Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional Manancial UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/4457 |
Resumo: | The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is responsible to terminate acetylcholine activity in the terminal nervous junctions with its effector organs or post-synaptic sites. The activity of this enzyme could be inhibited by organophosphorus (OP) compounds, and this inactivation leads to an accumulation of acetylcholine in the cholinergic receptors, leading to a cholinergic crisis that may result in death. In this way, the OP compound methamidophos has been related to its broad use in various agriculture cultures in Brazil, with high intoxication rate. Actually, the only compounds able to revert the AChE inhibition by OP, are the oxime, such compounds may reactive the enzyme activity due its high nuclephilic power, attacking the phosphoryl group of the inhibited enzyme and displacing it. However, such compounds show toxic effects, and its use is limited by the high specificity, with each oxime acting only in the reactivation of AChE induced by specific OP coumponds. These limitations raise the need of development of new compounds with AChE reactivator potency, with minor side effects. In this way, have been utilized a series of computational tools (in silico models), with the aim of understand the interaction occurring at a molecular level and, so, rationalize the new compounds development. Thus, the aim of this thesis is to evaluate the activity of three new compounds in reactivate the AChE inhibited by methamidophos, in comparison with two others oximes used in clinical (obidoxime and pralidoxime), both in in silico and in vitro models. Our work demonstrate that the newly synthesized compounds are able to reactivate human erythrocyte AChE, however less efficiently than pralidoxime and obidoxime, and reactivate human plasma butyrylcholinesterase (BChE), where the classical oximes failed. We also show that pralidoxime, which obtained the best reactivation constant among all tested compounds, attack the phosphorus-oxygen moiety (formed between the methamidophos and the AChE catalytic triad residue Ser203) via a region known as oxyanion-hole , composed by the residues Gly120, Gly121 and Ala204. Such found may help in the development of new compounds with better reactivatory activity on AChE inhibited by OP compounds. Furthermore, we show for the first time the individual contribution of each amino acid of AChE, in a radii of 14 Å from the ligand, to the oxime bonding to its active site, using quantum chemistry methods. Here, we demonstrate the important of a quaternary nitrogen to the stabilization of the oximes into the active site; as well as, we obtained evidences that the active form of oximes should be the unprotonated one, instead the protonated, which has been target of debate in the scientific society. Particularly important, we show the critical contribution of amino acids that lies distant from the ligand to the adopted conformation and stabilization of the compounds into the active site of AChE, which has been neglected until far. Finally, our study also evaluates the toxic effects of the compound isatin-3-N4-benzilthiosemicarbazone (IBTC) in mice, which presented low toxicity, with median lethal dose superior at 500 mg/kg. Concluding, this study contributes significantly to the development of new drugs able to restore the AChE activity with minor toxic effects. |
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2013-03-042013-03-042012-06-04LUGOKENSKI, Thiago Henrique. EVALUATION OF NEW COMPOUNDS ON CHOLINESTERASES ACTIVITY IN MODELS IN SILICO AND IN VITRO. 2012. 107 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2012.http://repositorio.ufsm.br/handle/1/4457The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is responsible to terminate acetylcholine activity in the terminal nervous junctions with its effector organs or post-synaptic sites. The activity of this enzyme could be inhibited by organophosphorus (OP) compounds, and this inactivation leads to an accumulation of acetylcholine in the cholinergic receptors, leading to a cholinergic crisis that may result in death. In this way, the OP compound methamidophos has been related to its broad use in various agriculture cultures in Brazil, with high intoxication rate. Actually, the only compounds able to revert the AChE inhibition by OP, are the oxime, such compounds may reactive the enzyme activity due its high nuclephilic power, attacking the phosphoryl group of the inhibited enzyme and displacing it. However, such compounds show toxic effects, and its use is limited by the high specificity, with each oxime acting only in the reactivation of AChE induced by specific OP coumponds. These limitations raise the need of development of new compounds with AChE reactivator potency, with minor side effects. In this way, have been