Determinação estrutural e busca por inibidores da enzima desoxi-hipusina sintase de organismos eucarióticos causadores de doenças negligenciadas
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2023-02-27
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Universidade Estadual Paulista (Unesp)
Resumo
O fator de tradução de eucariotos 5A (eIF5A) é altamente conservado em eucariotos e essencial para a proliferação celular. eIF5A é modificado pós-traducionalmente através de uma reação muito específica, chamada hipusinação, a qual é necessária para sua função. A hipusinação ocorre em duas etapas, envolvendo as enzimas desoxi-hipusina sintase (DHPS) e desoxi-hipusina hidroxilase (DOOH). Considerando a essencialidade da hipusinação e de eIF5A, a DHPS e a DOOH correspondem a alvos interessantes para o desenvolvimento de fármacos para inibir a proliferação de vários organismos. Neste contexto, o objetivo deste trabalho é determinar a estrutura tridimensional e buscar inibidores para as DHPSs dos seguintes organismos causadores de doenças tropicais negligenciadas: Paracoccidioides brasiliensis (Pb), Histoplasma capsulatum (Hc), Brugia malayi (Bm) e Leishmania sp. (duas isoformas: A e B). Para tanto, foram padronizadas as condições de clonagem, produção em Escherichia coli e purificação dessas proteínas. Isso possibilitou o estabelecimento de um ensaio bioquímico fácil de ser miniaturizado para a HcDHPSA, BmDHPSA e PbDHPSA, e a determinação da estrutura cristalográfica da BmDHPSA e da HcDHPSA. No entanto, essas estruturas apresentam uma arquitetura muito similar à da enzima humana (HsDHPS), o que dificulta o desenvolvimento de inibidores seletivos para as DHPSs desses patógenos. Portanto, novos esforços foram direcionados para a obtenção das Leishmania sp. DHPSs na forma solúvel a partir de E. coli, visto que essas enzimas provavelmente formam um heterocomplexo, diferentemente da HsDHPS. Após combinar uma série de estratégias, as DHPSs de L. donovani (LdDHPSAB) e de L. major (LmDHPSAB) foram produzidas na forma solúvel e ativa a partir de E. coli, o que permitiu a realização de um high-throughput drug screening in vitro com a LdDHPSAB, empregando a reação parcial das DHPSs acoplada ao ensaio NAD+/NADH-Glo™. Dos 26771 compostos testados inicialmente a 10 μM, 44 compostos (hits) inibiram a atividade da LdDHPSAB em mais de 60%. Uma triagem secundária confirmou que 24 (57%) dos hits exibiram efeitos inibitórios consistentes (maior do que 60%) sobre a atividade da LdDHPSAB. Depois de excluir os hits que também inibiram a atividade das enzimas do ensaio NAD+/NADH-Glo™, os 13 compostos restantes foram analisados quanto à concentração-resposta empregando o mesmo ensaio acoplado. Nove desses 13 compostos apresentaram um IC50 contra a atividade da LdDHPSAB na faixa micromolar baixa. Dentre esses, os compostos MMV1674024 e MMV904563 apresentaram um maior potencial para futuras otimizações com o intuito de melhorar a potência de inibição sobre a atividade das Leishmania sp. DHPSs. Além disso, os dados de inibição in vitro sugerem que a inibição das Leishmania sp. DHPSs sem afetar a atividade da HsDHPS corresponde a uma tarefa viável, confirmando a hipótese de que a DHPS é um alvo interessante para ser explorado em Leishmania sp. Ainda, empregando o ensaio baseado na detecção da fluorescência do NADH, foi possível determinar que o composto MMV1674024 exerce uma inibição sobre a atividade da LmDHPSAB do tipo mista em relação ao NAD+ e à espermidina. Finalmente, foram obtidos cristais para a LmDHPSAB preparada na presença de NAD+ e do composto MMV1674024. Entetanto, esses cristais não difrataram durante a coleta de dados de difração por raios X, mesmo após tentativas de otimização das condições de cristalização.
