O surgimento de novas substâncias psicoativas (NPS-New Psychoactive Substances) levantou muitas questões no contexto da aplicação da lei e políticas públicas de drogas. De acordo com o Escritório das Nações Unidas sobre Drogas e Crime (UNODC- United Nations Office on Drugs and Crime) como uma alternativa às drogas proibidas. Esses novos compostos foram projetados e formulados para escapar à legislação de controle de drogas, criando um fenômeno que se tornou um problema internacional. No Brasil, essas substâncias são controladas e penalmente puníveis, no pela Lei 11.343/2006, também conhecida como Lei de Drogas. Este trabalho traz estudos relativos às catinonas sintéticas com metodologia in silico para investigar mecanismos de detecção e tendência de atuação no organismo humano. No estudo relacionado à detecção utilizamos a reação dessas drogas com o isotiocianato de fluoresceína (FITC Fluorescein isothiocyanate). Para essa proposta foram feitos estudos de viabilidade de métodos de cálculo, análise conformacional do FITC, avaliação energética da reação com as catinonas e os espectros de emissão. Em relação à viabilidade dos métodos de cálculo temos que a otimização prévia dos compostos envolvidos com o semi-empírico PM6 e posterior refinamento com o método B3LYP/6-31G** foram adequados para os cálculos. A avaliação energética mostrou que a reação é favorável para anfetaminas, aminoácidos e catinonas, e os menores valores foram encontrados no último caso. Nos estudos de emissão obtivemos resultados semelhantes ao perfil energético; no entanto, observamos que os espectros são únicos, representando uma baixa probabilidade de falsos positivos. Avaliações de docking mostraram que as catinonas têm mais afinidade com o receptor dopaminérgico do que suas anfetaminas homólogas, confirmando dados experimentais relatados na literatura. Por fim, os estudos realizados neste trabalho demonstraram a importância e a capacidade dos métodos in silico que apresentam grau potencial na área e que podem ser amplamente utilizados em investigações com diferentes propósitos no campo forense. / The emergence of new psychoactive substances (NPSs) has raised many issues in the context of law enforcement and public drug policies. According to the United Nations Office on Drugs and Crime (UNODC), NPS were created as an alternative to forbidden drugs. These new compounds were designed and formulated to escape the drug control legislation, creating a phenomenon that has become an international problem. In Brazil, these substances are controlled and punishable by Law 11,343 / 2006, also known as the Drug Law. This work presents studies on synthetic cathinones with in silico methodology to investigate mechanisms of detection and tendency of action in the human organism. In the detection-related study, we used the reaction of these drugs with fluorescein isothiocyanate (FITC). For this proposal were made studies regarding to the viability of the calculation methods, FITC conformational analysis, energetic evaluation of the reaction with the cathinones and the emission spectra. In relation to the viability of the calculation methods we have that the previous optimization of the compounds involved with the semi-empirical PM6 and subsequent refinement with the B3LYP / 6-31G ** method were adequate for the calculations. The energetic evaluation showed that the reaction is favorable for amphetamines, amino acids and cathinones, and the lowest values were found in the last case. In the emission studies we obtained similar results to the energy profile; however, we observed that the spectra are unique representing a low probability of false positive. Docking evaluations have shown that cathinones have more affinity to the dopaminergic receptor than their homologous amphetamines, confirming experimental data reported in the literature. Finally, the studies carried out in this work demonstrated the importance and the capacity of the in silico methods that present with potential grade in the area and that can be widely used in investigations with different purposes in the forensic field.

