RESUMO
Mimics of antimicrobial peptides (AMPs) have been proposed as a promising class of antimicrobial agents. We report the analysis of five tetrasubstituted, cationic, amphipathic heterocycles as potential AMP mimics. The analysis showed that the heterocyclic scaffold had a strong influence on the haemolytic activity of the compounds, and the hydantoin scaffold was identified as a promising template for drug lead development. Subsequently, a total of 20 hydantoin derivatives were studied for their antimicrobial potency and haemolytic activity. We found 19 of these derivatives to have very low haemolytic toxicity and identified three lead structures, 2dA, 6cG, and 6dG with very promising broad-spectrum antimicrobial activity. Lead structure 6dG displayed minimum inhibitory concentration (MIC) values as low as 1 µg/mL against Gram-positive bacteria and 4-16 µg/mL against Gram-negative bacteria. Initial mode of action (MoA) studies performed on the amine derivative 6cG, utilizing a luciferase-based biosensor assay, suggested a strong membrane disrupting effect on the outer and inner membrane of Escherichia coli. Our findings show that the physical properties and structural arrangement induced by the heterocyclic scaffolds are important factors in the design of AMP mimics.
Assuntos
Anti-Infecciosos , Hidantoínas , Hidantoínas/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C8), decanoic acid (C10) or dodecanoic acid (C12). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C12-cTurg-1 and C8-cTurg-2. These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus, Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.
Assuntos
Anti-Infecciosos , Lipopeptídeos , Humanos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Ciclização , Peptídeos Antimicrobianos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli , HemóliseRESUMO
An amphipathic barbiturate mimic of the marine eusynstyelamides is reported as a promising class of antimicrobial agents. We hereby report a detailed analysis of the structure-activity relationship for cationic amphipathic N,N'-dialkylated-5,5-disubstituted barbiturates. The influence of various cationic groups, hydrocarbon linkers and lipophilic side chains on the compounds' antimicrobial potency and haemolytic activity was studied. A comprehensive library of 58 compounds was prepared using a concise synthetic strategy. We found cationic amine and guanidyl groups to yield the highest broad-spectrum activity and cationic trimethylated quaternary amine groups to exert narrow-spectrum activity against Gram-positive bacteria. n-Propyl hydrocarbon linkers proved to be the best compromise between potency and haemolytic activity. The combination of two different lipophilic side chains allowed for further fine-tuning of the biological properties. Using these insights, we were able to prepare both, the potent narrow-spectrum barbiturate 8a and the broad-spectrum barbiturates 11lG, 13jA and 13jG, all having low or no haemolytic activity. The guanidine derivative 11lG demonstrated a strong membrane disrupting effect in luciferase-based assays. We believe that these results may be valuable in further development of antimicrobial lead structures.
Assuntos
Anti-Infecciosos , Bactérias Gram-Negativas , Aminas , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Barbitúricos/farmacologia , Cátions/química , Cátions/farmacologia , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 µg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.
Assuntos
Antibacterianos/farmacologia , Barbitúricos/farmacologia , Produtos Biológicos/farmacologia , Guanidinas/farmacologia , Indóis/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Tensoativos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Barbitúricos/síntese química , Barbitúricos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Guanidinas/síntese química , Guanidinas/química , Indóis/síntese química , Indóis/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Citotóxicas Formadoras de Poros/síntese química , Proteínas Citotóxicas Formadoras de Poros/química , Relação Estrutura-Atividade , Tensoativos/síntese química , Tensoativos/químicaRESUMO
Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Produtos Biológicos/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Sequência de Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Organismos Aquáticos/genética , Produtos Biológicos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/síntese química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Análise de Sequência de Proteína , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys1-Cys6, Cys2-Cys5, and Cys3-Cys4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin AMox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos/farmacologia , Urocordados/química , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dissulfetos/química , Descoberta de Drogas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and high-resolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.
Assuntos
Naftoquinonas/análise , Strongylocentrotus/imunologia , Estruturas Animais/citologia , Animais , Separação Celular/métodos , Cromatografia Líquida de Alta Pressão , Grânulos Citoplasmáticos/química , Citometria de Fluxo/métodos , Microscopia de Fluorescência , Naftoquinonas/metabolismo , Imagem Óptica , Espectrofotometria Ultravioleta , Strongylocentrotus/citologia , Espectrometria de Massas em Tandem , Imagem com Lapso de TempoRESUMO
EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero-dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure-activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide (P6) consisted of the 12 N-terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C-terminal amidation. Results from the alanine scan indicated that the generated lead peptide (P6) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide (P6) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide (P6) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Ouriços-do-Mar/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted ß-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase - carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25-8⯵g/mL against Gram-positive and Gram-negative reference strains, and 2-32⯵g/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50â¯>â¯200⯵g/mL), human hepatocyte carcinoma cells (HepG2: EC50â¯>â¯64⯵g/mL), and human lung fibroblast cells (MRC-5: EC50â¯>â¯64⯵g/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.
