RESUMEN
Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers ("ampetoids") are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are "fast killers", which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Peptoides/farmacología , Secuencia de Aminoácidos , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Biomasa , Hemólisis/efectos de los fármacos , Humanos , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Rastreo , Peptoides/química , Peptoides/metabolismo , Permeabilidad , Ribosomas/metabolismoRESUMEN
Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Peptoides/química , Peptoides/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad Cuantitativa , Staphylococcus aureus/efectos de los fármacosRESUMEN
Antimicrobial peptides (AMPs) are integral components of innate immunity and are typically found in combinations in which they can synergize for broader-spectrum or more potent activity. Previously, we reported peptoid mimics of AMPs with potent and selective antimicrobial activity. Using checkerboard assays, we demonstrate that peptoids and AMPs can interact synergistically, with fractional inhibitory concentration indices as low as 0.16. These results strongly suggest that antimicrobial peptoids and peptides are functionally and mechanistically analogous.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Peptoides/farmacología , Sinergismo FarmacológicoRESUMEN
We report the creation of alkylated poly-N-substituted glycine (peptoid) mimics of antimicrobial lipopeptides with alkyl tails ranging from 5 to 13 carbons. In several cases, alkylation significantly improved the selectivity of the peptoids with no loss in antimicrobial potency. Using this technique, we synthesized an antimicrobial peptoid only 5 monomers in length with selective, broad-spectrum antimicrobial activity as potent as previously reported dodecameric peptoids and the antimicrobial peptide pexiganan.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Lipopéptidos/química , Lipopéptidos/farmacología , Peptoides/química , Peptoides/farmacología , Células Cultivadas , Hemólisis/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
Interactions with bacterial membranes are integral to the mechanisms of action of all antimicrobial peptides (AMPs), regardless of their final cellular targets. Here, we describe in detail two biophysical techniques that can be used to measure the membrane activities of AMPs and antimicrobial peptidomimetics: (1) a calcein leakage assay to investigate interactions between AMPs/peptidomimetics with large unilamellar vesicles and (2) a potential-sensitive dye-based depolarization assay to investigate interactions with the membranes of live bacteria. By comparing the membrane interactions of AMPs and their mimics, these techniques can provide insights into their extent of mimicry and their antimicrobial mechanisms.
Asunto(s)
Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/metabolismo , Materiales Biomiméticos/metabolismo , Péptidos/metabolismo , Liposomas Unilamelares/metabolismo , Permeabilidad de la Membrana Celular , Fluoresceínas/metabolismo , Potenciales de la MembranaRESUMEN
Surface modification techniques that create surfaces capable of killing adherent bacteria are promising solutions to infections associated with implantable medical devices. Antimicrobial (AM) peptoid oligomers (ampetoids) that were designed to mimic helical AM peptides were synthesised with a peptoid spacer chain to allow mobility and an adhesive peptide moiety for easy and robust immobilisation onto substrata. TiO(2) substrata were modified with the ampetoids and subsequently backfilled with an antifouling (AF) polypeptoid polymer in order to create polymer surface coatings composed of both AM (active) and AF (passive) peptoid functionalities. Confocal microscopy images showed that the membranes of adherent E. coli cells were damaged after 2-h exposure to the modified substrata, suggesting that ampetoids retain AM properties even when immobilised on substrata.
Asunto(s)
Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Peptoides/síntesis química , Peptoides/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Dicroismo Circular , Escherichia coli/ultraestructura , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Estructura Molecular , Propiedades de Superficie , Células 3T3 Swiss , Titanio/químicaRESUMEN
Antimicrobial peptides (AMPs) and their mimics are emerging as promising antibiotic agents. We present a library of "ampetoids" (antimicrobial peptoid oligomers) with helical structures and biomimetic sequences, several members of which have low-micromolar antimicrobial activities, similar to cationic AMPs like pexiganan. Broad-spectrum activity against six clinically relevant BSL2 pathogens is also shown. This comprehensive structure-activity relationship study, including circular dichroism spectroscopy, minimum inhibitory concentration assays, hemolysis and mammalian cell toxicity studies, and specular x-ray reflectivity measurements shows that the in vitro activities of ampetoids are strikingly similar to those of AMPs themselves, suggesting a strong mechanistic analogy. The ampetoids' antibacterial activity, coupled with their low cytotoxicity against mammalian cells, make them a promising class of antimicrobials for biomedical applications. Peptoids are biostable, with a protease-resistant N-substituted glycine backbone, and their sequences are highly tunable, because an extensive diversity of side chains can be incorporated via facile solid-phase synthesis. Our findings add to the growing evidence that nonnatural foldamers will emerge as an important class of therapeutics.