utilized a series of computational tools (in silico models), with the aim of understand the interaction occurring at a molecular level and, so, rationalize the new compounds development. Thus, the aim of this thesis is to evaluate the activity of three new compounds in reactivate the AChE inhibited by methamidophos, in comparison with two others oximes used in clinical (obidoxime and pralidoxime), both in in silico and in vitro models. Our work demonstrate that the newly synthesized compounds are able to reactivate human erythrocyte AChE, however less efficiently than pralidoxime and obidoxime, and reactivate human plasma butyrylcholinesterase (BChE), where the classical oximes failed. We also show that pralidoxime, which obtained the best reactivation constant among all tested compounds, attack the phosphorus-oxygen moiety (formed between the methamidophos and the AChE catalytic triad residue Ser203) via a region known as oxyanion-hole , composed by the residues Gly120, Gly121 and Ala204. Such found may help in the development of new compounds with better reactivatory activity on AChE inhibited by OP compounds. Furthermore, we show for the first time the individual contribution of each amino acid of AChE, in a radii of 14 Å from the ligand, to the oxime bonding to its active site, using quantum chemistry methods. Here, we demonstrate the important of a quaternary nitrogen to the stabilization of the oximes into the active site; as well as, we obtained evidences that the active form of oximes should be the unprotonated one, instead the protonated, which has been target of debate in the scientific society. Particularly important, we show the critical contribution of amino acids that lies distant from the ligand to the adopted conformation and stabilization of the compounds into the active site of AChE, which has been neglected until far. Finally, our study also evaluates the toxic effects of the compound isatin-3-N4-benzilthiosemicarbazone (IBTC) in mice, which presented low toxicity, with median lethal dose superior at 500 mg/kg. Concluding, this study contributes significantly to the development of new drugs able to restore the AChE activity with minor toxic effects.A enzima acetilcolinesterase (EC 3.1.1.7, AChE) é responsável por terminar a ação da acetilcolina nas junções das terminações nervosas com seus órgãos efetores ou sítios pós-sinaptícos. A atividade desta enzima pode ser inibida por compostos organofosforados (OP), e sua inativação resulta em um acúmulo de acetilcolina nos receptores colinérgicos, levando a crise colinérgica que pode levar a morte. No Brasil, se destaca o uso do composto OP metamidofós, que é largamente utilizado no controle de pragas em culturas agrícolas e tem sido relacionado com altas taxas de intoxicação. Atualmente, os únicos compostos capazes de reverter a inibição da AChE por OP são as oximas, tais compostos podem reativar a enzima devido a seu alto poder nucleofílico, podendo atacar e retirar o grupamento fosforil da enzima inibida. Porém, tais compostos apresentam efeitos tóxicos, e tem seu uso limitado pela alta especificidade, com cada oxima atuando na AChE inibida por apenas alguns compostos OP. Tais limitações criam a necessidade do desenvolvimento de novos fármacos com potencial reativador da AChE com menores efeitos colaterais. Neste sentido, se tem utilizado uma série de ferramentas computacionais (modelos in silico), com o objetivo de entender as interações que ocorrem em nível molecular e, desta forma, racionalizar o desenvolvimento de novos compostos. Sendo assim, o objetivo desta tese consiste em avaliar a atividade de três novos compostos, em comparação com duas oximas já utilizadas na clínica (obidoxima e pralidoxima), sobre a atividade da AChE inibida por metamidofós, tanto em modelos in silico como in vitro. Como resultados, observamos que os três novos compostos foram capazes de reativar a AChE de eritrócitos humanos inibida por metamidofós, contudo com menor eficiência que as oximas já utilizadas na clínica. Porém, todos os novos compostos foram capazes de reativar a enzima butirilcolinesterase (BChE), uma enzima acessória à AChE no sistema colinérgico, inibida por metamidofós, enquanto nenhumas das oximas clássicas tiveram qualquer atividade reativadora nesta enzima. Nosso trabalho também demonstrou que a pralidoxima, que obteve a melhor constante de reativação entre todos os compostos testados, ataca a ligação fosforo-oxigênio (formada entre o metamidofós e o resíduo Ser203, da tríade catalítica da AChE) via uma região conhecida como oxyanion-hole , que compreende os resíduos Gly120, Gly121 e Ala204. Tal achado pode ajudar no desenvolvimento de novos compostos com melhor atividade reativatória na AChE inibida por OPs. Além disso, mostramos aqui pela primeira vez, a contribuição de cada resíduo de aminoácido da AChE, num raio de 14 Å do ligante, para a ligação das oximas no seu sítio ativo, usando