There are two kinds of protein aggregates associated with Alzheimer’s disease: amyloid-beta and tau aggregates. Protein ligands are molecules with the ability to bind to these pathologicalprotein accumulations, and if the ligands are fluorescent, they can be used to detect the aggregates they’re bound to. The ligands can be selective and only bind to one kind of protein aggregate, or they can be general and bind to both kinds. PI-2620 is a ligand selective for tau aggregates, and in this thesis, three analogues of PI-2620 were synthesized and determined to be functional fluorescent protein ligands. Two of them, designated A1 and B1, were selective for amyloid-beta aggregates, while the third ligand, A2, obtained by ester-hydrolysis of A1, would bind to both tau and amyloid-beta aggregates. This finding suggests that introducing a charge to a ligand lowers its selectivity, since the staining experiments were carried out in a buffered solution at pH 7.4, where ligand A2 might be partially charged.

<p>Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.</p> / Dissertation

Ma thèse comporte deux volets: d’une part, le développement de ligands ciblant les protéines antiapoptotiques et d’autre part, l’étude par RMN des protéines CGC impliquées dans la biosynthèse de la congocidine, métabolite secondaire chez Streptomyces.La famille de protéines Bcl-2 est impliquée dans un des processus clé de la mort cellulaire programmée, appelée l’apoptose mitochondriale. Elle se divise en membres anti-apoptotiques (Bcl-2, Bcl-xL, Mcl-1) et pro-apoptotiques (Bak, Bax et les «BH3»).Ces molécules vont réguler l’apoptose en maintenant ou non l’intégrité de la membrane mitochondriale. En réponse à un signal de stress, les «BH3» neutralisent les antiapoptotiques et activent les pro-morts, leur permettant de former des pores sur la membrane mitochondriale. Ce phénomène aboutit au relargage du cytochrome c dans le cytosol et à l’activation de la cascade des caspases dont la finalité est la destruction de la celluleLa pertinence de l’étude des Bcl-2 s’observe de manière croissante depuis les années 1990. En effet, une surexpression des membres pro-survie de cette famille (Bcl-2, Bcl-xL, Mcl-1, BFL1 etc...) a été observée dans de nombreux cancers. Suite à ce constat, cibler ces molécules est devenue une piste prometteuse en cancérologie par le développement d’inhibiteurs des protéines pro-survie, l’objectif étant de restaurer l’apoptose dans les cellules tumorales.Dans cette perspective, différentes stratégies thérapeutiques ont été imaginées:(i) la thérapie génique avec l’Oblimersen, un ADN antisens développé par Genta, qui inhibe l’expression de la Bcl-2. Néanmoins, les résultats précliniques sont décevants.(ii) l’utilisation de peptides (ou peptidomimétiques) imitant les sentinelles «BH3» comme antagonistes de l’interaction Bcl-xL/Bak. Il faut souligner le concept des «stappled peptides», permettant la stabilisation des hélices par cyclisation des chaînes latérales. Si certaines de ces molécules synthétisées semblent très actives, aucune n’est encore en étude clinique.(iii) le développement de petites molécules non peptidiques, issues d’un criblage systématique in vitro ou in silico et se caractérisant par une grande variété structurale. Parmi ces molécules, certaines sont synthétiques comme l’ABT-737 et l’ABT-263 élaborés par les laboratoires Abbott, grâce à la méthode d’assemblage des fragments (fragment-based drug design) aidée par des études SAR by NMR (structure activity relationship). D’autres sont issues de produits naturels comme le (R)-Gossypol, le TW-37, la sanguinarine ou l’Obatoclax. En 2008, 11 composés étaient en phase préclinique ou clinique et les résultats pour certains d’entres eux semblaient plus prometteurs que pour l’Oblimersen.Ces stratégies ont permis de mettre au point un certain nombre de composés ciblant les protéines anti-apoptototiques. Si certains de ces composés sont actuellement en phase de tests cliniques, les plus prometteurs (ABT) ont démontré une efficacité uniquement vers certains des protéines anti-apoptotiques laissant place à un phénomène d’échappement des cellules cancéreuses.Un criblage réalisé à l’ICSN par l’équipe de F. Guéritte (Pôle Substances Naturelles Plantes) a permis d’identifier une nouvelle classe de molécules ayant une affinité de l’ordre du μM pour Bcl-xL. Parmi ces composés, deux présentent une attractivité d’un point de vue structural qui rend faisable leur synthèse chimique:(i) la meiogynine A, un sesquiterpène dimère de structure originale isolé des écorces de Meiogyne cylindrocarpa, une plante de Malaisie.(ii) le drimane, un sesquiterpène isolé en grandes quantités du genre zygogynum, une espècede Nouvelle-Calédonie.Ainsi, des collaborations ont été établies au sein de l’ICSN, réunissant diverses expertises (chimie, biologie, physicochimie et modélisation) afin de dégager les synergies souhaitables.Dans la perspective de la conception rationnelle d’analogues aux propriétés améliorées ciblant l’ensemble des membres anti-apoptotiques de la famille Bcl-2, ma contribution est de choisir les cibles biologiques (Bcl-xL et Mcl-1), de les obtenir pures et marquées en isotopes stables afin de réaliser par RMN et modélisation moléculaire une étude structurale des complexes protéines/ligands et de définir un modèle d’interaction.Le deuxième volet de ma thèse, à dominante fondamentale, a pour objectif de caractériser par RMN les médiateurs enzymatiques d’une voie de biosynthèse d’un métabolite secondaire, la congocidine issue des bactéries