La mesure d’interaction entre une molécule candidate et sa cible biologique est d’un intérêt majeur dans la recherche de nouveaux composés thérapeutiques. A l’heure actuelle, le marquage radioactif est la méthode de référence pour ce type d’étude pharmacologique, permettant une détection spécifique et extrêmement sensible de la molécule d’intérêt dans des matrices biologiques complexes. Cependant l’utilisation de la radioactivité présente de nombreux inconvénients, principalement en termes de sécurité pour les utilisateurs et problématiques environnementales induites par le stockage de déchets radioactifs. Au regard de ces limitations, des technologies alternatives ont été développées principalement basées sur une détection de fluorescence. Toutefois, l’incorporation de groupements encombrants tels que les fluorophores peut potentiellement perturber les propriétés physico-chimiques de la molécule et ainsi altérer son affinité envers le récepteur, ce qui limite l’application de ce type de stratégie pour les ligands de faible poids moléculaire. Dans ce contexte, nous souhaitons développer une méthodologie alternative pour quantifier les interactions récepteur-ligands. La spectrométrie de masse présente de remarquables capacités en termes de sensibilité et de spécificité d’analyse. Parmi les technologies existantes, l’ICP-MS est une technique extrêmement sensible permettant la quantification absolue d’hétéroéléments indépendamment de la matrice dans laquelle se trouve l’échantillon. Cette technique associée à un marquage approprié de la molécule d’intérêt a donc été évaluée. Le sélénium a été sélectionné comme un bon compromis entre la réponse obtenue en ICP-MS et la possibilité d’introduire ce marqueur au sein de la molécule d’intérêt selon des protocoles de chimie conventionnelle, sans perturbation de l’affinité pour le récepteur. La preuve de concept a été illustrée sur le récepteur à la vasopressine V1A impliquant des ligands de nature peptidique. Différentes stratégies ont été appliquées pour la conception de ligands marqués au sélénium basées sur la substitution d’acides aminés par leurs analogues séléniés (ex : remplacement de la cystéine (Cys) par la sélénocystéine (Sec) et de la proline (Pro) par la sélénazolidine (Sez)) ainsi que la dérivatisation en position N-terminale du peptide avec une petite molécule organique. La méthodologie analytique par ICP-MS a été développée de manière à obtenir une mesure du sélénium à la fois sensible et fiable. La forte proportion de sels inorganiques contenus dans la matrice pharmacologique a nécessité le recours à une séparation chromatographique et à une cellule de collision en amont de la détection par ICP-MS. Le protocole pharmacologique a également été adapté aux exigences analytiques. Ces modifications ont notamment porté sur la récupération de l’échantillon pour procéder à son injection ainsi que sur la quantité de cellules utilisées. La robustesse des expériences pharmacologiques ainsi conçues a été évaluée par la mesure de la constante d’affinité (Ki) de ligands caractéristiques du récepteur V1A présentant des affinités élevées et faibles pour ce dernier. Les résultats obtenus ont présenté une très bonne corrélation avec les valeurs de référence indiquées dans la littérature, permettant ainsi de valider la preuve de concept de cette méthodologie et de proposer une alternative viable au marquage radioactif. / Measuring interactions between a drug candidate and its specific target constitutes a parameter of upmost importance in drug discovery process. Because of its robustness and very high sensitivity, radio-isotopic labeling represents up to now the reference method for these pharmacological studies. However, the use of radioactivity confers several constraints about security, health hazards and environmental issues involved by radioactive waste storage. Regarding such limitations, non-radioactive technologies have been developed principally based on fluorescence detection. But the cost of such assay as well as the fact that incorporation of a bulky fluorescent block can affect the affinity for small ligand limit their use. In this context, we aim to develop a new methodology to quantify receptor-ligands interactions. Mass spectrometry (MS) presents very interesting features in terms of detection sensitivity and specificity, making this technique a challenging analytical tool to replace radioactivity and fluorescence measurements commonly used in pharmacology. Among all MS technologies, the capacity of inductively coupled plasma-mass spectrometry (ICP-MS) to provide metallic and hetero elements absolute quantification, whatever the nature of the sample medium, prompted us to investigate this technique in combination with an appropriate labeling of the molecule of interest. Selenium was selected as a good compromise between ICP-MS response and chemical tagging ability through the creation of covalent bonds using conventional organic sulfur chemistry without disturbance of the affinity toward targeted receptor. Proof of concept was illustrated on the vasopressin receptor (V1A), a GPCR involved in vasoconstriction and emotional behavior and implying peptide as native ligand. Different strategies were applied to design selenium labeled peptides relying in either conventional amino acid substitution by corresponding selenium containing residue into peptide sequence such as cysteine (Cys) replacement by selenocysteine (Sec) as well as proline (Pro) by selenazolidine (Sez), or N-terminal peptide derivatization with a selenium containing small organic entity. ICP-MS analytical methodology was carefully investigated to provide sensitive and reliable selenium signal measurement. High inorganic salts contents of pharmacological buffer along with polyatomic interferences from plasma interfering selenium detection necessitate chromatographic separation and collision reaction cell equipment before ICP-MS detection. The pharmacological protocol was also adapted to the analytical requirements, in particular quantity of cells and sample handling. Robustness of the designed competitive binding assay was evaluated through the affinity constant (Ki) measurement of several known V1A-R ligands exhibiting either high or poor affinity for the receptor. Experimental values were strongly correlated to literature data, enabling to validate the proof of concept of such methodology and to propose a suitable alternative to radioactive labeling.

