L,D-transpeptidases de Mycobacterium tuberculosis: estudo das interações com antibióticos β-lactâmicos e triagem de fragmentos
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Data
2018-07-26
Autores
Libreros-Zúñiga, Gerardo Andrés
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O reposicionamento de antibióticos e a busca novos fármacos constituem duas estratégias para abordar a resistência de Mycobacterium tuberculosis aos antimicrobianos. A parede celular de M. tuberculosis contém peptideoglicano (PG), estrutura essencial para a viabilidade celular e, portanto, um alvo farmacológico. O PG micobacteriano apresenta resíduos de açúcares altamente interconectados por ligações 3→3 catalisadas por L,D-transpeptidases (LdtsMt), enzimas sensíveis aos carbapenêmicos, que são reguladas positivamente durante a latência. M. tuberculosis codifica cinco LdtsMt (LdtMt1→LdtMt5), algumas delas sendo consideradas essenciais para estabilidade da parede micobacteriana, entretanto a LdtMt3 e LdtMt4 permanecem menos compreendidas. Uma vez que as LdtsMt podem ser inibidas por alguns β-lactâmicos, este trabalho busca conhecer seus mecanismos de interação com estes antibióticos. Adicionalmente, através da triagem de fragmentos, espera-se identificar compostos que possam servir como ponto de partida para a síntese de novos inibidores contra estas enzimas. LdtMt2, LdtMt3 e LdtMt5 foram produzidas em E. coli, purificadas e cristalizadas. Suas estruturas foram resolvidas a 1,8, 1,3 e 2,6 Å de resolução respectivamente. A estrutura de LdtMt3 é conservada com outras LdtsMt, embora diferenças no volume, topologia e carga da cavidade catalítica. LdtMt3 interage preferencialmente com faropenem e carbapenêmicos com diferentes afinidades e perfis termodinâmicos. A massa intacta da LdtMt3 na presença de carbapenêmicos indica que estes antibióticos permanecem covalentemente ligados a enzima sem alterações, enquanto que faropenem sofre degradação, possivelmente até um fragmento de acetila que fica covalentemente ligado à Cys246, como demostrado pela estrutura cristalográfica a 1.8 Å de resolução. Ensaios de docking revelaram os resíduos de LdtMt3 que provavelmente interagem com os β-lactâmicos intactos. Por outro lado, não foi possível identificar a afinidade de interação entre LdtMt5 e os β-lactâmicos testados. A triagem de 365 fragmentos confirmou dezoito que interagem com às LdtMt2, LdtMt3 e LdtMt5 com afinidades entre 10 e 3300 μM. Um conjunto de seis aminoácidos em cada enzima parece participar nas interações com esses fragmentos. Este trabalho proporciona novas informações sobre a estrutura de LdtMt3 e suas interações com antibióticos β-lactâmicos, assim como o possível mecanismo de degradação de faropenem posterior à acilação, o qual pode ser útil no melhoramento dos β-lactâmicos para tratamento da tuberculose. Adicionalmente, este é o primeiro estudo que identifica fragmentos que ligam às LdtsMt, informação relevante para a síntese de novos inibidores não β- lactâmicos das L,D-transpeptidases de M. tuberculosis.