Streptomyces Ambofaciens.La congocidine présente des propriétés antivirales et anticancéreuses de par sa capacité à se fixer à l’ADN. Cependant, du fait de sa forte toxicité, cette molécule ne peut pas être utilisée directement à des fins thérapeutiques.L’analyse des groupes de gènes impliqués dans la biosynthèse de la congocidine a mis en évidence 24 gènes. Seuls certains intermédiaires réactionnels ont été identifiés. Cependant, le rôle précis des produits de ces gènes n’est pas encore bien défini.Ainsi, en collaboration avec l’équipe de JL Pernodet à l’Université d’Orsay, nous nous sommes intéressés à deux enzymes en particulier intervenant dans la synthèse de la congocidine, les protéines CGC-10 et CGC-19.L’objectif de cette collaboration est d’utiliser la spectroscopie RMN couplée à la modélisation moléculaire sous contraintes expérimentales afin de déterminer la structure de ces deux protéines. Nous souhaitons apporter des informations sur l’éventuelle présence de motifs structuraux au sein de ces protéines afin de mieux comprendre leur fonction et de définir à quel moment de la voie de biosynthèse elles interviennent.Concernant la protéine CGC-10, nous avons conçu un plasmide optimisé que nous avons fait synthétiser. Le gène obtenu a été cloné dans un vecteur d’expression choisi par nos collaborateurs (pQE30).Concernant la protéine CGC-19, nous allons en décrire les étapes d’expression et de purification qui nous ont permis d’enregistrer l’ensemble des expériences 3D-triple résonnance nécessaires à la détermination de la structure de la protéine. De plus, il a été mis en évidence la présence d’une modification post-traductionnelle de type phosphopanthéténylation au sein de cette protéine. Nous avons produit l’enzyme responsable de cette modification, la sfp, afin de pouvoir effectuer la réaction et suivre l’effet de la modification sur les spectres RMN de la protéine et donc sur la structure.Ce projet, qui s’inscrit dans une perspective de recherche à plus long terme, a pour objectif de caractériser précisément le mécanisme de production de la congocidine. A travers cette démarche, il s’agit de combiner la biologie moléculaire (modification en amont les gènes) à la chimie afin d’obtenir des molécules différentes aux propriétés améliorées et non toxiques. / My PhD thesis contains two parts: development of ligands against anti-apoptotic proteins and structural study of CGC proteins involved in the biosynthesis of congocidine, a Streptomyces Ambofaciens secondary metabolite.The first project concerns the NMR study of the interactions between the anti-apoptotic proteins and two potential ligand candidates, the Meiogynine and the Drimane. These two terpenoïds, identified from ICSN’s chemical library screening against the Bcl-xL protein, have shown a significant inhibiting activity, thus opening promising perspectives for the treatment of cancer cells overexpressing anti-apoptotic proteins. In fact, as these compounds are considerably smaller than the binding site, our objective is to introduce modifications (such as elongation of their structure, functionalization with hydrophilic groups etc.) that may improve their binding properties as well as their delivery and bioavailability.Following to the successful recombinant expression and purification, necessary to obtain labelled targets (15N/13C), our preliminary NMR studies suggested a rather universal action of our candidates, capable to bind not only to Bcl-xL but also to the other major anti-apoptotic protein, Mcl-1. Titration experiments revealed significant perturbations of the HSQC protein NMR spectra with the progressive disappearance of several protein HN and ligand signals, confirming dissociation constants at the µM region for both targets. However, the intermediate chemical exchange NMR regime observed, associated with the weak ligand solubility, poses severe difficulties for the structural elucidation of the complexes by classical NMR methods.In this work alternative approaches for the localisation of the ligands in the hydrophobic cleft of both target proteins will be presented.Oligopyrroles are secondary metabolites synthesized by Streptomyces bacteria. This family of natural products, composed by one or more pyrrole-2-carboxamide groups is characterized by a variety of biological activities such as antiviral, antitumor and antibiotic functions.One of the best-known metabolites is the congocidine, extensively studied due to its capacity to bind into the minor groove of the DNA double helix, with strong sequence specificity. However, because of its strong toxicity, this molecule cannot be directly used for therapeutic purposes.The analysis of the groups of genes involved in congocidine biosynthesis brought to light 24 genes, but their precise role is not yet well defined. We were particularly interested in two enzymes: the proteins called CGC-10 and CGC-19. For the recombinant expression of the first one, we designed an optimized insert which was cloned in an expression vector pQE30.Concerning CGC-19, the stages of expression and purification, which allowed us to obtain doubly-labeled protein, as well as the 3D NMR experiments for spectral assignment and structure elucidation, will be discussed.Furthermore, we were interested in the holo- state of this protein obtained through a post-traductional modification (phosphopanthéténylation). To this, we produced the enzyme responsible for this modification, Sfp, carry out the reaction in vitro and follow the effect of the modification at the NMR spectra.

