Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Approche psychosociale de l’acceptation des terrains d’accueil pour les gens du voyage

International audienceSince 1990 and 2000, French laws imply specific places for gypsies locations in 5.000 at least inhabitants villages. Relations between gypsies and non gypsies people are quite difficult. Indeed, most of the time fear and threat guide these relations. Based on different social psychology theories, two studies are presented. The first aim concerns the link between perception of gypsies by sedentary people and acceptation of specific areas from them. Perceived humanity of the target, acculturative orientations and threat are assessed. Then, links with acceptance are examined. Results suggest first that gypsies are defined with primary emotions more than with secondary ones (study 1) and perceived as separatist people (study 1 and 2). Finally, correlations show that emotion attribution, acculturative perception and threat perception are linked with the acceptation of areas.En France, les communes de plus de 5.000 habitants doivent s’équiper de terrains d’accueil pour les gens du voyage. Or, les relations entre ces derniers et l’environnement local (dont les riverains) sont souvent appréhendées dans un contexte de craintes et de réticences. Étayées par différents champs de la psychologie sociale, les deux études portent sur l’acceptation des terrains d’accueil pour les gens du voyage. L’objectif de la recherche est premièrement de repérer l’effet de la «proximité» et de la «commune» d’installation du terrain sur l’acceptation et deuxièmement de repérer si les perceptions qu’ont les sédentaires vis‑à‑vis des gens du voyage (perception d’humanité, d’acculturation et menace perçue) sont corrélées avec l’acceptation des terrains d’accueil. Les résultats montrent que les gens du voyage sont bien définis avec plus d’émotions primaires que secondaires (étude 1), et qu’ils sont perçus comme séparatistes (études 1 et 2). Enfin, les corrélations montrent que l’attribution d’émotions, la perception d’adoption de la culture française et la menace sont liées à l’acceptation des terrains d’accuei

Estimation of optic nerve sheath diameter on an initial brain computed tomography scan can contribute prognostic information in traumatic brain injury patients.

International audienceINTRODUCTION: To evaluate the prognostic value of optic nerve sheath diameter (ONSD) measured on the initial brain computed tomography (CT) scan for intensive care unit (ICU) mortality in severe traumatic brain injury (TBI) patients. METHODS: A prospective observational study of all severe TBI patients admitted to a neurosurgical ICU (10-month period). Demographic and clinical data, and brain CT scan results were recorded. ONSD for each eye was measured on the initial CT scan. The group of ICU survivors was compared to non-survivors. Glasgow Outcome Scale (GOS) was evaluated 6 months after ICU discharge. RESULTS: 77 patients were included (age: 43 +/- 18; 81% males; mean Injury Severity Score: 35 +/- 15; ICU mortality: 28.5% [n=22]). Mean ONSD on the initial brain CT scan was 7.8 +/- 0.1 mm in non-survivors vs. 6.8 +/- 0.1 mm in survivors (p<0.001). The operative value of ONSD was a good predictor of mortality (area under the curve [AUC]: 0.805). An ONSD cut-off [greater than or equal to] 7.3 had a sensitivity of 86.4% and a specificity of 74.6% and was independently associated with mortality in this population (adjusted odds ratio [AOR] [95% confidence interval]: 22.7 [3.2-159.6], p=0.002). There was a relationship between initial ONSD values and 6-month GOS (p=0.03). CONCLUSIONS: ONSD measured on the initial brain CT scan is independently associated with ICU mortality rate (when [greater than or equal to]7.3 mm) in severe TBI patients

Discovery of novel N-phenylphenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining high-throughput screening and synthesis.

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis -infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Microvascular flow alterations in critically ill COVID-19 patients: A prospective study.

BackgroundData on microcirculatory pattern of COVID-19 critically ill patients are scarce. The objective was to compare sublingual microcirculation parameters of critically ill patients according to the severity of the disease.MethodsThe study is a single-center prospective study with critically ill COVID-19 patients admitted in ICU. Sublingual microcirculation was assessed by IDF microscopy within 48 hours of ICU admission. Microcirculatory flow index (MFI), proportion of perfused vessel (PPV), total vessel density (TVD), De Backer score (DBS), perfused vessel density (PVD) and heterogeneity index (HI) were assessed. Patients were divided in 2 groups (severe and critical) according to the World health organization definition.FindingsFrom 19th of March to 7th of April 2020, 43 patients were included. Fourteen patients (33%) were in the severe group and twenty-nine patients (67%) in the critical group. Patients in the critical group were all mechanically ventilated. The critical group had significantly higher values of MFI, DBS and PVD in comparison to severe group (respectively, PaCO2: 49 [44-45] vs 36 [33-37] mmHg; pConclusionCritical COVID-19 patients under mechanical ventilation seem to have higher red blood cell velocity than severe non-ventilated patients

Ethionamide boosters: Synthesis, biological activity, and structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors

We report in this article an extensive structure-activity relationships (SAR) study with 58 thiophen-2-yl-1,2,4-oxadiazoles as inhibitors of EthR, a transcriptional regulator controling ethionamide bioactivation in Mycobacterium tuberculosis. We explored the replacement of two key fragments of the starting lead BDM31343. We investigated the potency of all analogues to boost subactive doses of ethionamide on a phenotypic assay involving M. tuberculosis infected macrophages and then ascertained the mode of action of the most active compounds using a functional target-based surface plasmon resonance assay. This process revealed that introduction of 4,4,4-trifluorobutyryl chain instead of cyanoacetyl group was crucial for intracellular activity. Replacement of 1,4-piperidyl by (R)-1,3-pyrrolidyl scaffold did not enhance activity but led to improved pharmacokinetic properties. Furthermore, the crystal structures of ligand-EthR complexes were consistent with the observed SAR. In conclusion, we identified EthR inhibitors that boost antibacterial activity of ethionamide with nanomolar potency while improving solubility and metabolic stability. © 2011 American Chemical Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe