RESUMO
The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.
Assuntos
Cobre/química , Cobre/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Cátions Bivalentes , Humanos , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Domínios ProteicosRESUMO
Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that act through diverse mechanisms, this characteristic makes them suitable starting points for development of novel classes of antibiotics. We have previously reported the increase in activity of AMPs upon addition of the Amino Terminal Copper and Nickel (ATCUN) Binding Unit. Herein we synthesized the membrane active peptide, Anoplin and two ATCUN-Anoplin derivatives and show that the increase in activity is indeed due to the ROS formation by the Cu(II)-ATCUN complex. We found that the ATCUN-Anoplin peptides were up to four times more potent compared to Anoplin alone against standard test bacteria. We studied membrane disruption, and cellular localization and found that addition of the ATCUN motif did not lead to a difference in these properties. When helical content was calculated, we observed that ATCUN-Anoplin had a lower helical composition. We found that ATCUN-Anoplin are able to oxidatively damage lipids in the bacterial membrane and that their activity trails the rate at which ROS is formed by the Cu(II)-ATCUN complexes alone. This study shows that addition of a metal binding tripeptide motif is a simple strategy to increase potency of AMPs by conferring a secondary action.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Cobre/química , Espécies Reativas de Oxigênio/metabolismo , Venenos de Vespas/química , Motivos de Aminoácidos , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Escherichia coli/metabolismo , Humanos , Peroxidação de Lipídeos , Microscopia Confocal , Estresse Oxidativo , beta-Galactosidase/metabolismoRESUMO
Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition.
RESUMO
In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs with Cu2+ is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that prepared as Cu2+-complexes, the peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two- and seven-fold, respectively. Remarkably, P3-Cu2+ is particularly effective at inserting in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where complexing HDPs with Cu2+ to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Cobre/química , Bicamadas Lipídicas/química , Células A549 , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Células HeLa , Humanos , Peroxidação de Lipídeos , Modelos MolecularesRESUMO
Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Indóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Triptofano/biossíntese , Animais , Antituberculosos/química , Antituberculosos/metabolismo , Relação Dose-Resposta a Droga , Indóis/química , Indóis/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMO
Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.
Assuntos
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Ensaios de Triagem em Larga Escala , Mycobacterium tuberculosis/genética , Relação Estrutura-AtividadeRESUMO
With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a nonconserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Sistemas de Liberação de Medicamentos , Fumarato Hidratase/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Sítios de Ligação , Fumarato Hidratase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.
Assuntos
Antibacterianos/química , Desenvolvimento de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/enzimologia , tRNA Metiltransferases/antagonistas & inibidores , tRNA Metiltransferases/metabolismo , Antibacterianos/farmacologia , Cristalografia por Raios X/métodos , Humanos , Estrutura Secundária de ProteínaRESUMO
Copper (Cu) ions are critical in controlling bacterial infections, and successful pathogens like Mycobacterium tuberculosis (Mtb) possess multiple Cu resistance mechanisms. We report, as proof of concept, that a novel Cu hypersensitivity phenotype can be generated in mycobacteria, including Mtb, through a peptide, DAB-10, that is able to form reactive oxygen species (ROS) following Cu-binding. DAB-10 induces intramycobacterial oxidative stress in a Cu-dependent manner in vitro and during infection. DAB-10 penetrates murine macrophages and encounters intracellular mycobacteria. Significant intracellular Cu-dependent protection was observed when Mtb-infected macrophages were treated with DAB-10 alongside a cell-permeable Cu chelator. Treatment with the Cu chelator reversed the intramycobacterial oxidative shift induced by DAB-10. We conclude that DAB-10 utilizes the pool of phagosomal Cu ions in the host-Mtb interface to augment the mycobactericidal activity of macrophages while simultaneously exploiting the susceptibility of Mtb to ROS. DAB-10 serves as a model with which to develop next-generation, multifunctional antimicrobials.
Assuntos
Quelantes/farmacologia , Cobre/química , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos/farmacologia , Fagossomos/química , Animais , Antibacterianos/farmacologia , Quelantes/química , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Camundongos , Oxirredução , Estresse Oxidativo , Peptídeos/química , Estudo de Prova de Conceito , Células RAW 264.7 , Tuberculose/microbiologiaRESUMO
The biophysical properties of cellular membranes intimately influence the delivery of cargoes into cells by cell-penetrating peptides (CPPs) and the bactericidal activity of antimicrobial peptides (AMPs). Here, we discuss how lipid oxidation creates important chemical and biophysical changes in membranes, and hypothesize about the observed synergy between oxidized membranes and membrane-active peptides.
Assuntos
Membrana Celular/química , Peptídeos Penetradores de Células/química , Lipídeos de Membrana/química , Animais , Membrana Celular/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , OxirreduçãoRESUMO
Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargoes into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by antimicrobial peptides. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches.