<p>A new approach to analysis of protein-ligand interactions, termed proteochemometrics, has been developed. Contrary to traditional quantitative structure-activity relationship (QSAR) methods that aim to correlate a description of ligands to their interactions with one particular target protein, proteochemometrics considers many targets simultaneously.</p><p>Proteochemometrics thus analyzes the experimentally determined protein-ligand interaction activity data by correlating the data to a complex description of all interaction partners and; in a more general case even to interaction environment and assaying conditions, as well. In this way, a proteochemometric model analyzes an “interaction space,” from which only one cross-section would be contemplated by any one QSAR model.</p><p>Proteochemometric models reveal the physicochemical and structural properties that are essential for protein-ligand complementarity and determine specificity of molecular interactions. From a drug design perspective, models may find use in the design of drugs with improved selectivity and in the design of drugs for multiple targets, such as mutated proteins (e.g., drug resistant mutations of pathogens).</p><p>In this thesis, a general concept for creating of proteochemometric models and approaches for validation and interpretation of models are presented. Different types of physicochemical and structural description of ligands and macromolecules are evaluated; mathematical algorithms for proteochemometric modeling, in particular for analysis of large-scale data sets, are developed. Artificial chimeric proteins constructed according to principles of statistical design are used to derive high-resolution models for small classes of proteins.</p><p>The studies of this thesis use data sets comprising ligand interactions with several families of G protein-coupled receptors. The presented approach is, however, general and can be applied to study molecular recognition mechanisms of any class of drug targets.</p>

