RESUMO
The aims of this work were to optimize the production of Erwinia carotovoral-asparaginase II enzyme in Escherichia coli by different fed-batch cultivation strategies using a benchtop bioreactor and to evaluate the therapeutic potential of the recombinant enzyme against different acute lymphoblastic leukemia cell lines. The highest enzyme activities (â¼98,000 U/L) were obtained in cultures using the DO-stat feeding strategy with induction in 18 h of culture. Under these experimental conditions, the maximum values for recombinant l-asparaginase II (rASNase) yield per substrate, rASNase yield per biomass, and productivity were approximately 1204 U/gglucose, 3660 U/gcells, and 3260 U/(L·h), respectively. This condition was efficient for achieving high yields of the recombinant enzyme, which was purified and used in in vitro antileukemic potential tests. Of all the leukemic cell lines tested, RS4;11 showed the highest sensitivity to rASNase, with an IC50 value of approximately 0.0006 U/mL and more than 70% apoptotic cells. The study demonstrated that the cultivation strategies used were efficient for obtaining high yield and productivity of rASNase with therapeutic potential inasmuch as cytotoxic activity and induction of apoptosis were demonstrated for this protein.
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The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis (MtFolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of MtFolB. These compounds exhibited IC50 values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting MtFolB, an attractive molecular target for TB drug development.
Assuntos
Aldeído Liases , Antituberculosos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Antituberculosos/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Relação Estrutura-Atividade , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/metabolismo , Aldeído Liases/química , Células Vero , Estrutura Molecular , Cristalografia por Raios X , Chlorocebus aethiops , Animais , Guanina/farmacologia , Guanina/química , Guanina/análogos & derivados , Guanina/síntese química , Simulação de Acoplamento Molecular , Células Hep G2 , Modelos MolecularesRESUMO
Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.
Assuntos
Antibacterianos , Proteínas de Bactérias , Biofilmes , Staphylococcus aureus , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Brasil , Poríferos/microbiologiaRESUMO
Utilizing a scaffold-hopping strategy from the drug candidate telacebec, a novel series of 2-(quinolin-4-yloxy)acetamides was synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (Mtb) growth. These compounds demonstrated potent activity against drug-sensitive and multidrug-resistant strains (MIC ≤ 0.02 µM). Leading compounds were evaluated against a known qcrB resistant strain (T313A), and their loss in activity suggested that the cytochrome bc1 complex is the likely target. Additionally, these structures showed high selectivity regarding mammalian cells (selectivity index > 500) and stability across different aqueous media. Furthermore, some of the synthesized quinolines demonstrated aqueous solubility values that exceeded those of telacebec, while maintaining low rates of metabolism. Finally, a selected compound prevented Mtb growth by more than 1.7 log10 colony forming units in a macrophage model of tuberculosis (TB) infection. These findings validate the proposed design and introduce new 2-(quinolin-4-yloxy)acetamides with potential for development in TB drug discovery campaigns.
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We analyzed the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) itself and SARS-CoV-2-IgG immune complexes to trigger human monocyte necroptosis. SARS-CoV-2 was able to induce monocyte necroptosis dependently of MLKL activation. Necroptosis-associated proteins (RIPK1, RIPK3 and MLKL) were involved in SARS-CoV-2N1 gene expression in monocytes. SARS-CoV-2 immune complexes promoted monocyte necroptosis in a RIPK3- and MLKL-dependent manner, and Syk tyrosine kinase was necessary for SARS-CoV-2 immune complex-induced monocyte necroptosis, indicating the involvement of Fcγ receptors on necroptosis. Finally, we provide evidence that elevated LDH levels as a marker of lytic cell death are associated with COVID-19 pathogenesis.
Assuntos
Complexo Antígeno-Anticorpo , COVID-19 , Humanos , Complexo Antígeno-Anticorpo/metabolismo , SARS-CoV-2 , Proteínas Quinases/metabolismo , Monócitos , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Simulação de Acoplamento Molecular , Antituberculosos/farmacologia , Antituberculosos/química , Tuberculose/tratamento farmacológicoRESUMO
Using cycloalkyl and electron-donating groups to decrease the carbonyl electrophilicity, a novel series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Structure-activity relationship studies led to selective and potent antitubercular agents with minimum inhibitory concentrations in the submicromolar range against drug-sensitive and drug-resistant Mtb strains. An evaluation of the activity of the lead compounds against a spontaneous qcrB mutant strain indicated that the structures targeted the cytochrome bc 1 complex. In addition, selected molecules inhibited Mtb growth in a macrophage model of tuberculosis infection. Furthermore, the leading compound was chemically stable depending on the context and showed good kinetic solubility, high permeability, and a low rate of in vitro metabolism. Finally, the pharmacokinetic profile of the compound was assessed after oral administration to mice. To the best of our knowledge, for the first time, a 2-(quinoline-4-yloxy)acetamide was obtained with a sufficient exposure, which may enable in vivo effectiveness and its further development as an antituberculosis drug candidate.
RESUMO
Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.
Assuntos
COVID-19 , SARS-CoV-2 , Brasil , Genoma Viral , Humanos , Mutação , Filogenia , SARS-CoV-2/genéticaRESUMO
Flexibility and function are related properties in the study of protein dynamics. Flexibility reflects in the conformational potential of proteins and thus in their functionalities. The presence of interactions between protein-ligands and protein-protein complexes, substrates, and environmental changes can alter protein plasticity, acting from the rearrangement of the side chains of amino acids to the folding/unfolding of large structural motifs. To evaluate the effects of the flexibility in protein systems, we defined the enzyme 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis, or MtInhA, as our target system. MtInhA is biologically active as a tetramer in solution; however, computational studies commonly use the monomer justifying the independence of its active sites due to their distances. However, differences in flexibility between tertiary and quaternary structures could present impact on the size of the active site, influencing the drug discovery process. In this study, we investigated the influence of flexibility restrictions in A- and B-loops of the MtInhA in order to suggest a monomeric structure that describes the conformational behavior of the tetrameric system. Overall, we observed that simulations where restrictions were applied to the A- and B-loops present a more similar behavior to the native structure when compared to unrestricted simulations. Therefore, our work presents a monomeric model of MtInhA, which has conformational characteristics of the biologically active structure. Thus, the data obtained in this work can be applied to the MtInhA system for the generation of more reliable flexible models for molecular docking experiments, and also for the performance of longer simulations by molecular dynamics and with a lower computational cost.
Assuntos
Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Oxirredutases/metabolismoRESUMO
Tuberculosis remains a global health problem that affects millions of people around the world. Despite recent efforts in drug development, new alternatives are required. Herein, a series of 27 N-(4-(benzyloxy)benzyl)-4-aminoquinolines were synthesized and evaluated for their ability to inhibit the M. tuberculosis H37Rv strain. Two of these compounds exhibited minimal inhibitory concentrations (MICs) similar to the first-line drug isoniazid. In addition, these hit compounds were selective for the bacillus with no significant change in viability of Vero and HepG2 cells. Finally, chemical stability, permeability and metabolic stability were also evaluated. The obtained data show that the molecular hits can be optimized aiming at the development of drug candidates for tuberculosis treatment.