The repositioning of antibiotics and the search for new drugs are two strategies to address the antibiotic resistance of Mycobacterium tuberculosis. The cell wall of M. tuberculosis contains peptidoglycan (PG), a structure essential for cell viability and therefore is considered a pharmacological target. Mycobacterial PG presents highly interconnected sugars by 3→3 bonds catalyzed by L, D-transpeptidases (LdtsMt). LdtsMt are a group of carbapenems sensitive enzymes, which are upregulated during latency. M. tuberculosis encodes five LdtsMt (LdtMt1→LdtMt5) and most of them are considered essential for mycobacterial wall stability; however, LdtMt3 and LdtMt4 remain less understood. Since LdtsMt can be inhibited by some β-lactams, this work seeks to know its mechanisms of interaction with these antibiotics. Additionally, by screening of fragments, it is expected to identify novel compounds that may serve as a starting point for the synthesis of new inhibitors against LdtsMt. LdtMt2, LdtMt3 and LdtMt5 were produced in E. coli, purified and crystallized. Their structures were solved at 1.8, 1.3 and 2.6 Å resolution, respectively. The structure of LdtMt3 is similar to other LdtsMt, despite differences in volume, topology and charge of the catalytic cavity. LdtMt3 interacts preferentially with faropenem and carbapenems with different affinities and thermodynamic profiles. The intact mass of LdtMt3 in the presence of carbapenems indicates that these antibiotics remain unaltered after LdtMt3 acylation, whereas faropenem undergoes degradation, possibly even an acetyl fragment which is covalently bound to Cys246 as demonstrated by the crystallographic structure at 1.8 Å resolution. Docking calculations revealed LdtMt3 residues that probably interact with the intact β-lactams. On the other hand, we could not identify interaction affinity between LdtMt5 and any tested β-lactams. Screening of 365 fragments confirmed eighteen compounds that bind to LdtMt2, LdtMt3 and LdtMt5 with affinities between 10 and 3300 μM. A set of six amino acids in each enzyme appears to participate in interactions with these fragments. This work provides novel information on the structure of LdtMt3 and its interactions with β-lactam antibiotics, as well as the possible mechanism of faropenem degradation after acylation. These information are useful to the improvement of β-lactams for tuberculosis treatment. Additionally, this is the first study identifying fragments binding to LdtsMt, providing relevant information for the synthesis of novel non-β-lactam inhibitors against L, D-transpeptidases from M. tuberculosis.
The repositioning of antibiotics and the search for new drugs are two strategies to address the antibiotic resistance of Mycobacterium tuberculosis. The cell wall of M. tuberculosis contains peptidoglycan (PG), a structure essential for cell viability and therefore is considered a pharmacological target. Mycobacterial PG presents highly interconnected sugars by 3→3 bonds catalyzed by L, D-transpeptidases (LdtsMt). LdtsMt are a group of carbapenems sensitive enzymes, which are upregulated during latency. M. tuberculosis encodes five LdtsMt (LdtMt1→LdtMt5) and most of them are considered essential for mycobacterial wall stability; however, LdtMt3 and LdtMt4 remain less understood. Since LdtsMt can be inhibited by some β-lactams, this work seeks to know its mechanisms of interaction with these antibiotics. Additionally, by screening of fragments, it is expected to identify novel compounds that may serve as a starting point for the synthesis of new inhibitors against LdtsMt. LdtMt2, LdtMt3 and LdtMt5 were produced in E. coli, purified and crystallized. Their structures were solved at 1.8, 1.3 and 2.6 Å resolution, respectively. The structure of LdtMt3 is similar to other LdtsMt, despite differences in volume, topology and charge of the catalytic cavity. LdtMt3 interacts preferentially with faropenem and carbapenems with different affinities and thermodynamic profiles. The intact mass of LdtMt3 in the presence of carbapenems indicates that these antibiotics remain unaltered after LdtMt3 acylation, whereas faropenem undergoes degradation, possibly even an acetyl fragment which is covalently bound to Cys246 as demonstrated by the crystallographic structure at 1.8 Å resolution. Docking calculations revealed LdtMt3 residues that probably interact with the intact β-lactams. On the other hand, we could not identify interaction affinity between LdtMt5 and any tested β-lactams. Screening of 365 fragments confirmed eighteen compounds that bind to LdtMt2, LdtMt3 and LdtMt5 with affinities between 10 and 3300 μM. A set of six amino acids in each enzyme appears to participate in interactions with these fragments. This work provides novel information on the structure of LdtMt3 and its interactions with β-lactam antibiotics, as well as the possible mechanism of faropenem degradation after acylation. These information are useful to the improvement of β-lactams for tuberculosis treatment. Additionally, this is the first study identifying fragments binding to LdtsMt, providing relevant information for the synthesis of novel non-β-lactam inhibitors against L, D-transpeptidases from M. tuberculosis.
Descrição
Palavras-chave
Mycobacterium tuberculosis, L,D-transpeptidases, β-lactâmicos, Faropenem, Cristalografia de raios X, Triagem de fragmentos, β-lactams, X-ray crystallography, Fragment screening