O surgimento de novas substâncias psicoativas (NPS-New Psychoactive Substances) levantou muitas questões no contexto da aplicação da lei e políticas públicas de drogas. De acordo com o Escritório das Nações Unidas sobre Drogas e Crime (UNODC- United Nations Office on Drugs and Crime) como uma alternativa às drogas proibidas. Esses novos compostos foram projetados e formulados para escapar à legislação de controle de drogas, criando um fenômeno que se tornou um problema internacional. No Brasil, essas substâncias são controladas e penalmente puníveis, no pela Lei 11.343/2006, também conhecida como Lei de Drogas. Este trabalho traz estudos relativos às catinonas sintéticas com metodologia in silico para investigar mecanismos de detecção e tendência de atuação no organismo humano. No estudo relacionado à detecção utilizamos a reação dessas drogas com o isotiocianato de fluoresceína (FITC Fluorescein isothiocyanate). Para essa proposta foram feitos estudos de viabilidade de métodos de cálculo, análise conformacional do FITC, avaliação energética da reação com as catinonas e os espectros de emissão. Em relação à viabilidade dos métodos de cálculo temos que a otimização prévia dos compostos envolvidos com o semi-empírico PM6 e posterior refinamento com o método B3LYP/6-31G** foram adequados para os cálculos. A avaliação energética mostrou que a reação é favorável para anfetaminas, aminoácidos e catinonas, e os menores valores foram encontrados no último caso. Nos estudos de emissão obtivemos resultados semelhantes ao perfil energético; no entanto, observamos que os espectros são únicos, representando uma baixa probabilidade de falsos positivos. Avaliações de docking mostraram que as catinonas têm mais afinidade com o receptor dopaminérgico do que suas anfetaminas homólogas, confirmando dados experimentais relatados na literatura. Por fim, os estudos realizados neste trabalho demonstraram a importância e a capacidade dos métodos in silico que apresentam grau potencial na área e que podem ser amplamente utilizados em investigações com diferentes propósitos no campo forense. / The emergence of new psychoactive substances (NPSs) has raised many issues in the context of law enforcement and public drug policies. According to the United Nations Office on Drugs and Crime (UNODC), NPS were created as an alternative to forbidden drugs. These new compounds were designed and formulated to escape the drug control legislation, creating a phenomenon that has become an international problem. In Brazil, these substances are controlled and punishable by Law 11,343 / 2006, also known as the Drug Law. This work presents studies on synthetic cathinones with in silico methodology to investigate mechanisms of detection and tendency of action in the human organism. In the detection-related study, we used the reaction of these drugs with fluorescein isothiocyanate (FITC). For this proposal were made studies regarding to the viability of the calculation methods, FITC conformational analysis, energetic evaluation of the reaction with the cathinones and the emission spectra. In relation to the viability of the calculation methods we have that the previous optimization of the compounds involved with the semi-empirical PM6 and subsequent refinement with the B3LYP / 6-31G ** method were adequate for the calculations. The energetic evaluation showed that the reaction is favorable for amphetamines, amino acids and cathinones, and the lowest values were found in the last case. In the emission studies we obtained similar results to the energy profile; however, we observed that the spectra are unique representing a low probability of false positive. Docking evaluations have shown that cathinones have more affinity to the dopaminergic receptor than their homologous amphetamines, confirming experimental data reported in the literature. Finally, the studies carried out in this work demonstrated the importance and the capacity of the in silico methods that present with potential grade in the area and that can be widely used in investigations with different purposes in the forensic field.

