Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The organotellurium compounds have been the subject of research due to their pharmacological and toxicological properties. It is believed that the main mechanism involved in the toxicity of these compounds is the ability to interact with sulfhydryl groups from molecules biologically active. Beyond the toxicological effects, some pharmacological properties have been attributed to organotellurium compounds. This study aimed to investigate the potential toxicological and pharmacologic of 2-phenylethynyl-butyltellurium (PEBT) through experiments in vitro and in vivo in rodents. To evaluate the toxicological effect the PEBT was used at different concentrations in the oxidation of mono and dithiols and analysis of enzyme Na+K+ -ATPase and lactate dehydrogenase in vitro. Furthermore, lethality studies were performed to calculate the LC50 LD50 of this compound and for better understanding their toxicity. To evaluate the pharmacological effect the PEBT was used at a dose of 1mg/kg 30 minutes before the behavioral experiments, evaluation of locomotor activity, forced swim test (FST) and the tail suspension test (TST), immediately after testing the cerebral cortex was removed for analysis of monoamine oxidase (MAO) enzyme. The results showed that the PEBT oxidized thiols of low molecular weight and inhibits the activity of the enzyme Na+K+ - ATPase by oxidation of sulfhydryl groups, and such oxidation is dependent on the tellurium atom in the structure of this compound. Moreover, the acute administration of PEBT showed an antidepressant-like effect on TNF in mice, as well inhibits the activity of MAO-A enzyme in the cerebral cortex, demonstrating the involvement of this enzyme in its antidepressant-like effect. / Os compostos orgânicos de telúrio têm despertado o interesse dos pesquisadores, devido as suas propriedades farmacológicas e toxicológicas. Acredita-se que o principal mecanismo envolvido na toxicidade desses compostos, seja a capacidade de interagir com os grupamentos sulfidrílicos de moléculas biologicamente ativas. Além dos efeitos toxicológicos, propriedades farmacológicas vêm sendo atribuídas aos compostos orgânicos de telúrio. Esse estudo teve como objetivo investigar o potencial toxicológico e farmacológico do 2- feniletinilbutil-telúrio (PEBT), através de experimentos in vitro e in vivo em roedores. Para a avaliação toxicológica, o PEBT foi utilizado em diferentes concentrações na oxidação de mono e ditiois e na determinação da atividade das enzimas Na+K+ -ATPase e lactato desidrogenase, in vitro. Ainda nesse sentido estudos de letalidade foram realizados para calcular a CL50 e DL50 desse composto para melhor compreender a sua toxicidade. Para a avaliação farmacológica, o PEBT foi administrado em camundongos, na dose de 1 mg/kg 30 minutos antes dos experimentos comportamentais, avaliação da atividade locomotora, teste do nado forçado (TNF) e teste de suspensão da cauda (TSC). Após os testes comportamentais, os animais foram mortos e o córtex cerebral foi retirado para determinação da atividade da enzima monoamina oxidase (MAO). Os resultados mostraram que o PEBT oxida tióis de baixo peso molecular e inibe a atividade da enzima Na+K+ -ATPase, pela oxidação de seus grupamentos sulfidrilicos, sendo essa oxidação depende da presença do átomo de telúrio na estrutura do composto. Além disso, a administração aguda do PEBT produz um efeito do tipo antidepressivo no TNF em camundongos, bem como inibe a atividade da enzima MAO-A em córtex cerebral, demonstrando o envolvimento dessa enzima no seu efeito do tipo antidepressivo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Monoamines appear to play an important modulatory role on pain descending pathways and are involved in the antinociceptive mechanism of several drugs commonly used for the management of pain. In this study, we assessed the involvement of monoamine oxidase B (MAO-B), a key enzyme implicated in monoamine metabolism, on models of postsurgical and neuropathic pain in mice. For this purpose, we evaluated the effects of the selective and irreversible MAO-B inhibitor selegiline on mechanical sensitivity and ex vivo MAO-B activity in different central nervous system regions in mice submitted to incisional and partial sciatic nerve ligation (PSNL) pain models. Mice subjected to plantar incision showed a significant decrease in mechanical threshold when compared with sham-operated mice, characterizing the development of mechanical allodynia. Selegiline, at a dose sufficient to inhibit selectively the MAO-B activity (10 mg/kg), showed an anti-allodynic effect from 0.5 until 6 h after incision. The MAO-B activity was