84 research outputs found
Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design
The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor
Facile production of the Pseudomonas aeruginosa virulence factor LasB in Escherichia coli for structure‐based drug design
The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high‐level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor
An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay
Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships
An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay
Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model
microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising
and underexplored target may have important pharmacological potential through modulation of
vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an
alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane
transporters in a native biological environment rather than using a low-throughput in vitro assay
employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell
screening approach, we demonstrated the optimization of a weak hit compound (2) into a small
molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to
quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity
relationships
Bacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor
Liposomes have been studied for decades as nanoparticulate drug delivery systems for
cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial
efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work,
we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound
(HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored
antimicrobial target. The liposomes were composed of DOPG (18:1 (∆9-cis) phosphatidylglycerol)
and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and
Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of
the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 ◦C. Loading of
the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation
efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine
liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis
revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL
at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm.
Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably
hydrophobic, antibiotics
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Bacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor
Liposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics
Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH)
Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc‐binding group (ZBG) variants of this thiol‐derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development
Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors
Extracellular virulence factors have emerged as
attractive targets in the current antimicrobial resistance crisis.
The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an
important role in the infection process. Here, we report a submicromolar, non-peptidic, fragment-like inhibitor of LasB
discovered by careful visual inspection of structural data.
Inspired by the natural LasB substrate, the original fragment
was successfully merged and grown. The optimized inhibitor
is accessible via simple chemistry and retained selectivity with
a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the
inhibitor. We also demonstrate an improved in vivo efficacy
of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as
promising drug candidates
Not Every Hit-Identification Technique Works on 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXPS):Making the Most of a Virtual Screening Campaign
In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.</p
Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach
Pharmacophore searches that include anchors, fragments
contributing above average to receptor binding, combined with
one-step syntheses are a powerful approach for the fast
discovery of novel bioactive molecules. Here, we are presenting
a pipeline for the rapid and efficient discovery of aspartyl
protease inhibitors. First, we hypothesized that hydrazine could
be a multi-valent warhead to interact with the active site Asp
carboxylic acids. We incorporated the hydrazine anchor in a
multicomponent reaction and created a large virtual library of
hydrazine derivatives synthetically accessible in one-step. Next,
we performed anchor-based pharmacophore screening of the
libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an
enzyme activity assay and the binding mode confirmed by
several soaked crystal structures supporting the validity of the
hypothesis and approach. The herein reported pipeline of tools
will be of general value for the rapid generation of receptor
binders beyond Asp proteases
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