RESUMO
The GE81112 series, consisting of three naturally occurring tetrapeptides and synthetic derivatives, is evaluated as a potential lead structure for the development of a new antibacterial drug. Although the first total synthesis of GE81112A reported by our group provided sufficient amounts of material for an initial in depth biological profiling of the compound, improvements of the routes toward the key building blocks were needed for further upscaling and structure-activity relationship studies. The major challenges identified were poor stereoselectivity in the synthesis of the C-terminal ß-hydroxy histidine intermediate and a concise access to all four isomers of the 3-hydroxy pipecolic acid. Herein, we report a second-generation synthesis of GE81112A, which is also applicable to access further representatives of this series. Based on Lajoie's ortho-ester-protected serine aldehydes as key building blocks, the described route provides both a satisfactory improvement in stereoselectivity of the ß-hydroxy histidine intermediate synthesis and a stereoselective approach toward both orthogonally protected cis and trans-3-hydroxy pipecolic acid.
Assuntos
Histidina , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , IsomerismoRESUMO
Nine new hydroxyphenyloxazolines, madurastatin B4, C2, D3 and D4, E1 and E2, F1 as well as G1 and G2 (8-16), along with two new enantiomers of madurastatin D1 (ent-6) and D2 (ent-7) and two known congeners, madurastatin B1 (2) and C1 (5), were isolated from the liquid culture of Actinomadura sp. ST100801 based on the initial activity against Escherichia coli screened in bicarbonate-supplemented Mueller Hinton II medium and identification via molecular networking. Structure elucidation was achieved by comprehensive 1D and 2D NMR as well as MS/MS fragmentation analyses. Their absolute configuration was determined by Marfey's analysis. Complemented with functionalized hydroxyphenyloxazolines (2, 4, 17-18) obtained by total synthesis, the isolated compounds were evaluated for antibacterial activities revealing MICs down to 4 µg ml-1 against Moraxella catarrhalis. Therefore, this study enlarges the family of madurastatin siderophores.
Assuntos
Actinomadura , Sideróforos , Antibacterianos/química , Escherichia coli , Testes de Sensibilidade Microbiana , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining-using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating-has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.
Assuntos
Produtos Biológicos , Bacteroidetes/genética , Genoma Bacteriano , Genômica , Família MultigênicaRESUMO
Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.
Assuntos
Isópteros , Micobioma , Trichoderma , Animais , Isópteros/microbiologia , Espectrometria de Massas em TandemRESUMO
Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.
Assuntos
Antimaláricos , Cisteína Proteases , Malária , Parasitos , Animais , Antimaláricos/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Plasmodium falciparum , Espectrometria de Massas em TandemRESUMO
High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.
Assuntos
Produtos Biológicos , Microfluídica , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microfluídica/métodos , Extratos Vegetais , Espectrometria de Massas em TandemRESUMO
The 'core' metabolome of the Bacteroidetes genus Chitinophaga was recently discovered to consist of only seven metabolites. A structural relationship in terms of shared lipid moieties among four of them was postulated. Here, structure elucidation and characterization via ultra-high resolution mass spectrometry (UHR-MS) and nuclear magnetic resonance (NMR) spectroscopy of those four lipids (two lipoamino acids (LAAs), two lysophosphatidylethanolamines (LPEs)), as well as several other undescribed LAAs and N-acyl amino acids (NAAAs), identified during isolation were carried out. The LAAs represent closely related analogs of the literature-known LAAs, such as the glycine-serine dipeptide lipids 430 (2) and 654. Most of the here characterized LAAs (1, 5-11) are members of a so far undescribed glycine-serine-ornithine tripeptide lipid family. Moreover, this study reports three novel NAAAs (N-(5-methyl)hexanoyl tyrosine (14) and N-(7-methyl)octanoyl tyrosine (15) or phenylalanine (16)) from Olivibacter sp. FHG000416, another Bacteroidetes strain initially selected as best in-house producer for isolation of lipid 430. Antimicrobial profiling revealed most isolated LAAs (1-3) and the two LPE 'core' metabolites (12, 13) active against the Gram-negative pathogen M. catarrhalis ATCC 25238 and the Gram-positive bacterium M. luteus DSM 20030. For LAA 1, additional growth inhibition activity against B. subtilis DSM 10 was observed.