A new approach to analysis of protein-ligand interactions, termed proteochemometrics, has been developed. Contrary to traditional quantitative structure-activity relationship (QSAR) methods that aim to correlate a description of ligands to their interactions with one particular target protein, proteochemometrics considers many targets simultaneously. Proteochemometrics thus analyzes the experimentally determined protein-ligand interaction activity data by correlating the data to a complex description of all interaction partners and; in a more general case even to interaction environment and assaying conditions, as well. In this way, a proteochemometric model analyzes an “interaction space,” from which only one cross-section would be contemplated by any one QSAR model. Proteochemometric models reveal the physicochemical and structural properties that are essential for protein-ligand complementarity and determine specificity of molecular interactions. From a drug design perspective, models may find use in the design of drugs with improved selectivity and in the design of drugs for multiple targets, such as mutated proteins (e.g., drug resistant mutations of pathogens). In this thesis, a general concept for creating of proteochemometric models and approaches for validation and interpretation of models are presented. Different types of physicochemical and structural description of ligands and macromolecules are evaluated; mathematical algorithms for proteochemometric modeling, in particular for analysis of large-scale data sets, are developed. Artificial chimeric proteins constructed according to principles of statistical design are used to derive high-resolution models for small classes of proteins. The studies of this thesis use data sets comprising ligand interactions with several families of G protein-coupled receptors. The presented approach is, however, general and can be applied to study molecular recognition mechanisms of any class of drug targets.

<p>In this dissertation a novel approach to protein-ligand docking is presented. First an existing method to predict putative active sites is employed. These predictions are then used to cut down the search space of an algorithm that uses the fast Fourier transform to calculate the geometrical and electrostatic complementarity between a protein and a small organic ligand. A simplified hydrophobicity score is also calculated for each active site. The docking method could be applied either to dock ligands in a known active site or to rank several putative active sites according to their biological feasibility. The method was evaluated on a set of 310 protein-ligand complexes. The results show that with respect to docking the method with its initial parameter settings is too coarse grained. The results also show that with respect to ranking of putative active sites the method works quite well.</p>

A central goal of computational biophysics and biochemistry is to understand the behavior, interactions, and reactions of molecules, and to interpret and facilitate experimental design. The objective of this thesis research is to use the molecular modeling and simulation techniques to advance our understanding of principles in molecular structure properties, recognition and interaction at the atomic level. First, a physical molecular mechanics model is built to study the conformational properties of depsipeptide, which shows potential for engineered protein mimetics with controllable structure and function. We explore the possible kinase-substrate binding modes and the likelihood of an [alpha]-helix docking interaction within a kinase active site. Finally, efficient physical models based on a polarizable potential function are developed to describe the structural properties and calculate protein-ligand binding affinities accurately for both trypsin and matrix metalloproteinase. / text

<p>While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment. </p><p>The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment. </p><p>The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.</p> / Dissertation