Assuntos
Amidas/química , Anti-Infecciosos/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Amidas/síntese química , Amidas/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Halogenação , Humanos , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
There is an urgent need for novel antimicrobial agents to address the threat of bacterial resistance to modern society. We have used a structural motif found in antimicrobial marine hit compounds as a basis for synthesizing a library of antimicrobial sulfonamidobenzamide lead compounds. Potent in vitro antimicrobial activity against clinically relevant bacterial strains was demonstrated for two compounds, G6 and J18, with minimal inhibitory concentrations (MIC) of 4-16⯵g/ml against clinical methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). The two compounds G6 and J18, together with several other compounds of this library, also caused ≥90% eradication of pre-established biofilm of methicillin-resistant S. epidermidis (MRSE) at 40⯵g/ml. Using a luciferase assay, the mechanism of action of G6 was shown to resemble the biocide chlorhexidine by targeting the bacterial cell membrane.
Assuntos
Antibacterianos/farmacologia , Benzamidas/farmacologia , Biofilmes/efeitos dos fármacos , Produtos Biológicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Sulfonamidas/farmacologia , Antibacterianos/química , Benzamidas/química , Produtos Biológicos/química , Farmacorresistência Bacteriana Múltipla , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Água do Mar/química , Sulfonamidas/químicaRESUMO
The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-ß2,2 -Xaa-Lys) containing one lipophilic ß2,2 -amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum ß-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8 µg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic ß2,2 -amino acids form a valuable scaffold for designing novel antimicrobial agents.
Assuntos
Antibacterianos/síntese química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Peptídeos Cíclicos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Halogenação , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Relação Estrutura-AtividadeRESUMO
As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, Paralithodes camtschaticus, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1-3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38-51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1-Cys8, Cys2-Cys6, Cys3-Cys5, and Cys4-Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1-3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5-100 µM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs.
Assuntos
Anomuros/química , Antibacterianos/química , Dissulfetos/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Cisteína/química , Cisteína/farmacologia , Dissulfetos/farmacologia , Peptídeos/farmacologiaRESUMO
A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.
Assuntos
Analgésicos , Antibacterianos , Anti-Inflamatórios , Peptídeos , Anêmonas-do-Mar , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Analgésicos/química , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dissulfetos/química , Edema/tratamento farmacológico , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/uso terapêuticoRESUMO
A library of small aminobenzamide derivatives was synthesised to explore a cationic amphipathic motif found in marine natural antimicrobials. The most potent compound E23 displayed minimal inhibitory concentrations (MICs) of 0.5-2µg/ml against several Gram-positive bacterial strains, including methicillin resistant Staphylococcus epidermidis (MRSE).E23 was also potent against 275 clinical isolates including Staphylococcus aureus, Enterococcus spp., Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), and ESBL-CARBA producing multi-resistant Gram-negative bacteria. The study demonstrates how structural motifs found in marine natural antimicrobials can be a valuable source for making novel antimicrobial lead-compounds.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Benzamidas/farmacologia , Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Benzamidas/síntese química , Benzamidas/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Peptídeos Catiônicos Antimicrobianos/química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ouriços-do-MarRESUMO
Arasin 1 from the spider crab Hyas araneus is a proline-rich antimicrobial peptide (PR-AMP), which kills target bacteria by a non-membranolytic mechanism. By using a fluorescent derivative of the peptide, we showed that arasin 1 rapidly penetrates into Escherichia coli cells without membrane damage. To unravel its mode of action, a knockout gene library of E. coli was screened and two types of mutants with a less susceptible phenotype to the arasin 1 fragment (1-23) were found. The first bore the mutation of sbmA, a gene coding for an inner membrane protein involved in the uptake of different antibiotic peptides. The second mutation was located in the ygdD gene, coding for a conserved inner membrane protein of unknown function. Functional studies showed that YgdD is required for the full susceptibility to arasin 1(1-25), possibly by supporting its uptake and/or intracellular action. These results indicated that different bacterial proteins are exploited by arasin 1(1-25) to exert its antibacterial activity and add new insights on the complex mode of action of PR-AMPs.
