Caracterização dos compostos derivados de furoxano como inibidores da atividade de bombas de efluxo em Mycobacterium tuberculosis e estudo da influência do efluxo e da halotolerância nos perfis de resistência do bacilo
Data
2016-02-29
Autores
Marino, Leonardo Biancolino [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Tuberculose (TB), com principal agente etiológico o bacilo Mycobacterium tuberculosis, é uma doença infecciosa passível de cura. Entretanto, é detentora de uma estatística mundial preocupante, e em 2014 apresentou 9,5 milhões de novos casos e 1,5 milhão de mortes. Além dos altos índices estatísticos, a TB aparece ainda associada a dois fatores agravantes: a co-infecção com HIV (“Human Immunodeficiency Virus”) e a crescente emergência de linhagens resistentes aos quimioterápicos como “multi-drug resistant” (MDR) e “extensively drug resistant” (XDR), que dificultam sobremaneira o tratamento, indicando premência na pesquisa de novos fármacos. A resistência do bacilo manifesta-se a partir de eventos adquiridos (como aquisição de mutações cromossomais em genes específicos relacionados ao metabolismo de fármacos) ou intrínsecos, como uma parede celular altamente hidrofóbica (devido à grande quantidade de ácidos micólicos). Estes mecanismos de resistência são discutidos amplamente na literatura. Por outro lado, pouco se sabe a respeito da resistência devido as alterações na expressão dos genes das bombas de efluxo, responsáveis pela exclusão de fármacos, bem como dos mecanismos de resistência relacionados a halotolerância bacteriana. Neste sentido, três assuntos de relevância no combate à tuberculose foram abordados nesta tese. Como primeira frente, avaliou-se inicialmente a expressão de 5 genes codificadores de bombas de efluxo pela técnica de RT-qPCR em 12 isolados clínicos frente ao tratamento com rifampicina, principal fármaco do regime terapêutico da TB. Resultados indicaram que essas proteínas auxiliam na emergência da resistência, sendo indicados pela superexpressão dos genes frente ao tratamento. Como segunda frente avaliou-se a atividade de 15 compostos inéditos de furoxânicos e benzofuroxânicos quanto à capacidade inibitória frente ao M. tuberculosis, citotoxicidade em células VERO, além de ensaios para avaliar seus mecanismos de ação (acumulação de brometo de etídio / inibição de efluxo, liberação de óxido nítrico e microarray). Resultados indicaram que a classe de compostos é promissora para o tratamento, com boa atividade inibitória contra o bacilo e considerado grau de seletividade. Ensaios de mecanismo de ação, indicaram atividade independente da inibição do efluxo, da subunidade de isoniazida ou da liberação de óxido nítrico. Microarray apontou para alteração em grande quantidade de proteínas ribossomais, semelhante ao que ocorre com aminoglicosídeos. Por fim, na terceira frente do estudo avaliou-se os efeitos da halotolerância na emergência de bacilos resistentes a fármacos, tendo como base as diferentes concentrações de cloreto de sódio a que o bacilo é submetido durante o seu ciclo de vida. Os resultados apontaram para aumento da resistência bacteriana com consequente aumento nas concentrações inibitórias de vários fármacos usados para a terapia da TB, quando o bacilo é submetido à concentração de cloreto de sódio encontrada no interior dos macrófagos, indicando uma necessidade de reflexão sobre as novas diretrizes para testes de novos fármacos contra TB.
Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis bacillus, presenting 9.5 million of new cases and 1.5 million of deaths in 2014. Two aggravating factors are associated with these high statistical indexes: a co-infection with HIV ("Human Immunodeficiency Virus") and the increasing emergence of resistant strains represented by MDR ("multi-drug resistant") and XDR ("extreme drug resistant") strains. The bacillus resistance manifests from acquired events (such as acquisition of chromosomal mutations in specific genes related to drug metabolism) or intrinsic, as a highly hydrophobic cell wall (due to the large amount of mycolic acids) and the action of efflux pumps (responsible for drug extrusion). Thus, the thesis project evaluated the expression of five genes encoding efflux pumps by RT-qPCR technique in 12 clinical isolates against rifampicin, the main anti-TB drug used in the standard treatment. Results indicated that these proteins contribute to the emergence of resistance, indicated by overexpression of these genes after treatment. The second part of the study evaluated the activity of 15 furoxan and benzofuroxan derivative compounds for inhibitory capacity against the M. tuberculosis, cytotoxicity in VERO cells, as well as ethidium bromide accumulation and efflux assays. Aiming evaluate their mechanism of action, microarray and assays of nitric oxide release were also done, as they are supposed nitric oxide donors and may present an action by oxidative stress and/or the activity efflux pumps inhibition. Results indicated that the class of compounds is promising for the treatment, with good inhibitory activity against bacillus and substantial degree of selectivity. Mechanism of action assays indicated activity independent of efflux inhibition, of isoniazid subunit’ or nitric oxide release. Microarray pointed to changes in expression of ribosomal proteins, similar to what occurs with aminoglycosides. Finally, the third part of the study demonstrated that the halotolerance developed by M. tuberculosis might cause the emergence of drug resistant bacilli, based on the different concentrations of sodium chloride that the bacillus faces during its cycle. The results indicate a need for reflection on the new guidelines for testing new TB drugs. The results pointed to an increase in the minimal inhibitory concentrations of various anti-TB drugs, when the bacillus was subjected to the sodium chloride concentration found in macrophages, indicating a need for reflection on the guidelines for discovery of new anti-TB drugs.
Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis bacillus, presenting 9.5 million of new cases and 1.5 million of deaths in 2014. Two aggravating factors are associated with these high statistical indexes: a co-infection with HIV ("Human Immunodeficiency Virus") and the increasing emergence of resistant strains represented by MDR ("multi-drug resistant") and XDR ("extreme drug resistant") strains. The bacillus resistance manifests from acquired events (such as acquisition of chromosomal mutations in specific genes related to drug metabolism) or intrinsic, as a highly hydrophobic cell wall (due to the large amount of mycolic acids) and the action of efflux pumps (responsible for drug extrusion). Thus, the thesis project evaluated the expression of five genes encoding efflux pumps by RT-qPCR technique in 12 clinical isolates against rifampicin, the main anti-TB drug used in the standard treatment. Results indicated that these proteins contribute to the emergence of resistance, indicated by overexpression of these genes after treatment. The second part of the study evaluated the activity of 15 furoxan and benzofuroxan derivative compounds for inhibitory capacity against the M. tuberculosis, cytotoxicity in VERO cells, as well as ethidium bromide accumulation and efflux assays. Aiming evaluate their mechanism of action, microarray and assays of nitric oxide release were also done, as they are supposed nitric oxide donors and may present an action by oxidative stress and/or the activity efflux pumps inhibition. Results indicated that the class of compounds is promising for the treatment, with good inhibitory activity against bacillus and substantial degree of selectivity. Mechanism of action assays indicated activity independent of efflux inhibition, of isoniazid subunit’ or nitric oxide release. Microarray pointed to changes in expression of ribosomal proteins, similar to what occurs with aminoglycosides. Finally, the third part of the study demonstrated that the halotolerance developed by M. tuberculosis might cause the emergence of drug resistant bacilli, based on the different concentrations of sodium chloride that the bacillus faces during its cycle. The results indicate a need for reflection on the new guidelines for testing new TB drugs. The results pointed to an increase in the minimal inhibitory concentrations of various anti-TB drugs, when the bacillus was subjected to the sodium chloride concentration found in macrophages, indicating a need for reflection on the guidelines for discovery of new anti-TB drugs.
Descrição
Palavras-chave
Tuberculose, Efluxo, Resistência bacteriana, Halotolerância, Furoxanos