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Alexandre Wohlkonig

Bio: Alexandre Wohlkonig is an academic researcher from Vrije Universiteit Brussel. The author has contributed to research in topics: Ethionamide & Mycobacterium tuberculosis. The author has an hindex of 19, co-authored 46 publications receiving 1975 citations. Previous affiliations of Alexandre Wohlkonig include Université libre de Bruxelles & VU University Amsterdam.

Papers
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Journal ArticleDOI
19 Aug 2010-Nature
TL;DR: This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Abstract: Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

614 citations

Journal ArticleDOI
TL;DR: A general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof.
Abstract: There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months.

548 citations

Journal ArticleDOI
TL;DR: A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in theDNA cleavage domain through chelation of a noncatalytic magnesium ion.
Abstract: Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.

249 citations

Journal ArticleDOI
17 Mar 2017-Science
TL;DR: The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethion chloride-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.
Abstract: Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

108 citations

Journal ArticleDOI
TL;DR: From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP (C) intoPrP(Sc).
Abstract: Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrPC, into its misfolded oligomeric form, known as prion or PrPSc. The canonical human PrPC (HuPrP) fold features an unstructured N-terminal part (residues 23–124) and a well-defined C-terminal globular domain (residues 125–231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113–120) plays an important role in the conversion to PrPSc. The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form...

101 citations


Cited by
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Journal ArticleDOI
TL;DR: This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quInolone action against their enzyme targets, and suggests approaches to designing new drugs that display improved activity against resistant strains.
Abstract: Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone–enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

839 citations

Journal ArticleDOI
20 Aug 2015-Nature
TL;DR: A 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment is reported, revealing an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.
Abstract: Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 A X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

704 citations

Journal ArticleDOI
TL;DR: The acquisition of antimicrobial resistance genes by ESKAPE pathogens has reduced the treatment options for serious infections, increased the burden of disease, and increased death rates due to treatment failure and requires a coordinated global response for antim antibiotic resistance surveillance.
Abstract: Antimicrobial-resistant ESKAPE ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens represent a global threat to human health. The acquisition of antimicrobial resistance genes by ESKAPE pathogens has reduced the treatment options for serious infections, increased the burden of disease, and increased death rates due to treatment failure and requires a coordinated global response for antimicrobial resistance surveillance. This looming health threat has restimulated interest in the development of new antimicrobial therapies, has demanded the need for better patient care, and has facilitated heightened governance over stewardship practices.

674 citations

Journal ArticleDOI
TL;DR: This review discusses how topoisomerase inhibitors kill cells by trapping topoisomersases on DNA rather than by classical enzymatic inhibition, and extends to a novel mechanism of action of PARP inhibitors and could be applied to the targeting of transcription factors.
Abstract: Topoisomerases are ubiquitous enzymes that control DNA supercoiling and entanglements. They are essential during transcription and replication, and topoisomerase inhibitors are among the most effective and most commonly used anticancer and antibacterial drugs. This review consists of two parts. In the first part (“Lessons”), it gives background information on the catalytic mechanisms of the different enzyme families (6 different genes in humans and 4 in most bacteria), describes the “interfacial inhibition” by which topoisomerase-targeted drugs act as topoisomerase poisons, and describes clinically relevant topoisomerase inhibitors. It generalizes the interfacial inhibition principle, which was discovered from the mechanism of action of topoisomerase inhibitors, and discusses how topoisomerase inhibitors kill cells by trapping topoisomerases on DNA rather than by classical enzymatic inhibition. Trapping protein–DNA complexes extends to a novel mechanism of action of PARP inhibitors and could be applied to...

671 citations

Journal ArticleDOI
TL;DR: New Delhi metallo-β-lactamase (NDM) enzymes are the latest carbapenemases to be recognized and since 2008 have been reported worldwide, mostly in bacteria from patients epidemiologically linked to the Indian subcontinent, where they occur widely in hospital and community infections, and also in contaminated urban water.

574 citations