RESUMO
Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.
Assuntos
Brucelose/imunologia , Carboxiliases/imunologia , Pneumopatias/imunologia , Macrófagos Alveolares/imunologia , Animais , Isocitrato Liase/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lactate dehydrogenases (LDHs) are tetrameric enzymes of therapeutic relevance for cancer therapy due to their important implications in cancer cell metabolism. LDH active site inhibition suffers from different drawbacks due to several features such as high cellular concentration and a shared active site among the dehydrogenase family. Conversely, targeting the LDH oligomeric state is an exciting strategy that could provide a suitable alternative to active-site inhibition. In the present study, we developed a biophysical screening cascade to probe the LDHs tetrameric interface. Using nanoscale differential fluorimetry (nanoDSF) as a primary screening method, we identified a series of hits that destabilize the tetrameric protein. From this primary screening, we validated selected hits using saturation transfer difference nuclear magnetic resonance (STD NMR) and microscale thermophoresis (MST) as a combination of orthogonal biophysical techniques. Finally, we characterized the validated hits and demonstrated that they specifically interact at the tetrameric interface of LDH-1 and LDH-5 and can inhibit the LDH tetramerization process. Overall, this work provides a convenient method for screening ligands at the LDH tetrameric interface and has identified promising hits suitable for further optimization. We believe that this biophysical screening cascade, especially the use of (nano)DSF, could be extended to other homomeric proteins.
Assuntos
Lactato Desidrogenases , Fluorometria , Lactato Desidrogenases/antagonistas & inibidores , Ligantes , Espectroscopia de Ressonância MagnéticaRESUMO
OBJECTIVES: The spread of antibiotic resistant bacteria is an important threat for human health. Acinetobacter baumannii bacteria impose such a major issue, as multidrug- to pandrug-resistant strains have been isolated, rendering some infections untreatable. In this context, carbapenem-resistant A. baumannii bacteria were ranked as top priority by both WHO and CDC. In addition, A. baumannii bacteria survive in harsh environments, being capable of resisting to disinfectants and to persist prolonged periods of desiccation. Due to the high degree of variability found in A. baumannii isolates, the search for new antibacterials is very challenging because of the requirement of drug target conservation amongst the different strains. Here, we screened a chemical library to identify compounds active against several reference strains and carbapenem-resistant A. baumannii bacteria. METHODS: A repurposing drug screen was undertaken to identify A. baumannii growth inhibitors. One hit was further characterized by determining the IC50 and testing the activity on 43 modern clinical A. baumannii isolates, amongst which 40 are carbapenem-resistant. RESULTS: The repurposing screen led to the identification of a harmine-derived compound, called HDC1, which proves to have bactericidal activity on the multidrug-resistant AB5075-VUB reference strain with an IC50 of 48.23 µM. In addition, HDC1 impairs growth of 43 clinical A. baumannii isolates. CONCLUSIONS: We identified a compound with inhibitory activity on all tested strains, including carbapenem-resistant clinical A. baumannii isolates.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Carbapenêmicos/farmacologia , Harmina/farmacologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Hypoxia is one of the most important biological phenomena that influences cancer agressiveness and chemotherapy resistance. Cancer cells display dysregulated pathways notably resulting from oncogene expression. Tumors also show modifications in extracellular pH, extracellular matrix remodeling, neo-angiogenesis, hypoxia compared to normal tissues. Classically, the conventional anticancer therapies are efficient in cancer cells in normoxic conditions but under hypoxia, chemoresistance may occur. The addition of compounds that potentiate their activity in low oxygen environment could be a strategy to counteract this resistance. To identify new compounds active in hypoxia, we screened one hundred molecules with different chemical structures from an internal chemolibrary. Their potential ability to increase the activity of taxol and etoposide independently of their mechanism of action has been assayed. After a first step of selection, based on biological/pharmacological properties and chemical structure analysis, we identified three potential hits. Two hits are closely related amides/ureas and the third is a thiosemicarbazone. The compounds present no activity in cancer and normal cells when used alone but demonstrate chemosensitizing activity under hypoxia. Finally, by analyzing cell death, the indole thiosemicarbazone was shown to be able to significantly potentiate apoptosis induced by taxol and etoposide in two models of cancer cell lines. This new compound could lead to the development of an original series of chemosensitizers active under hypoxia.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Tiossemicarbazonas/administração & dosagem , Antineoplásicos/toxicidade , Apoptose/fisiologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Impressões Digitais de DNA/métodos , Etoposídeo/administração & dosagem , Etoposídeo/toxicidade , Células Hep G2 , Humanos , Paclitaxel/administração & dosagem , Paclitaxel/toxicidade , Tiossemicarbazonas/toxicidadeRESUMO
Direct access to the protonated merocyanine forms of two substituted spiropyrans by mechanosynthesis or sonochemistry was explored. The compounds were formed by the condensation reaction of the methyleneindolium iodide salt with salicylaldehyde derivatives. X-ray crystallography, 1Hâ NMR spectroscopy, abâ initio geometry optimization, and absorption spectroscopy were combined to provide a better understanding of the four-state molecular switch system in which the newly synthesized protonated merocyanines were found to play a central role. The results of this study suggest that the stability of the protonated merocyanines requires acidic conditions, as treatment with base led to the corresponding unprotonated merocyanines, which in turn spontaneously converted into photochromic closed spiropyrans.