métodos de química quântica. Tais achados mostraram a importância da presença de um nitrogênio quaternário para a estabilização das oximas no sítio ativo; assim como colheu evidências que a forma ativa das oximas seria a sua forma desprotonada, ao invés da forma protonada, o que tem sido alvo de algum debate no meio cientifico. Particularmente importante, foi demonstrado a contribuição fundamental de aminoácidos que se encontram distantes do ligante, para a estabilização e conformação adotada pelos compostos, e que até o momento tem sido negligenciados em estudos in silico. Por fim, nosso estudo também avaliou os efeitos tóxicos do composto isatina-3-N4-benziltiosemicarbazona (IBTC) em camundongos, o qual apresentando baixa toxicidade, com valores de dose letal mediana (LD50) superiores a 500 mg/kg. Desta forma, este estudo contribui para a desenvolvimento de novos fármacos capazes de reativar a AChE e que apresentem menos efeitos tóxicos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de Santa MariaPrograma de Pós-Graduação em Ciências Biológicas: Bioquímica ToxicológicaUFSMBRBioquímicaAcetilcolinesteraseOrganofosforadosMetamidofósOximasModelos computacionaisAcetylcholinesteraseOrganophosphorusMethamidophosOximesComputational modelsCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAAvaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitroEvaluation of new compounds on cholinesterases activity in models in silico and in vitroinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisSoares, Félix Alexandre Antuneshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769181A8Monserrat, José Maríahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769169H6Franco, Jeferson Luishttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4705814T8Schetinger, Maria Rosa ChitolinaLoro, Vania Luciahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796333D7http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4266777U6Lugokenski, Thiago Henrique2008000000024005003005003005005003aef53b7-b173-4c56-a944-bed2de5e5dda31545eab-c86c-4b7a-8a80-ac940a0e681fb5472080-62c6-4a3d-95a4-32cdf82b1007423a65fa-1ad8-43d9-b50e-5041a255d6a0a5d2f3d1-0aa0-4c3b-89d3-b20a63b19b248c4258ae-c78b-4a63-868b-639544b7b5d5info:eu-repo/semantics/openAccessreponame:Repositório Institucional Manancial UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALLUGOKENSKI, THIAGO HENRIQUE.pdfTese de Doutoradoapplication/pdf33448791http://repositorio.ufsm.br/bitstream/1/4457/1/LUGOKENSKI%2c%20THIAGO%20HENRIQUE.pdfefc3120c2028f2e4d69d46b6425dddd6MD51TEXTLUGOKENSKI, THIAGO HENRIQUE.pdf.txtLUGOKENSKI, THIAGO HENRIQUE.pdf.txtExtracted texttext/plain131370http://repositorio.ufsm.br/bitstream/1/4457/2/LUGOKENSKI%2c%20THIAGO%20HENRIQUE.pdf.txtf55820a33934a98ae0f2529a398d2033MD52THUMBNAILLUGOKENSKI, THIAGO HENRIQUE.pdf.jpgLUGOKENSKI, THIAGO HENRIQUE.pdf.jpgIM Thumbnailimage/jpeg4755http://repositorio.ufsm.br/bitstream/1/4457/3/LUGOKENSKI%2c%20THIAGO%20HENRIQUE.pdf.jpgc11ddca5bc5b624bb0a0a4066d3bd6a6MD531/44572017-08-31 14:06:46.194oai:repositorio.ufsm.br:1/4457Repositório Institucionalhttp://repositorio.ufsm.br/PUBhttp://repositorio.ufsm.br/oai/requestouvidoria@ufsm.bropendoar:39132017-08-31T17:06:46Repositório Institucional Manancial UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.por.fl_str_mv |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
dc.title.alternative.eng.fl_str_mv |
Evaluation of new compounds on cholinesterases activity in models in silico and in vitro |
title |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
spellingShingle |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro Lugokenski, Thiago Henrique Acetilcolinesterase Organofosforados Metamidofós Oximas Modelos computacionais Acetylcholinesterase Organophosphorus Methamidophos Oximes Computational models CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
title_short |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
title_full |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
title_fullStr |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
title_full_unstemmed |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
title_sort |
Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro |
author |
Lugokenski, Thiago Henrique |
author_facet |
Lugokenski, Thiago Henrique |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Soares, Félix Alexandre Antunes |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769181A8 |
dc.contributor.referee1.fl_str_mv |
Monserrat, José María |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769169H6 |
dc.contributor.referee2.fl_str_mv |
Franco, Jeferson Luis |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4705814T8 |
dc.contributor.referee3.fl_str_mv |
Schetinger, Maria Rosa Chitolina |
dc.contributor.referee4.fl_str_mv |
Loro, Vania Lucia |
dc.contributor.referee4Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796333D7 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4266777U6 |