Les lactones macrocycliques (LM) sont des anthelminthiques (AH) à effet paralysant très utilisés chez les animaux et les humains contre les nématodes parasites. Cependant, leur succès thérapeutique est compromis par la résistance croissante aux LM, qui pourrait être en partie dû aux ABC transporteurs P-glycoprotéines (Pgps) sélectionnés et surexprimés chez les nématodes résistants aux LM. Dans ce travail, nous avons étudié plus précisément la P-glycoprotéine 13 du parasite de petits ruminants, Haemonchus contortus. Son orthologue chez le modèle nématode C. elegans, Cel-Pgp-13, est exprimé dans les amphides, structures qui ont été associées à la sensibilité aux AH chez C. elegans et H. contortus. Pour prédire la capacité des Pgps de nematode à transporter des drogues, incluant des LM et autres AH, nous avons développé un modèle de docking in silico. Nous avons utilisé la structure cristallographique de C. elegans Pgp-1 (Cel-Pgp-1), et nous avons montré la liaison avec une forte affinité de plusieurs ligands décrits comme activateurs de sa fonction ATPasique. Nous avons aussi décrit une forte affinité des LM, et un site spécifique de liaison de ces composés à Cel-Pgp-1. Cette approche représente un outil important pour prédire les interactions entre AH, et pour concevoir rationnellement de nouveaux inhibiteurs compétitifs des Pgps de nématode, dans le but d'améliorer les stratégies thérapeutiques. Sur la base de cette approche, nous avons prédit la structure 3D de Hco-Pgp-13 à partir du cristal de Cel-Pgp-1 afin d'étudier son intéraction avec des substrats potentiels, en particulier les LM. Nous avons trouvé des affinités similaires pour différents composés précédemment testés sur Cel-Pgp-1. In vitro, la mesure de l'activité ATPasique montre que l'actinomycine D est un substrat de Hco-Pgp-13. Nos données démontrent la présence possible d'un domaine de reconnaissance multispécifique sur ce transporteur de parasite. La détermination par immunofluorescence de l'expression de Hco-Pgp-13 a montré une distribution tissulaire large indiquant que Hco-Pgp-13 pourrait jouer un role important dans le transport de substrats endogènes et/ou exogènes. En conclusion, ce travail permet de mieux comprendre le rôle des Pgps de nématodes dans le transport de médicaments AH, tant au niveau de l'organisme modèle C. elegans que du nématode parasite H. contortus. Cette étude suggère la conservation de la fonction de tranporteur ABC multidrogue dans ces espèces. La localisation de Hco-Pgp-13 sur les structures amphidiales, et son éventuelle implication dans la résistance aux médicaments et à la survie de H. contortus à l'exposition à des composés AH, restent à préciser. / Macrocyclic lactones (ML) are paralyzing anthelmintics used in animals and humans against parasite nematodes. However, their therapeutic success is compromised by the spread of ML resistance. This might be at least partly due to P-glycoproteins (Pgps) ABC transporters that are selected and overexpressed in ML-resistant nematodes. Deciphering the role of the 10 Pgps expressed in the parasite of small ruminants Haemonchus contortus is thus of major importance to guaranty anthelmintic (AH) efficacy of various drugs. Here we focused on Hco-Pgp-13 due to the expression in the amphids of its closest ortholog in the model nematode C. elegans. Indeed, the amphids represent a putative entry route of drugs to reach AH targets in the nervous system and have been linked to AH susceptibility in C. elegans and H. contortus. In order to predict the capacity of nematode Pgps to transport drugs, including ML and otherAH, we have developed an in silico drug docking model. We have used C. elegans Pgp-1 (Cel-Pgp-1) crystal structure and have showed a high affinity binding of several ligands that have been shown to be activators of its ATPase function. ML were also found to bind with high affinity to Cel-Pgp-1, on a specific binding site. This approach provides a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of nematode Pgps, in order to improve anthelmintic therapeutics. We then predicted a putative 3D structure of Hco-Pgp-13 based on the recently released crystal of Cel-Pgp-1, with which it presented a high homology. This allowed the study of the interaction of Hco-Pgp-13 with potential substrates, in particular ML. We found similar affinities for various drugs previously tested on Cel-Pgp-1, supporting the good homology of these two proteins. Together with in vitro ATPase assay experiments that confirmed the substrate status of actinomycin D, this indicates a possible multispecifc recognition capacity of this parasitic transporter. The determination of Hco-Pgp-13 localization using immunohistochemistry showed a wide tissue expression consistent with a critical role for Hco-Pgp-13 in endogenous and/or exogenous substrate transport. In conclusion, this work provides insights into the role of nematode Pgps in transporting AH drugs, both at the level of the model organism C. elegans and of the parasitic nematode H. contortus. This suggests a high homology of function conserved between ABC tranporters in these species. The localization of such protein on amphidial structures and its possible involvement in drug resistance and survival of H. contortus to exposure to AH compounds remain to be precised.