| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- Gray, Declan Alan, et al.
(författare)
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More than a pore: A current perspective on the in vivo mode of action of the lipopeptide antibiotic daptomycin
- 2020
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 9:1
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Forskningsöversikt (refereegranskat)abstract
- Daptomycin is a cyclic lipopeptide antibiotic, which was discovered in 1987 and entered the market in 2003. To date, it serves as last resort antibiotic to treat complicated skin infections, bacteremia, and right-sided endocarditis caused by Gram-positive pathogens, most prominently methicillin-resistant Staphylococcus aureus. Daptomycin was the last representative of a novel antibiotic class that was introduced to the clinic. It is also one of the few membrane-active compounds that can be applied systemically. While membrane-active antibiotics have long been limited to topical applications and were generally excluded from systemic drug development, they promise slower resistance development than many classical drugs that target single proteins. The success of daptomycin together with the emergence of more and more multi-resistant superbugs attracted renewed interest in this compound class. Studying daptomycin as a pioneering systemic membrane-active compound might help to pave the way for future membrane-targeting antibiotics. However, more than 30 years after its discovery, the exact mechanism of action of daptomycin is still debated. In particular, there is a prominent discrepancy between in vivo and in vitro studies. In this review, we discuss the current knowledge on the mechanism of daptomycin against Gram-positive bacteria and try to offer explanations for these conflicting observations.
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| 5. |
- Gray, Declan Alan, et al.
(författare)
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Multitarget Approaches against Multiresistant Superbugs
- 2020
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Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:6, s. 1346-1365
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Forskningsöversikt (refereegranskat)abstract
- Despite efforts to develop new antibiotics, antibacterial resistance still develops too fast for drug discovery to keep pace. Often, resistance against a new drug develops even before it reaches the market. This continued resistance crisis has demonstrated that resistance to antibiotics with single protein targets develops too rapidly to be sustainable. Most successful long-established antibiotics target more than one molecule or possess targets, which are encoded by multiple genes. This realization has motivated a change in antibiotic development toward drug candidates with multiple targets. Some mechanisms of action presuppose multiple targets or at least multiple effects, such as targeting the cytoplasmic membrane or the carrier molecule bactoprenol phosphate and are therefore particularly promising. Moreover, combination therapy approaches are being developed to break antibiotic resistance or to sensitize bacteria to antibiotic action. In this Review, we provide an overview of antibacterial multitarget approaches and the mechanisms behind them.
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| 6. |
- 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. ; 2601, 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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| 7. |
- 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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| 8. |
- Saaki, Terrens N.V., et al.
(författare)
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SepF supports the recruitment of the DNA translocase SftA to the Z-ring
- 2022
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 117:5, s. 1263-1274
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Tidskriftsartikel (refereegranskat)abstract
- In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA. SftA is active during vegetative growth whereas SpoIIIE is primarily active during sporulation and pumps the chromosome into the spore compartment. FtsK and SpoIIIE contain several transmembrane helices, however, SftA is assumed to be a cytoplasmic protein. It is unknown how SftA is recruited to the cell division site. Here we show that SftA is a peripheral membrane protein, containing an N-terminal amphipathic helix that reversibly anchors the protein to the cell membrane. Using a yeast two-hybrid screen we found that SftA interacts with the conserved cell division protein SepF. Based on extensive genetic analyses and previous data we propose that the septal localization of SftA depends on either SepF or the cell division protein FtsA. Since SftA seems to interfere with the activity of SepF, and since inactivation of SepF mitigates the sensitivity of a ∆sftA mutant for ciprofloxacin, we speculate that SftA might delay septum synthesis when chromosomal DNA is in the vicinity.
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| 9. |
- 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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| 10. |
- 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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