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T11:28:25Z (GMT). No. of bitstreams: 1 Figueiredo_IsisMartins_D.pdf: 1626484 bytes, checksum: 2c55f1a5b794f6f6e1c4a4931c394c4d (MD5) Previous issue date: 2006 / Resumo: Durante as últimas décadas, muitos métodos de RMN de H foram desenvolvidos e aplicados para triagem e caracterização de interações intermoleculares e para a metabonômica. Estes são temas recentes da RMN e ambos serão abordados em dois capítulos distintos neste trabalho. No Capítulo 1 foi realizada a implantação e otimização de técnicas de RMN como (STD, WaterLOGSY, NOE pumping e DOSY-NOESY). Para tanto, utilizou-se um sistema composto por albumina de soro bovino BSA e uma mistura de cinco compostos (ác. salicílico, cafeína, ác. cítrico, ác. adipico e D-glucose) dentre os quais, apenas o ácido salicílico e a cafeína interagiram com a BSA. Além disso, uma análise comparativa entre as técnicas permitiu afirmar que os experimentos de STD e WaterLOGSY são os mais sensíveis e rápidos fornecendo complementarmente o domínio hidrofóbico e hidrofílico de ligação com o ligante. Com intuito de confirmar nossa habilidade na aplicação destas técnicas, as mesmas foram aplicadas a um sistema composto por uma Chaperone Hsp70, substratos (ATP e ADP) e um polipeptíteo Angiotensina 2 (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). A análise desse sistema por WaterLOGSY e a comparação com os resultados obtidos por STD permitiu a obtenção do epitopo 1 o qual é formado pela porção adenosina do A TP ou ADP quando estes estão complexados a Hsp70 e do epitopo 2 formado pela porção hidrofóbica da Angiotensina 2 (Val-Tyr-Ile-His-Pro-Phe) que interage com a Hsp70. Já no Capitulo 2, a RMN de H foi aplicada na investigação da metabonômica do liquido cerebroespinal de pacientes com Esclerose Múltipla (EM). A análise dos dados de RMN através de métodos quimiométricos (HCA, PCA e PLS-DA) revelou alguns metabólitos importantes, dentre os quais o b-hidroxibutirato (1,17 ppm) e um sinal de proteína (0,065 ppm) foram detectados apenas em amostras EM podendo ser considerados marcadores de reações bioquímicas de degradação de mielina. Portanto, este estudo alcançou com êxito os objetivos traçados de implementar novas técnicas de RMN aplicadas a sistemas biológicos além de trazer novas informações sobre a Hsp70 e EM / Abstract: Over the past years H NMR methods have been developed and applied to the screening and characterization of protein epitopes in ligand receptor complexes and metabonomics. These are recent NMR methods issues of the present PhD thesis. To investigate proteinligand complexes we first optimized techniques that were unavailable at IQ/UNICAMP such as STD, WaterLOGSY, NOE pumping and DOSY-NOESY which were specially designed for epitope mapping. In order to optimized these techniques we employed a mixture of five compounds (salicilic ac., caffeine, citric ac., adipic ac. and D-glucose) and bovine serum albumine (BSA). Among the studied ligands salicilic acid and caffeine were the best. From these experiments we additionally concluded that STD and WaterLOGSY were most sensitive and appropriate for epitope mapping. A second system was investigated consisting of Chaperone Hsp70, cofactor (ATP and ADP) and polypeptide Angiotensine 2 (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). Epitope I was characterized as containing a lipophylic domain in which the adenosine portion of ATP or ADP was bound to Hsp70. Epitope 2 was the polypeptide-binding site in which the apoIar portion of Angiotensine 2 (Val-Tyr-Ile-His-Pro-Phe) was tightly bound to Hsp70. In chapter 2, H NMR was the major tool employed to investigate the metabonomics of CSF of Multiple Sclerosis patients. Analyses of the H NMR data applying quimiometric methods (HCA, PCA and PLS-DA) revealed that some metabolites, from which b-hydroxybutirate (1,17 ppm) and a protein signal (0,065 ppm) were detected in EM patients only. These signals were never described as EM biomarkers before. To match these observations a full set of lipolytic and proteolytic biochemical reactions were proposed which are responsible for myelin degradation. Therefore, in this study we describe the successful implementation of these new NMR techniques that were applied to biological systems revealing new aspects of the Hsp70 and MS / Doutorado / Quimica Organica / Doutor em Ciências

Virtually all processes in living organisms are conducted by proteins. Proteins perform their function by binding to other proteins (protein-protein interactions) or small molecules - so called ligands (protein-ligand interactions). Active sites for protein-ligand interactions are pockets in protein structure where ligand can bind. Predicting of ligand binding sites is the first step to study and predict protein functions and structure based drug-design. In this thesis we reviewed current approaches for binding site prediction and proposed our own improvement. We have developed a novel pocket ranking function based on prediction model that predicts ligandability (ability to bind a ligand) of a given point inside of a pocket. Prediction is done considering only a local physicochemical and geometric properties derived from neighbourhood.