Os avanços tecnológicos vêm contribuindo cada vez mais nas áreas biológicas e científicas oferecendo ferramentas computacionais e sistemas específicos que em análise de dados in silico fornecem resultados rápidos e confiáveis. O presente projeto propõe o desenvolvimento de um portal na Internet para instalação e utilização de um banco de dados laboratoriais das principais serpentes brasileiras com seus respectivos venenos e antivenenos naturais, e a análise dos dados obtidos em ensaios farmacológicos, e bioquímicos. Utilizando a via de comunicação e interação mais simples atualmente, a Internet permite o compartilhamento de dados entre comunidades de pesquisadores, viabilizando recursos e tempo, além de permitir uma significante interação entre pesquisadores de todo o mundo, principalmente brasileira, na troca de informações e compartilhamento de dados. Dados elementares relacionados às serpentes foram armazenados no banco de dados, bem como as atividades tóxicas, farmacológicas e enzimáticas dos componentes dos venenos, e ainda, as aplicações biotecnológicas dos produtos que podem ser obtidos destes venenos, abrangendo ainda dados clínicos e valores estatísticos dos acidentes ofídicos. Aspectos bioquímicos dos ensaios realizados em laboratório permitiram a construção de uma ferramenta para análise comparativa de estruturas primárias de PLA2s, depositadas em bancos de dados internacionais. Além da interatividade entre pesquisadores, em Fóruns de Discussões, o sistema conta com listas dos principais artigos publicados em periódicos indexados, e devidamente atualizados periodicamente, com revisões bibliográficas. / Technological advances have been contributing, more and more, with biological and scientific areas, offering computational tools and specific systems which in silico data analysis supply reliable and fast results. The present project considers the development of an Internet portal for installation and use of laboratory data base for the main Brazilian serpents, with its respective venom and natural anti-venom, and the analysis of obtained data in pharmacological assays and biochemists. Using the easiest way of communication and interaction, the Internet allows sharing of data and information between communities of researchers around the world, especially for Brazilian researchers, making resources and time possible. Elementary data about serpents have been stored in the data base, as well as toxic, pharmacological and enzymatic activities of venom components, besides biotechnological applications of the products that can be obtained from these venom, enclosing clinical data and statistical values of ophidian accidents. Biochemists aspects of the assays carried through in laboratory allowed the construction of a comparative analysis tool for primary structures of PLA2s, deposited in international data bases. Beyond interactivity between researchers, in discussion forums, the system counts with lists of main articles published in indexed periodic, duly and constantly updated with bibliographical revisions.

Os avanços tecnológicos vêm contribuindo cada vez mais nas áreas biológicas e científicas oferecendo ferramentas computacionais e sistemas específicos que em análise de dados in silico fornecem resultados rápidos e confiáveis. O presente projeto propõe o desenvolvimento de um portal na Internet para instalação e utilização de um banco de dados laboratoriais das principais serpentes brasileiras com seus respectivos venenos e antivenenos naturais, e a análise dos dados obtidos em ensaios farmacológicos, e bioquímicos. Utilizando a via de comunicação e interação mais simples atualmente, a Internet permite o compartilhamento de dados entre comunidades de pesquisadores, viabilizando recursos e tempo, além de permitir uma significante interação entre pesquisadores de todo o mundo, principalmente brasileira, na troca de informações e compartilhamento de dados. Dados elementares relacionados às serpentes foram armazenados no banco de dados, bem como as atividades tóxicas, farmacológicas e enzimáticas dos componentes dos venenos, e ainda, as aplicações biotecnológicas dos produtos que podem ser obtidos destes venenos, abrangendo ainda dados clínicos e valores estatísticos dos acidentes ofídicos. Aspectos bioquímicos dos ensaios realizados em laboratório permitiram a construção de uma ferramenta para análise comparativa de estruturas primárias de PLA2s, depositadas em bancos de dados internacionais. Além da interatividade entre pesquisadores, em Fóruns de Discussões, o sistema conta com listas dos principais artigos publicados em periódicos indexados, e devidamente atualizados periodicamente, com revisões bibliográficas. / Technological advances have been contributing, more and more, with biological and scientific areas, offering computational tools and specific systems which in silico data analysis supply reliable and fast results. The present project considers the development of an Internet portal for installation and use of laboratory data base for the main Brazilian serpents, with its respective venom and natural anti-venom, and the analysis of obtained data in pharmacological assays and biochemists. Using the easiest way of communication and interaction, the Internet allows sharing of data and information between communities of researchers around the world, especially for Brazilian researchers, making resources and time possible. Elementary data about serpents have been stored in the data base, as well as toxic, pharmacological and enzymatic activities of venom components, besides biotechnological applications of the products that can be obtained from these venom, enclosing clinical data and statistical values of ophidian accidents. Biochemists aspects of the assays carried through in laboratory allowed the construction of a comparative analysis tool for primary structures of PLA2s, deposited in international data bases. Beyond interactivity between researchers, in discussion forums, the system counts with lists of main articles published in indexed periodic, duly and constantly updated with bibliographical revisions.