| 1. |
- El-sagheir, Ahmed M. Kamal, et al.
(författare)
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Rational design, synthesis, molecular modeling, biological activity, and mechanism of action of polypharmacological norfloxacin hydroxamic acid derivatives
- 2023
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Ingår i: RSC Medicinal Chemistry. - 2632-8682. ; 14:12, s. 2593-2610
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Tidskriftsartikel (refereegranskat)abstract
- Fluoroquinolones are broad-spectrum antibiotics that target gyrase and topoisomerase IV, involved in DNA compaction and segregation. We synthesized 28 novel norfloxacin hydroxamic acid derivatives with additional metal-chelating and hydrophobic pharmacophores, designed to enable interactions with additional drug targets. Several compounds showed equal or better activity than norfloxacin against Gram-positive, Gram-negative, and mycobacteria, with MICs as low as 0.18 mu M. The most interesting derivatives were selected for in silico, in vitro, and in vivo mode of action studies. Molecular docking, enzyme inhibition, and bacterial cytological profiling confirmed inhibition of gyrase and topoisomerase IV for all except two tested derivatives (10f and 11f). Further phenotypic analysis revealed polypharmacological effects on peptidoglycan synthesis for four derivatives (16a, 17a, 17b, 20b). Interestingly, compounds 17a, 17b, and 20b, showed never seen before effects on cell wall synthetic enzymes, including MreB, MurG, and PonA, suggesting a novel mechanism of action, possibly impairing the lipid II cycle. Addition of metal-chelating and lipophilic groups to norfloxacin yielded dual-action compounds inhibiting DNA gyrase/topoisomerase IV and bacterial cell wall synthesis.
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| 2. |
- Kamal El-Sagheir, Ahmed M., et al.
(författare)
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N4-Substituted Piperazinyl Norfloxacin Derivatives with Broad-Spectrum Activity and Multiple Mechanisms on Gyrase, Topoisomerase IV, and Bacterial Cell Wall Synthesis
- 2023
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Ingår i: ACS Bio and Med Chem Au. - 2694-2437. ; 3:6, s. 494-506
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Tidskriftsartikel (refereegranskat)abstract
- Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.
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| 3. |
- El-sagheir, Ahmed M. Kamal, et al.
(författare)
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Design, Synthesis, Molecular Modeling, Biological Activity, and Mechanism of Action of Novel Amino Acid Derivatives of Norfloxacin
- 2023
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Ingår i: ACS Omega. - 2470-1343. ; 8:45, s. 43271-43284
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Tidskriftsartikel (refereegranskat)abstract
- Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.
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| 4. |
- Humphrey, Madeleine, et al.
(författare)
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Tracking Global and Local Changes in Membrane Fluidity Through Fluorescence Spectroscopy and Microscopy
- 2023
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; , s. 203-229
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Membrane fluidity is a critical parameter of cellular membranes, which cells continuously strive to maintain within a viable range. Interference with the correct membrane fluidity state can strongly inhibit cell function. Triggered changes in membrane fluidity and associated impacts on lipid domains have been postulated to contribute to the mechanism of action of membrane targeting antimicrobials, but the corresponding analyses have been hampered by the absence of readily available analytical tools. Here, we expand upon the protocols outlined in the first edition of this book, providing further and alternative protocols that can be used to measure changes in membrane fluidity. We provide detailed protocols, which allow straightforward in vivo and in vitro measurement of antibiotic compound-triggered changes in membrane fluidity and fluid membrane microdomains. Furthermore, we summarize useful strains constructed by us and others to characterize and confirm lipid specificity of membrane antimicrobials directly in vivo.
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| 5. |
- Beyer, Luisa I., et al.
(författare)
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Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides
- 2023
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Ingår i: ACS Infectious Diseases. - 2373-8227. ; 10:1, s. 79-92
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Tidskriftsartikel (refereegranskat)abstract
- Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.
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| 6. |
- Schäfer, Ann-Britt, 1993, et al.
(författare)
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A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides
- 2020
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Ingår i: Frontiers in cellular and infection microbiology. - : Frontiers Media SA. - 2235-2988. ; 10
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Forskningsöversikt (refereegranskat)abstract
- Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.
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| 7. |
- Schäfer, Ann-Britt, 1993
(författare)
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Characterization of the in vivo mechanisms of action of cell envelope-targeting antibiotics
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Multidrug-resistant bacteria pose a major threat to society. Once easily treatable diseases are now challenging to cure. With the golden age of antibiotics being over, the discovery of new antibiotics has been stagnant, and no new antibiotic class has been introduced into the clinic since 2003. Therefore, it is crucial to discover new antibiotics and to optimize existing antimicrobial compounds. This thesis aims to provide more information on the mechanisms of antibiotics on living bacteria and can be divided into two parts. One part provides detailed information on techniques that can be utilized to study the mode of action of new antibiotic compounds. Here, methods are discussed in detail and curated to a method set that can be easily utilized by other researchers. The curated assays were then applied, and a phenotypic reference set was created. In the second part, these methods were used to investigate the mechanisms of new antimicrobial compounds and lead structures to provide important information for their further development. The fluorescence dyes laurdan, Nile red, and DiIC12 that are able to track fluidity changes in the bacterial membranes in response to antibiotic treatment were described. In addition to the detailed protocols and their evaluation, the methods were applied to investigate the effects of daptomycin. These methods were able to detect that daptomycin changes the overall membrane fluidity differently in B. subtilis and S. aureus . Furthermore, I have successfully applied the curated methods, consisting of BCP, membrane potential (DiSC3(5), and cell wall synthesis assays (MreB mobility and PliaI ) to study several new compounds, e.g., antimicrobial peptides from marine streptomycetes. Two of these new antimicrobial peptides led to the consecutive permeabilization of the outer and inner membrane in E. coli . Interestingly, they also affected DNA packing. Further, an in-depth study of a new promising drug candidate, ES24, was conducted, showing its unique target of the Sec-secretion pathway and an additional DNA-damaging effect. Overall, this thesis provides important insights into the techniques used in mode of action studies, describes an easily accessible method set for other researchers, and provides important information on the modes of action of new antimicrobial compounds. Mode of action, cell-envelope targeting, antibiotics, antimicrobial peptides
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| 8. |
- Schäfer, Ann-Britt, 1993, et al.
(författare)
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Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA
- 2023
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Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 9:2, s. 253-269
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Tidskriftsartikel (refereegranskat)abstract
- Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.
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| 9. |
- Sidarta, Margareth, 1992, et al.
(författare)
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Lipid phase separation impairs membrane thickness sensing by the Bacillus subtilis sensor kinase DesK
- 2024
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Ingår i: Microbiology spectrum. - 2165-0497. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions. IMPORTANCE The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.
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