not altered in incision submitted mice when compared with sham-operated animals in any analyzed structure. Likewise, PSNL submitted mice also developed mechanical allodynia, which was reversed by selegiline (10 mg/kg) from 2 until 6 h after treatment. In addition, a significant increase on striatal MAO-B activity was observed in mice subjected to PSNL when compared with sham-operated animals, which was reversed by selegiline treatment. Taken together, our results showed that selegiline presented an antinociceptive effect on mice models of both acute and chronic pain, suggesting a potential involvement of MAO-B on pain mechanisms. / As monoaminas possuem uma função modulatória importante nas vias descendentes do controle da dor e estão envolvidas no mecanismo antinociceptivo de diversos fármacos comumente utilizados no tratamento de síndromes dolorosas. Nesse estudo, nós avaliamos a participação da monoamina oxidase B (MAO-B), uma enzima chave envolvida no metabolismo das monoaminas, em modelos de dor pós-operatória e neuropática em camundongos. Para esse propósito, foram avaliados os efeitos da selegilina, um inibidor seletivo e irreversível da MAO-B, na sensibilidade mecânica e na atividade ex vivo da MAO-B em diferentes regiões do sistema nervoso central (córtex cerebral, estriado e medula espinhal) de camundongos submetidos à incisão plantar ou à ligação parcial do nervo ciático (PSNL). Os camundongos que foram submetidos à incisão plantar apresentaram uma diminuição significativa no limiar mecânico quando comparados aos animais falso-operados, caracterizando o desenvolvimento de alodínia mecânica. Tanto o pré quanto o pós-tratamento com selegilina, em uma dose capaz de inibir seletivamente a atividade da MAO-B (10 mg/kg, p.o.), apresentaram efeito anti-alodínico a partir de 0,5 até 6 h após o tratamento. A atividade da MAO-B, medida 4 h após o procedimento cirúrgico, não foi alterada nos camundongos submetidos à incisão quando comparada com a atividade dos animais falso-operados em nenhuma das estruturas analisadas. Os camundongos submetidos à PSNL também desenvolveram alodínia mecânica, a qual foi revertida pela selegilina (10 mg/kg, p.o.) de 2 até 6 h após o tratamento. Além disso, os camundongos submetidos à PSNL apresentaram um aumento significativo na atividade da MAO-B no estriado 4 h após o tratamento, o qual foi revertido pela selegilina. Foi observado também que a selegilina, em uma dose sem efeito antinociceptivo (1 mg/kg, p.o.) em ambos os modelos de dor utilizados, não foi capaz de inibir a atividade da MAO-B. Nossos resultados mostram que a selegilina apresentou um efeito antinociceptivo tanto em um modelo de dor aguda quanto em um modelo de dor crônica, sugerindo um possível envolvimento da MAO-B nos mecanismos da dor.

Depression is a psychiatric disorder that affects 2-15% of the world population. It is a heterogeneous neuropathology, it includes psychological, behavioral and physiological symptoms. Despite having several treatments for depression, they are not as effective and have many undesirable side effects. Therefore, it is necessary the development of new therapeutic strategies to improve current treatments. Therefore, open up doors to the use of plant medicines, which has already been used for thousands of years by various populations for different diseases. The Bauhinia variegata, popularly known as "pata-de-vaca", has beneficial health effects, such as anti-inflammatory activity, antidiabetic, antidiarrheal. In the present study, we investigated the antidepressant-like effect of the crude hydroethanolic extract of leaves of Bauhinia variegata in two predictive tests of antidepressant property, the tail suspension test (TST) and the forced swimming test (FST) in mice. Furthermore, the involvement of monoaminergic system in its antidepressant-like effect was investigated and the effect of extract on monoamine oxidase A (MAO-A) activity was also evaluated. The acute treatment with the extract of Bauhinia variegate (300 mg/kg, p.o.) significantly reduced the immobility time in the TST. Moreover, the repeated administration (once a day for 7 days) of the extract by p.o. route also produced an antidepressant-like effect in the TST (100 and 300 mg/kg) and FST (300 mg/kg).The pretreatment of mice with ketanserin (5 mg/kg, i.p. a preferential 5-HT2A receptor antagonist), prazosin (1 mg/kg, i.p. an α1-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p. an α2-adrenoceptor antagonist), propranolol (2 mg/kg, i.p. a β-adrenoceptor antagonist), SCH23390 (0.05 mg/kg, i.p. a dopamine D1 receptor antagonist) or sulpiride (50 mg/kg, i.p. a dopamine D2 receptor antagonist) was able to reverse the anti-immobility