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Leiba J.,Montpellier University | Leiba J.,Center Medical Universersitaire | Carrere-Kremer S.,Montpellier University | Blondiaux N.,University of Lille Nord de France | And 6 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb. © 2014 Elsevier Inc. All rights reserved. Source

Wohlkonig A.,Structural Biology Brussels and Molecular and Cellular Interactions | Huet J.,Free University of Colombia | Looze Y.,Free University of Colombia | Wintjens R.,Free University of Colombia | Wintjens R.,French National Center for Scientific Research
PLoS ONE | Year: 2010

Background:Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. Results:Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-a domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a b-hairpin pointed towards the substrate binding cleft. The invariant b-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. Conclusions: The seven families and subfamilies of the lysozyme superfamily all have in common a b-hairpin structure which displays a family-specific sequence motif. These GH b-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily. © 2010 Wohlkönig et al. Source

Dubar F.,University of Lille Nord de France | Wintjens R.,Free University of Colombia | Martins-Duarte E.S.,Federal University of Rio de Janeiro | Vommaro R.C.,Federal University of Rio de Janeiro | And 7 more authors.
MedChemComm | Year: 2011

Novel ester prodrugs of ciprofloxacin were synthesized, and tested for their antimalarial and antitoxoplasma activity. These new compounds proved to be extremely efficient against these parasites. Molecular modeling and computational calculations were used to understand the mechanisms of action of these drugs. © The Royal Society of Chemistry 2011. Source

Villemagne B.,University of Lille Nord de France | Villemagne B.,French Institute of Health and Medical Research | Villemagne B.,Institute Pasteur Of Lille | Flipo M.,University of Lille Nord de France | And 47 more authors.
Journal of Medicinal Chemistry | Year: 2014

Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide, and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein, we have developed fragment-based approaches. In the current study, we combined surface plasmon resonance assay, X-ray crystallography, and ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, and in vitro and ex vivo activities of these compounds will be discussed. © 2014 American Chemical Society. Source

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