scholarly journals N1-Benzofused Modification of Fluoroquinolones Reduces Activity Against Gram- Negative Bacteria

2019 ◽  
Author(s):  
Mark Laws ◽  
Charlotte Hind ◽  
Andrea Favaron ◽  
Shirin Jamshidi ◽  
J Mark Sutton ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Dna Gyrase ◽  
Polymyxin B ◽  
Membrane Penetration ◽  
Gram Negative ◽  
New Information ◽  
A Cell ◽  
Modelling Techniques ◽  
Free Environment ◽  
Solubility Profiles

<p>The fluoroquinolone class of antibiotics have a well-established structure-activity relationship (SAR) and a long history in the clinic, but the effect of electron-rich benzofused substituents at the N1 position remains poorly explored. Groups at this position are part of the topoisomerase-DNA binding complex and form a hydrophobic interaction with the major groove of DNA. Molecular modelling techniques were employed to select benzofused substituents that were shown to enhance this interaction of fluoroquinolones for the DNA gyrase target. Seven N1-modified fluoroquinolones were synthesised and tested against a panel of Gram-negative pathogens to determine minimum inhibitory concentration (MIC) values. Gram-negative outer membrane penetration was investigated using the membrane permeabiliser polymyxin B nonapeptide (PMBN) and the target affinity of representative compound <b>6e</b> was determined in a cell-free environment. A correlation between N1 substituent hydrophobicity and activity was observed across the MIC panel, with compound activity decreasing with increased hydrophobicity. Those compounds with highest hydrophobicity were inactive due to poor solubility profiles whereas compounds with intermediate hydrophobicity were inactive due to impaired outer membrane penetration and reduced inhibition for the DNA gyrase target, the latter in contrast to modelling predictions. This study adds new information to the fluoroquinolone SAR and suggests limited utility of large hydrophobic substitution at the N1 position of fluoroquinolones.</p>

scholarly journals N1-Benzofused Modification of Fluoroquinolones Reduces Activity Against Gram- Negative Bacteria

2019 ◽  
Author(s):  
Mark Laws ◽  
Charlotte Hind ◽  
Andrea Favaron ◽  
Shirin Jamshidi ◽  
J Mark Sutton ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Dna Gyrase ◽  
Polymyxin B ◽  
Membrane Penetration ◽  
Gram Negative ◽  
New Information ◽  
A Cell ◽  
Modelling Techniques ◽  
Free Environment ◽  
Solubility Profiles

<p>The fluoroquinolone class of antibiotics have a well-established structure-activity relationship (SAR) and a long history in the clinic, but the effect of electron-rich benzofused substituents at the N1 position remains poorly explored. Groups at this position are part of the topoisomerase-DNA binding complex and form a hydrophobic interaction with the major groove of DNA. Molecular modelling techniques were employed to select benzofused substituents that were shown to enhance this interaction of fluoroquinolones for the DNA gyrase target. Seven N1-modified fluoroquinolones were synthesised and tested against a panel of Gram-negative pathogens to determine minimum inhibitory concentration (MIC) values. Gram-negative outer membrane penetration was investigated using the membrane permeabiliser polymyxin B nonapeptide (PMBN) and the target affinity of representative compound <b>6e</b> was determined in a cell-free environment. A correlation between N1 substituent hydrophobicity and activity was observed across the MIC panel, with compound activity decreasing with increased hydrophobicity. Those compounds with highest hydrophobicity were inactive due to poor solubility profiles whereas compounds with intermediate hydrophobicity were inactive due to impaired outer membrane penetration and reduced inhibition for the DNA gyrase target, the latter in contrast to modelling predictions. This study adds new information to the fluoroquinolone SAR and suggests limited utility of large hydrophobic substitution at the N1 position of fluoroquinolones.</p>

scholarly journals Outer Membrane Interaction Kinetics of New Polymyxin B Analogs in Gram-Negative Bacilli

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Noushin Akhoundsadegh ◽  
Corrie R. Belanger ◽  
Robert E. W. Hancock
Keyword(s):  
Outer Membrane ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Intact Cells ◽  
Membrane Interaction ◽  
Gram Negative ◽  
Content Type ◽  
Interaction Kinetics ◽  
Hill Numbers ◽  
Multiple Strains

ABSTRACT Infections caused by drug-resistant Gram-negative bacilli are a severe global health threat, limiting effective drug choices for treatment. In this study, polymyxin analogs designed to have reduced nephrotoxicity, direct activity, and potentiating activity were assessed for inhibition and outer membrane interaction kinetics against wild-type (WT) and polymyxin or multidrug-resistant (MDR) Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In MIC assays, two polymyxin B (PMB) analogs (SPR1205 and SPR206) and a polymyxin E analog (SPR946), with shortened peptide side chains and branched aminobutyryl N termini, exhibited promising activity compared with PMB and previously tested control polymyxin analogs SPR741 and polymyxin B nonapeptide (PMBN). Using dansyl-polymyxin (DPX) binding to assess the affinity of interaction with lipopolysaccharide (LPS), purified or in the context of intact cells, SPR206 exhibited similar affinities to PMB but higher affinities than the other SPR analogs. Outer membrane permeabilization measured by the 1-N-phenyl-napthylamine (NPN) assay did not differ significantly between the polymyxin analogs. Moreover, Hill numbers were greater than 1 for most of the compounds tested on E. coli and P. aeruginosa strains which indicates that the disruption of the outer membrane by one molecule of compound cooperatively enhances the subsequent interactions of other molecules against WT and MDR strains. The high activity demonstrated by SPR206 as well as its ability to displace LPS and permeabilize the outer membrane of multiple strains of Gram-negative bacilli while showing cooperative potential with other membrane disrupting compounds supports further research with this polymyxin analog.

scholarly journals Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane

2019 ◽  
Vol 6 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
Shawn French ◽  
Maya Farha ◽  
Michael J. Ellis ◽  
Zaid Sameer ◽  
Jean-Philippe Côté ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Gram Negative

scholarly journals Direct Observation of Conversion From Walled Cells to Wall-Deficient L-Form and Vice Versa in Escherichia coli Indicates the Essentiality of the Outer Membrane for Proliferation of L-Form Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Taiki Chikada ◽  
Tomomi Kanai ◽  
Masafumi Hayashi ◽  
Taishi Kasai ◽  
Taku Oshima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Polymyxin B ◽  
Cell Deformation ◽  
Gram Negative Bacteria ◽  
Physical Force ◽  
Synthesis Inhibitor ◽  
Hypertonic Medium ◽  
Gram Negative ◽  
E Coli

Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

scholarly journals Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors

2018 ◽  
Vol 115 (26) ◽  
pp. 6834-6839 ◽  
Author(s):  
Ge Zhang ◽  
Vadim Baidin ◽  
Karanbir S. Pahil ◽  
Eileen Moison ◽  
David Tomasek ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bacterial Infections ◽  
Cell Envelope ◽  
New Drugs ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Cellular Targets ◽  
Outer Leaflet ◽  
A Cell ◽  
Membrane Treatment

New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway inAcinetobacter. We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

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