dc.contributor.author.fl_str_mv |
Lugokenski, Thiago Henrique |
contributor_str_mv |
Soares, Félix Alexandre Antunes Monserrat, José María Franco, Jeferson Luis Schetinger, Maria Rosa Chitolina Loro, Vania Lucia |
dc.subject.por.fl_str_mv |
Acetilcolinesterase Organofosforados Metamidofós Oximas Modelos computacionais |
topic |
Acetilcolinesterase Organofosforados Metamidofós Oximas Modelos computacionais Acetylcholinesterase Organophosphorus Methamidophos Oximes Computational models CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.subject.eng.fl_str_mv |
Acetylcholinesterase Organophosphorus Methamidophos Oximes Computational models |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
description |
The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is responsible to terminate acetylcholine activity in the terminal nervous junctions with its effector organs or post-synaptic sites. The activity of this enzyme could be inhibited by organophosphorus (OP) compounds, and this inactivation leads to an accumulation of acetylcholine in the cholinergic receptors, leading to a cholinergic crisis that may result in death. In this way, the OP compound methamidophos has been related to its broad use in various agriculture cultures in Brazil, with high intoxication rate. Actually, the only compounds able to revert the AChE inhibition by OP, are the oxime, such compounds may reactive the enzyme activity due its high nuclephilic power, attacking the phosphoryl group of the inhibited enzyme and displacing it. However, such compounds show toxic effects, and its use is limited by the high specificity, with each oxime acting only in the reactivation of AChE induced by specific OP coumponds. These limitations raise the need of development of new compounds with AChE reactivator potency, with minor side effects. In this way, have been utilized a series of computational tools (in silico models), with the aim of understand the interaction occurring at a molecular level and, so, rationalize the new compounds development. Thus, the aim of this thesis is to evaluate the activity of three new compounds in reactivate the AChE inhibited by methamidophos, in comparison with two others oximes used in clinical (obidoxime and pralidoxime), both in in silico and in vitro models. Our work demonstrate that the newly synthesized compounds are able to reactivate human erythrocyte AChE, however less efficiently than pralidoxime and obidoxime, and reactivate human plasma butyrylcholinesterase (BChE), where the classical oximes failed. We also show that pralidoxime, which obtained the best reactivation constant among all tested compounds, attack the phosphorus-oxygen moiety (formed between the methamidophos and the AChE catalytic triad residue Ser203) via a region known as oxyanion-hole , composed by the residues Gly120, Gly121 and Ala204. Such found may help in the development of new compounds with better reactivatory activity on AChE inhibited by OP compounds. Furthermore, we show for the first time the individual contribution of each amino acid of AChE, in a radii of 14 Å from the ligand, to the oxime bonding to its active site, using quantum chemistry methods. Here, we demonstrate the important of a quaternary nitrogen to the stabilization of the oximes into the active site; as well as, we obtained evidences that the active form of oximes should be the unprotonated one, instead the protonated, which has been target of debate in the scientific society. Particularly important, we show the critical contribution of amino acids that lies distant from the ligand to the adopted conformation and stabilization of the compounds into the active site of AChE, which has been neglected until far. Finally, our study also evaluates the toxic effects of the compound isatin-3-N4-benzilthiosemicarbazone (IBTC) in mice, which presented low toxicity, with median lethal dose superior at 500 mg/kg. Concluding, this study contributes significantly to the development of new drugs able to restore the AChE activity with minor toxic effects. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-06-04 |
dc.date.accessioned.fl_str_mv |
2013-03-04 |
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2013-03-04 |
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info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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LUGOKENSKI, Thiago Henrique. EVALUATION OF NEW COMPOUNDS ON CHOLINESTERASES ACTIVITY IN MODELS IN SILICO AND IN VITRO. 2012. 107 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2012. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/4457 |
identifier_str_mv |
LUGOKENSKI, Thiago Henrique. EVALUATION OF NEW COMPOUNDS ON CHOLINESTERASES ACTIVITY IN MODELS IN SILICO AND IN VITRO. 2012. 107 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2012. |
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