effect of the extract (300 mg/kg, p.o) in TST. The combination of subeffective dose of fluoxetine (5 mg/kg, p.o.), imipramine (5 mg/kg, v.o.) or bupropion (5 mg/kg, v.o.) with a subffective dose of the extract (100 mg/kg) produced a synergistic antidepressant-like effect in the TST. The extract did not cause motor alteration in the open-field test at effective doses in both TST and FST. We also showed that the extract inhibited the MAO-A activity of mice brain in vitro. These results suggest that the extract from Bauhinia variegata present an antidepressanloke potential, which may be dependent on the monoaminergic system and its MAO-A inhibitory properties. / A depressão é um transtorno psiquiátrico que atinge 2-15% da população mundial. É uma neuropatologia heterogênea, pois inclui sintomas psicológicos, comportamentais e fisiológicos. Apesar de possuir diversos tratamentos para depressão, eles ainda não são tão efetivos e possuem muitos efeitos colaterais indesejáveis. Por isso, faz-se necessário o desenvolvimento de novas estratégias terapêuticas a fim de aperfeiçoar os tratamentos atuais. Diante disso, abrem-se possibilidades para o uso de plantas medicinas, uma vez que estas vem sendo utilizadas há milhares de anos por muitas populações para tratar diversas enfermidades. A Bauhinia variegata, conhecida popularmente como pata-de-vaca , possui efeitos benéficos à saúde, como: atividade anti-inflamatória, antidiabética, antidiarréica, entre outras. No presente estudo, foi investigado o efeito tipo-antidepressivo do extrato hidroetanólico bruto de folhas de Bauhinia variegata em dois testes preditivos de propriedade antidepressiva, o teste de suspensão pela cauda (TSC) e do teste do nado forçado (TNF) em camundongos machos adultos. Além disso, foi investigado o envolvimento do sistema monoaminérgico no efeito do extrato e sua atividade sobre a monoamina-oxidase A (MAO-A). O extrato bruto hidroetanólico foi administrado por via oral (vo) por gavagem. O tratamento agudo com o extrato de Bauhinia variegata (300 mg/kg) reduziu o tempo de imobilidade no TST. Além disso, a administração repetida (uma vez por dia durante 7 dias) do extrato também produziu um efeito tipo-antidepressivo no TSC (100 e 300 mg/kg) e TNF (300 mg/kg). O pré-tratamento de camundongos com cetanserina (5 mg/kg, ip, antagonista do receptor serotoninérgico 5-HT2A), prazosina (1 mg/kg, ip, antagonista α1-adrenérgico), ioimbina (1 mg/kg, ip, antagonista α2-adrenérgico), propranolol (2 mg/kg, ip, antagonista β-adrenérgico), SCH23390 (0,05 mg/kg, ip, antagonista do receptor dopaminérgico D1) ou sulpirida (50 mg/kg, ip, antagonista do receptor dopaminérgico D2) reverteu o efeito anti-imobilidade do extrato (300 mg/kg) no TSC. A combinação de doses sub-efetivas dos antidepressivos clássicos, fluoxetina (5 mg/kg, vo), imipramina (5 mg/kg vo) ou bupropiona (5 mg/kg, vo) com uma dose sub-efetiva do extrato (100 mg/kg) produziu um efeito antidepressivo sinérgico no TSC. O extrato não causou alteração locomotora no teste de campo aberto em doses eficazes, tanto no TSC como no TNF. Além disso, o extrato inibiu a atividade da MAO-A em preparações de mitocôndria encefálicas in vitro. Estes resultados sugerem que o extrato hidroetanólico bruto de folhas de Bauhinia variegata apresenta um potencial efeito tipo-antidepressivo, que pode ser dependente do sistema monoaminérgico.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Depression is a psychiatric disorder associated with a negative impact on quality of life. Monoaminergic system has been involved in this disease and in the action of antidepressants. This study aimed to investigate the potential antidepressant-like of 7-fluoro-1,3-diphenylisoquinoline-1-amine (FDPI) and the possible involvement of monoaminergic system. Results showed that FDPI (1, 10 and 20 mg/kg, intragastric (i.g.)) reduced the immobility time, increased swimming time, but did not alter climbing time of mice in the modified forced swimming test (FST). These effects were similar to those of paroxetine (8 mg/kg, intraperitoneally (i.p.)), a selective serotonin reuptake inhibitor, which was used as positive control. Pretreatments with p-chlorophenylalanine (pCPA, an inhibitor of serotonin (5-HT) synthesis, 100 mg/kg, i.p., once a day for 4 consecutive days), N-[1]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY 100635, a 5-HT1A receptor antagonist, 0.1 mg/kg, subcutaneous injection (s.c.)) and ondansetron (a 5-HT3 receptor antagonist, 1 mg/kg, i.p.) reversed the antidepressant-like effect of FDPI at the dose 1 mg/kg in FST, this did not occurs with ritanserin (a 5-HT2A/2C receptor antagonist, 1 mg/kg, i.p.). Antagonist related with dopaminergic