RESUMO
Antimicrobial peptides (AMPs) are important effector molecules in innate immunity. Here we briefly summarize characteristic traits of AMPs and their mechanisms of antimicrobial activity. Echinoderms live in a microbe-rich marine environment and are known to express a wide range of AMPs. We address two novel AMP families from coelomocytes of sea urchins: cysteine-rich AMPs (strongylocins) and heterodimeric AMPs (centrocins). These peptide families have conserved preprosequences, are present in both adults and pluteus stage larvae, have potent antimicrobial properties, and therefore appear to be important innate immune effectors. Strongylocins have a unique cysteine pattern compared to other cysteine-rich peptides, which suggests a novel AMP folding pattern. Centrocins and SdStrongylocin 2 contain brominated tryptophan residues in their native form. This review also includes AMPs isolated from other echinoderms, such as holothuroidins, fragments of beta-thymosin, and fragments of lectin (CEL-III). Echinoderm AMPs are crucial molecules for the understanding of echinoderm immunity, and their potent antimicrobial activity makes them potential precursors of novel drug leads.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Resistência à Doença/imunologia , Equinodermos/imunologia , Imunidade Inata/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Resistência à Doença/genética , Equinodermos/genética , Equinodermos/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Dados de Sequência Molecular , Ouriços-do-Mar/genética , Ouriços-do-Mar/imunologia , Ouriços-do-Mar/microbiologia , Água do Mar/microbiologia , Homologia de Sequência de AminoácidosRESUMO
The current study describes the antifouling properties of four members belonging to the recently discovered synoxazolidinone and pulmonarin families, isolated from the sub-Arctic sessile ascidian Synoicum pulmonaria collected off the Norwegian coast. Four simplified synthetic analogues were also prepared and included in the study. Several of the studied compounds displayed MIC values in the micro-nanomolar range against 16 relevant marine species involved in both the micro- and macrofouling process. Settlement studies on Balanus improvisus cyprids indicated a deterrent effect and a low toxicity for selected compounds. The two synoxazolidinones displayed broad activity and are shown to be among the most active natural antifouling bromotyrosine derivatives described. Synoxazolidinone C displayed selected antifouling properties comparable to the commercial antifouling product Sea-Nine-211. The pulmonarins prevented the growth of several bacterial strains at nanomolar concentrations but displayed a lower activity toward microalgae and no effect on barnacles. The linear and cyclic synthetic peptidic mimics also displayed potent antifouling activities mainly directed against bacterial adhesion and growth.
Assuntos
Incrustação Biológica , Bromobenzenos/isolamento & purificação , Guanidina/análogos & derivados , Oxazolidinonas/isolamento & purificação , Urocordados/química , Animais , Bromobenzenos/síntese química , Bromobenzenos/química , Bromobenzenos/farmacologia , Guanidina/síntese química , Guanidina/química , Guanidina/isolamento & purificação , Guanidina/farmacologia , Guanidinas , Larva/efeitos dos fármacos , Biologia Marinha , Estrutura Molecular , Oxazolidinonas/síntese química , Oxazolidinonas/química , Oxazolidinonas/farmacologia , Thoracica/fisiologiaRESUMO
The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described. All major stages of the fouling process are investigated. The effect of ianthelline on adhesion and growth of marine bacteria and microalgae is tested to investigate its influence on the initial microfouling process comparing with the known marine antifoulant barettin as a reference. Macrofouling is studied via barnacle (Balanus improvisus) settlement assays and blue mussel (Mytilus edulis) phenoloxidase inhibition. Ianthelline is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (minimum inhibitory concentration (MIC) values 0.1-10 µg/mL) and barnacle larval settlement (IC50 = 3.0 µg/mL). Moderate effects are recorded on M. edulis (IC50 = 45.2 µg/mL) and microalgae, where growth is more affected than surface adhesion. The effect of ianthelline is also investigated against human pathogenic bacteria. Ianthelline displayed low micromolar MIC values against several bacterial strains, both Gram positive and Gram negative, down to 2.5 µg/mL. In summary, the effect of ianthelline on 20 different representative marine antifouling organisms and seven human pathogenic bacterial strains is presented.
Assuntos
Incrustação Biológica/prevenção & controle , Imidazóis/farmacologia , Poríferos/química , Tirosina/análogos & derivados , Adesividade/efeitos dos fármacos , Animais , Regiões Árticas , Bactérias/efeitos dos fármacos , Imidazóis/química , Microalgas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Mytilus edulis/enzimologia , Thoracica/efeitos dos fármacos , Tirosina/química , Tirosina/farmacologiaRESUMO
Pulmonarins A and B are two new dibrominated marine acetylcholinesterase inhibitors that were isolated and characterized from the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast. The structures of natural pulmonarins A and B were tentatively elucidated by spectroscopic methods and later verified by comparison with synthetically prepared material. Both pulmonarins A and B displayed reversible, noncompetitive acetylcholinesterase inhibition comparable to several known natural acetylcholinesterase inhibitiors. Pulmonarin B was the strongest inhibitor, with an inhibition constant (Ki) of 20 µM. In addition to reversible, noncompetitive acetylcholinesterase inhibition, the compounds displayed weak antibacterial activity but no cytotoxicity or other investigated bioactivities.