Assuntos
Aminoácidos/química , Aminoácidos/farmacologia , Bacteroidetes/metabolismo , Glicerofosfolipídeos/química , Glicerofosfolipídeos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Técnicas de Tipagem Bacteriana/métodosRESUMO
Background: The ability of MDR Gram-negative bacteria to evade even antibiotics of last resort is a severe global challenge. The development pipeline for conventional antibiotics cannot address this issue, but antimicrobial peptides (AMPs) offer an alternative solution. Objectives: Two insect-derived AMPs (LS-sarcotoxin and LS-stomoxyn) were profiled to assess their suitability for systemic application in humans. Methods: The peptides were tested against an extended panel of 114 clinical MDR Gram-negative bacterial isolates followed by time-kill analysis, interaction studies and assays to determine the likelihood of emerging resistance. In further in vitro studies we addressed cytotoxicity, cardiotoxicity and off-target interactions. In addition, an in vivo tolerability and pharmacokinetic study in mice was performed. Results: LS-sarcotoxin and LS-stomoxyn showed potent and selective activity against Gram-negative bacteria and no cross-resistance with carbapenems, fluoroquinolones or aminoglycosides. Peptide concentrations of 4 or 8 mg/L inhibited 90% of the clinical MDR isolates of Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Salmonella enterica isolates tested. The 'all-d' homologues of the peptides displayed markedly reduced activity, indicating a chiral target. Pharmacological profiling revealed a good in vitro therapeutic index, no cytotoxicity or cardiotoxicity, an inconspicuous broad-panel off-target profile, and no acute toxicity in mice at 10 mg/kg. In mouse pharmacokinetic experiments LS-sarcotoxin and LS-stomoxyn plasma levels above the lower limit of quantification (1 and 0.25 mg/mL, respectively) were detected after 5 and 15 min, respectively. Conclusions: LS-sarcotoxin and LS-stomoxyn are suitable as lead candidates for the development of novel antibiotics; however, their pharmacokinetic properties need to be improved for systemic administration.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dípteros/química , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Larva/química , Animais , Anti-Infecciosos/efeitos adversos , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/efeitos adversos , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Masculino , CamundongosRESUMO
The spread of antibiotic-resistant human pathogens and the declining number of novel antibiotics in the development pipeline is a global challenge that has fueled the demand for alternative options. The search for novel drug candidates has expanded to include not only antibiotics but also adjuvants capable of restoring antibiotic susceptibility in multidrug-resistant (MDR) pathogens. Insect-derived antimicrobial peptides (AMPs) can potentially fulfil both of these functions. We tested two coleoptericins and one coleoptericin-like peptides from the invasive harlequin ladybird Harmonia axyridis against a panel of human pathogens. The AMPs displayed little or no activity when tested alone but were active even against clinical MDR isolates of the Gram-negative ESKAPE strains when tested in combination with polymyxin derivatives, such as the reserve antibiotic colistin, at levels below the minimal inhibitory concentration. Assuming intracellular targets of the AMPs, our data indicate that colistin potentiates the activity of the AMPs. All three AMPs achieved good in vitro therapeutic indices and high intrahepatic stability but low plasma stability, suggesting they could be developed as adjuvants for topical delivery or administration by inhalation for anti-infective therapy to reduce the necessary dose of colistin (and thus its side effects) or to prevent development of colistin resistance in MDR pathogens.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Besouros , Bactérias Gram-Negativas , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Besouros/química , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Proteínas de Insetos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The total synthesis of the naturally occurring antibiotic GE81112A, a densely functionalized tetrapeptide, is reported. Comparison of spectral data with those of the natural product and the lack of biological activity of the synthesized compound led us to revise the published configuration of the 3-hydroxypipecolic acid moiety. This hypothesis was fully validated by the synthesis of the corresponding epimer.
Assuntos
Antibacterianos/síntese química , Oligopeptídeos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Escherichia coli/efeitos dos fármacos , Histidina/síntese química , Histidina/química , Testes de Sensibilidade Microbiana , Oligopeptídeos/química , Oligopeptídeos/farmacologia , EstereoisomerismoRESUMO
Seven new halogenated peptides termed svetamycins A-G (1-7) have been isolated from laboratory cultures of a Streptomyces sp. Svetamycins A-D, F, and G are cyclic depsipeptides, whereas svetamycin E is a linear analogue of svetamycin C. Their structures were determined using extensive spectroscopic analysis, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and chemical derivatizations. Svetamycins are characterized by the presence of a hydroxyl acetic acid and five amino acids including a rare 4,5-dihydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid, a γ-halogenated piperazic acid, and a novel δ-methylated piperazic acid in svetamycins B-C, E, and G. Moreover, isotope-labeled substrate feeding experiments demonstrated ornithine as the precursor of piperazic acid and that methylation at the δ position of the piperazyl scaffold is S-adenosyl-l-methionine (SAM)-dependent. Svetamycin G, the most potent antimicrobial of this suite of compounds, inhibited the growth of Mycobacterium smegmatis with an MIC80 value of 2 µg/mL.