly toxic without further photomodification. The data presented highlight the effects of UV radiation on mediating PAH toxicity. The importance of the role of photomodification is also stressed, as several oxyPAHs were highly toxic to <i>D. magna</i>, a key bioindicator species in aquatic ecosystems. <br /><br /> A QSAR model previously developed for <i>Lemna gibba</i> showed that a photosensitization factor (PSF) and a photomodification factor (PMF) could be combined to describe toxicity. To determine whether it was predictive for <i>D. magna</i>, toxicity was assessed as both EC50 and ET50. As with <i>L. gibba</i> and <i>Vibrio fischeri</i>, neither the PSF nor the PMF alone correlated to D. magna toxicity. However, a PSF modified for <i>D. magna</i> did in fact exhibit correlation with toxicity, which was further improved when summed with a modified PMF. The greatest correlation was observed with EC50 toxicity data. This research provides further evidence that models that include factors for photosensitization and photomodification will likely be applicable across a broad range of species. To gain further knowledge of the roles that the variables contributing to the photosensitization and photomodification, a structural equation model was constructed based on the <i>D. magna</i> QSAR. This model accounted for a high amount of variance in six sets of toxicity data, as well as insight into the mechanisms of phototoxicity affecting different aquatic organisms.