system, as haloperidol (a D2 receptor antagonist, 0.2 mg/kg, i.p.) and SCH23390 (a D1 receptor antagonist, 0.05 mg/kg, s.c.) were able to reverse the antidepressant-like effect of FDPI at the dose 1 mg/kg in FST, this did not occurs with sulpiride (a D2 and D3 receptors antagonist, 50 mg/kg, i.p.). FDPI, at doses of 10 and 20 mg/kg, inhibited monoamine oxidase-B activity in prefrontal cortex of mice. These results suggest that FDPI produced an antidepressant-like action in the FST in mice, possibly by an involvement of the monoaminergic system. Additional studies are necessary in order to propose FDPI as a drug for depression treatment. / A depressão é uma doença psiquiátrica associada com um impacto negativo na qualidade de vida. O sistema monoaminérgico parece estar envolvido nessa doença e na ação dos antidepressivos. Esse estudo teve como objetivo investigar o potencial do tipo antidepressivo do 7-flúor- 1,3 difenilisoquinolina-1-amino (FDPI) e o possível envolvimento do sistema monoaminérgico. Os resultados mostraram que o FDPI (1, 10 e 20 mg/kg, intragástrico (i.g.)) reduziu o tempo de imobilidade, aumentou o tempo de nado, mas não alterou o tempo de escalada dos camundongos durante o teste do nado forçado (TNF) modificado. Esses efeitos foram similares aos da paroxetina (8 mg/kg, intraperitoneal (i.p.)), um inibidor seletivo da recaptação de serotonina, o qual foi usado como controle positivo. Os pré-tratamentos com p-clorofenilalanina (pCPA, um inibidor da síntese de serotonina (5-HT), 100 mg/kg, i.p., uma vez por dia, por 4 dias consecutivos), N-{2-[4-(2-metoxifenil)-1-piperazinil]etil}-N-(2-piridinil) ciclohexanocarboxamida (WAY 100635, um antagonista dos receptores 5-HT1A, 0,1 mg/kg, subcutâneo (s.c.)) e ondansetrona (um antagonista dos receptores 5-HT3, 1 mg/kg, i.p.) conseguiram reverter o efeito do tipo antidepressivo do FDPI na dose de 1 mg/kg no TNF, o que não aconteceu com a ritanserina (um antagonista do receptores 5-HT2A/2C, 1 mg/kg, i.p.). Antagonistas relacionados com o sistema dopaminérgico, como haloperidol (um antagonista do receptor D2, 0,2 mg/kg, i.p.), e SCH23390 (um antagonista do receptor D1, 0,05 mg/kg, s.c.) foram capazes de reverter o efeito do tipo antidepressivo do FDPI na dose de 1 mg/kg no TNF, o que não aconteceu com o sulpiride (um antagonista dos receptores D2 e D3, 50mg/kg, i.p.). O composto FDPI nas doses de 10 e 20 mg/kg inibiu a atividade da monoamino oxidase B em córtex pré-frontal de camundongos. Estes resultados sugerem que o FDPI apresentou uma ação do tipo antidepressiva no TNF em camundongos, possivelmente por um envolvimento do sistema monoaminérgico. Mais estudos se fazem necessários antes que se possa propor o FDPI como uma droga para o tratamento da depressão.

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Molecular modeling is a term referring to the study of proteins, nucleic acids, lipids, and other bio- or macro- or small molecules at the atomistic level using a combination of computational methods, physico-chemical principles, and mathematical functions. It can be generally sub-divided into two areas: molecular mechanics, which is the treatment of atoms and bonds as Newtonian particles and springs, and quantum mechanics, which models electronic behaviors using the Schrödinger equation and wavefunctions. Each technique is a powerful tool that, when used alone or in combination with wet lab experiments, can yield useful results, the products of which have broad applications in studying human disease models, oxidative damage, and other biomolecular processes that are otherwise not easily observed by experiment alone. Within this document, we study seven different such systems. This includes the mode of inhibitor binding to the enzyme monoamine oxidase B, the active site mechanism of that same enzyme, the dynamics of the unstructured p53 C-terminal domain in complex with globular, structured proteins, the process of the viral protein B2 unbinding from double-stranded RNA, and a focus on the dynamics of a variable loop in the antigenic peanut protein Ara h 2. In addition to those conventional molecular modeling studies, several of which were done in tandem with wet lab experiment, we also discuss the validation of charges and charge group parameters for small molecules used in molecular mechanics, and the development of software for the analysis of lipid bilayer systems in molecular mechanics simulations. As computational resources continue to evolve, and as more structural information becomes available, these methods are becoming an integral part of the study of biomolecules in the context of disease. / Ph. D.

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015