Assuntos
Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Antígenos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/metabolismo , Morte Celular , Membrana Celular/química , Permeabilidade da Membrana Celular , Células Dendríticas/metabolismo , Humanos , OxirreduçãoRESUMO
Host-defense peptides (HDPs) feature evolution-tested potency against life-threatening pathogens. While piscidin 1 (p1) and piscidin 3 (p3) are homologous and potent fish HDPs, only p1 is strongly membranolytic. Here, we hypothesize that another mechanism imparts p3 strong potency. We demonstrate that the N-termini of both peptides coordinate Cu2+ and p3-Cu cleaves isolated DNA at a rate on par with free Cu2+ but significantly faster than p1-Cu. On planktonic bacteria, p1 is more antimicrobial but only p3 features copper-dependent DNA cleavage. On biofilms and persister cells, p3-Cu is more active than p1-Cu, commensurate with stronger peptide-induced DNA damage. Molecular dynamics and NMR show that more DNA-peptide interactions exist with p3 than p1, and the peptides adopt conformations simultaneously poised for metal- and DNA-binding. These results generate several important conclusions. First, homologous HDPs cannot be assumed to have identical mechanisms since p1 and p3 eradicate bacteria through distinct relative contributions of membrane and DNA-disruptive effects. Second, the nuclease and membrane activities of p1 and p3 show that naturally occurring HDPs can inflict not only physicochemical but also covalent damage. Third, strong nuclease activity is essential for biofilm and persister cell eradication, as shown by p3, the homolog more specific toward bacteria and more expressed in vascularized tissues. Fourth, p3 combines several physicochemical properties (e.g., Amino Terminal Copper and Nickel binding motif; numerous arginines; moderate hydrophobicity) that confer low membranolytic effects, robust copper-scavenging capability, strong interactions with DNA, and fast nuclease activity. This new knowledge could help design novel therapeutics active against hard-to-treat persister cells and biofilms.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biofilmes/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Clivagem do DNA/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Peixes/isolamento & purificação , Peixes , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/citologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the amino-terminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen- and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.
Assuntos
Motivos de Aminoácidos/efeitos dos fármacos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Artrópodes/síntese química , Imunidade Inata/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/farmacologia , Membrana Celular , Cobre , Ixodes , Testes de Sensibilidade Microbiana , Níquel , Glândulas Salivares , Relação Estrutura-AtividadeRESUMO
The emergence of antibiotic resistant strains of bacteria has resulted in the need to develop more potent antimicrobials that target microorganisms in a novel manner. Antimicrobial Peptides (AMPs) show great potential for drug development because of their broad activity and unique mechanism of action. Several AMPs contain an Amino Terminal Copper and Nickel (ATCUN) binding motif; however, its function has not yet been determined. We have previously demonstrated that the activity of a truncated version of Buforin II (sh-Buforin, RAGLQFPVGRVHRLLRK-NH2) increases by the addition of an ATCUN motif. We now focus our current studies on understanding the effect of: 1) a positively charged ATCUN sequence, and 2) l-to-d amino acid substitution on the hybrid peptides. We identified that the addition of a positively charged ATCUN motif increases the affinity of the ATCUN-AMP for DNA but does not always result in an enhanced antimicrobial activity over a neutral ATCUN motif. The all-d peptides exhibited up to a 32-fold increase in antimicrobial activity compared to the all-l peptides. The larger activity of the all-d peptides is the result of a larger DNA cleavage activity and higher stability towards proteolysis. Cytotoxicity assays determined that, at their MIC, these peptides caused less than 8% hemolysis and, at 128 µM, no toxicity to HeLa and HEK293 cell lines. These results indicate that the ATCUN-AMP hybrids are an attractive alternative for treating infectious diseases and provide key insights into the role of the ATCUN motif in naturally-occurring AMPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Escherichia coli/crescimento & desenvolvimento , Proteínas , Motivos de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células HEK293 , Células HeLa , Humanos , Proteínas/síntese química , Proteínas/química , Proteínas/farmacologiaRESUMO
Piscidins were the first antimicrobial peptides discovered in the mast cells of vertebrates. While two family members, piscidin 1 (p1) and piscidin 3 (p3), have highly similar sequences and α-helical structures when bound to model membranes, p1 generally exhibits stronger antimicrobial and hemolytic activity than p3 for reasons that remain elusive. In this study, we combine activity assays and biophysical methods to investigate the mechanisms underlying the cellular function and differing biological potencies of these peptides, and report findings spanning three major facets. First, added to Gram-positive (Bacillus megaterium) and Gram-negative (Escherichia coli) bacteria at sublethal concentrations and imaged by confocal microscopy, both p1 and p3 translocate across cell membranes and colocalize with nucleoids. In E. coli, translocation is accompanied by nonlethal permeabilization that features more pronounced leakage for p1. Second, p1 is also more disruptive than p3 to bacterial model membranes, as quantified by a dye-leakage assay and (2)H solid-state NMR-monitored lipid acyl chain order parameters. Oriented CD studies in the same bilayers show that, beyond a critical peptide concentration, both peptides transition from a surface-bound state to a tilted orientation. Third, gel retardation experiments and CD-monitored titrations on isolated DNA demonstrate that both peptides bind DNA but p3 has stronger condensing effects. Notably, solid-state NMR reveals that the peptides are α-helical when bound to DNA. Overall, these studies identify two polyreactive piscidin isoforms that bind phosphate-containing targets in a poised amphipathic α-helical conformation, disrupt bacterial membranes, and access the intracellular constituents of target cells. Remarkably, the two isoforms have complementary effects; p1 is more membrane active, while p3 has stronger DNA-condensing effects. Subtle differences in their physicochemical properties are highlighted to help explain their contrasting activities.