The eukaryotic translation factor 5A (eIF5A) is highly conserved in eukaryotes and is essential for cell proliferation. eIF5A undergoes an extremely specific post-translational modification, called hypusination, required for eIF5A function. Hypusination occurs in two steps that involve the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOOH). Considering the essentiality of hypusination for eIF5A function, DHPS and DOHH are promising targets for developing new drugs to stop cell proliferation in various organisms. In this context, the aim of this work is to determine the three-dimensional structure and search for DHPS inhibitors of the following organisms that cause neglected tropical diseases: Paracoccidioides brasiliensis (Pb), Histoplasma capsulatum (Hc), Brugia malayi (Bm) and Leishmania sp. (two isoforms: A and B). For this purpose, the conditions for cloning, production in Escherichia coli and purification of these proteins were established. This enabled the adaptation of an easy-to-miniaturize biochemical assay for HcDHPSA, BmDHPSA and PbDHPSA, and the determination of the crystallographic structure of BmDHPSA and HcDHPSA. However, these structures present a very similar architecture to the human enzyme (HsDHPS), which hinders the development of selective inhibitors for the DHPSs from those pathogens. Therefore, all the efforts were directed towards obtaining Leishmania sp. DHPSs in a soluble form from E.coli, as these enzymes probably form a heterocomplex, unlike HsDHPS. After combining a series of strategies, it was possible to produce recombinant L. donovani (LdDHPSAB) and L. major (LmDHPSAB) DHPSs in a soluble and active form from E. coli. A high-throughput screening assay was established in vitro with the LdDHPSAB, in which the LdDHPSAB activity was quantified using NAD+/NADH-Glo™ assay coupled with the partial reaction of the DHPSs. Of the 26771 compounds initially screened at 10 μM, 44 compounds (hits) inhibited LdDHPSAB activity by over 60%. The secondary screening confirmed that 24 (57%) hits exhibited consistent inhibitory effects (inhibition above 60%) on LdDHPSAB activity. Then, 11 hit compounds that also inhibited the enzymes from the NAD+/NADH-Glo™ assay were excluded from further analysis. The remaining 13 hit compounds were analyzed for dose-response using the same coupled assay. Nine out of those 13 compounds presented IC50 against LdDHPSAB in the lower micromolar range. Among these, the compounds MMV1674024 and MMV904563 showed increased potential for future optimizations to improve the inhibition potency against Leishmania sp. DHPSs activity. Furthermore, the in vitro data suggested that inhibiting Leishmania sp. DHPS targets without inhibiting the human counterpart is a feasible task, confirming the hypothesis that DHPS is an interesting target to be explored in Leishmania sp. Also, using the assay based on the detection of NADH fluorescence, it was possible to determine that the compound MMV1674024 exerts a mixed-type inhibition on the activity of the LmDHPSAB in relation to NAD+ and spermidine. Finally, crystals were obtained for LmDHPSAB prepared in the presence of NAD+ and the compound MMV1674024. However, these crystals did not diffract during x-ray diffraction data collection, even after attempts to optimize the crystallization conditions.
The eukaryotic translation factor 5A (eIF5A) is highly conserved in eukaryotes and is essential for cell proliferation. eIF5A undergoes an extremely specific post-translational modification, called hypusination, required for eIF5A function. Hypusination occurs in two steps that involve the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOOH). Considering the essentiality of hypusination for eIF5A function, DHPS and DOHH are promising targets for developing new drugs to stop cell proliferation in various organisms. In this context, the aim of this work is to determine the three-dimensional structure and search for DHPS inhibitors of the following organisms that cause neglected tropical diseases: Paracoccidioides brasiliensis (Pb), Histoplasma capsulatum (Hc), Brugia malayi (Bm) and Leishmania sp. (two isoforms: A and B). For this purpose, the conditions for cloning, production in Escherichia coli and purification of these proteins were established. This enabled the adaptation of an easy-to-miniaturize biochemical assay for HcDHPSA, BmDHPSA and PbDHPSA, and the determination of the crystallographic structure of BmDHPSA and HcDHPSA. However, these structures present a very similar architecture to the human enzyme (HsDHPS), which hinders the development of selective inhibitors for the DHPSs from those pathogens. Therefore, all the efforts were directed towards obtaining Leishmania sp. DHPSs in a soluble form from E.coli, as these enzymes probably form a heterocomplex, unlike HsDHPS. After combining a series of strategies, it was possible to produce recombinant L. donovani (LdDHPSAB) and L. major (LmDHPSAB) DHPSs in a soluble and active form from E. coli. A high-throughput screening assay was established in vitro with the LdDHPSAB, in which the LdDHPSAB activity was quantified using NAD+/NADH-Glo™ assay coupled with the partial reaction of the DHPSs. Of the 26771 compounds initially screened at 10 μM, 44 compounds (hits) inhibited LdDHPSAB activity by over 60%. The secondary screening confirmed that 24 (57%) hits exhibited consistent inhibitory effects (inhibition above 60%) on LdDHPSAB activity. Then, 11 hit compounds that also inhibited the enzymes from the NAD+/NADH-Glo™ assay were excluded from further analysis. The remaining 13 hit compounds were analyzed for dose-response using the same coupled assay. Nine out of those 13 compounds presented IC50 against LdDHPSAB in the lower micromolar range. Among these, the compounds MMV1674024 and MMV904563 showed increased potential for future optimizations to improve the inhibition potency against Leishmania sp. DHPSs activity. Furthermore, the in vitro data suggested that inhibiting Leishmania sp. DHPS targets without inhibiting the human counterpart is a feasible task, confirming the hypothesis that DHPS is an interesting target to be explored in Leishmania sp. Also, using the assay based on the detection of NADH fluorescence, it was possible to determine that the compound MMV1674024 exerts a mixed-type inhibition on the activity of the LmDHPSAB in relation to NAD+ and spermidine. Finally, crystals were obtained for LmDHPSAB prepared in the presence of NAD+ and the compound MMV1674024. However, these crystals did not diffract during x-ray diffraction data collection, even after attempts to optimize the crystallization conditions.
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Palavras-chave
eIF5A, desoxi-hipusina sintase, doenças tropicais negligenciadas, leishmaniose, descoberta de fármacos, deoxyhypusine synthase, neglected tropical diseases, leishmaniasis, drug discovery