RESUMO
Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.
Assuntos
Produtos Biológicos , Burkholderia , Humanos , Burkholderia/genética , Peptídeo Sintases/genética , Lipopeptídeos/química , DNA , Produtos Biológicos/química , Serina/genética , Família MultigênicaRESUMO
High-throughput chemical analysis of natural product mixtures lags behind developments in genome sequencing technologies and laboratory automation, leading to a disconnect between library-scale chemical and biological profiling that limits new molecule discovery. Here, we report a new orthogonal sample multiplexing strategy that can increase mass spectrometry-based profiling up to 30-fold over traditional methods. Profiled pooled samples undergo subsequent computational deconvolution to reconstruct peak lists for each sample in the set. We validated this approach using in silico experiments and demonstrated a high assignment precision (>97%) for large, pooled samples (r = 30), particularly for infrequently occurring metabolites of relevance in drug discovery applications. Requiring only 5% of the previously required MS acquisition time, this approach was repeated in a recent biological activity profiling study on 925 natural product extracts, leading to the rediscovery of all previously reported bioactive metabolites. This new method is compatible with MS data from any instrument vendor and is supported by an open-source software package: https://github.com/liningtonlab/MultiplexMS.
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Produtos Biológicos , Software , Espectrometria de Massas , Descoberta de Drogas , TecnologiaRESUMO
Two new lipopeptaibols, tolypocaibols A (1) and B (2), and the mixed NRPS-polyketide-shikimate natural product maximiscin [(P/M)-3)] were isolated from a Tolypocladium sp. fungal endophyte of the marine alga Spongomorpha arcta. Analysis of NMR and mass spectrometry data revealed the amino acid sequences of the lipopeptaibols, which both comprise 11 residues with a valinol C-terminus and a decanoyl acyl chain at the N-terminus. The configuration of the amino acids was determined by Marfey's analysis. Tolypocaibols A (1) and B (2) showed moderate, selective inhibition against Gram-positive and acid-fast bacterial strains, while maximiscin [(P/M)-3)] showed moderate, broad-spectrum antibiotic activity.
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Endófitos , Alga Marinha , Bactérias , Antibacterianos/químicaRESUMO
The total synthesis of the natural product coralmycin A/epi-coralmycin A, as well as a desmethoxy analogue is described. Synthesis was achieved via a divergent, bidirectional solid-phase strategy, including a key on-resin O-acylation, O to N acyl shift, and O-alkylation protocol to incorporate the unusual 4-amino-2-hydroxy-3-isopropoxybenzoic acid motifs. The synthetic natural product was generated as a 1 : 1 mixture of epimers at the central ß-methoxyasparagine residue and exhibited potent antibacterial activity against a panel of ten Gram-negative and seven Gram-positive organisms. The desmethoxy analogue possessed significantly more potent antimicrobial activity against this panel with minimal inhibitory concentrations (MICs) as low as 50 nM.
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DepsipeptídeosRESUMO
The development of new "omics" platforms is having a significant impact on the landscape of natural products discovery. However, despite the advantages that such platforms bring to the field, there remains no straightforward method for characterizing the chemical landscape of natural products libraries using two-dimensional nuclear magnetic resonance (2D-NMR) experiments. NMR analysis provides a powerful complement to mass spectrometric approaches, given the universal coverage of NMR experiments. However, the high degree of signal overlap, particularly in one-dimensional NMR spectra, has limited applications of this approach. To address this issue, we have developed a new data analysis platform for complex mixture analysis, termed MADByTE (Metabolomics and Dereplication by Two-Dimensional Experiments). This platform employs a combination of TOCSY and HSQC spectra to identify spin system features within complex mixtures and then matches spin system features between samples to create a chemical similarity network for a given sample set. In this report we describe the design and construction of the MADByTE platform and demonstrate the application of chemical similarity networks for both the dereplication of known compound scaffolds and the prioritization of bioactive metabolites from a bacterial prefractionated extract library.
Assuntos
Produtos Biológicos/química , Misturas Complexas/química , Espectroscopia de Ressonância Magnética/métodos , Metabolômica , Software , Interface Usuário-ComputadorRESUMO
Antibiotics are essential for numerous medical procedures, including the treatment of bacterial infections, but their widespread use has led to the accumulation of resistance, prompting calls for the discovery of antibacterial agents with new targets. A majority of clinically approved antibacterial scaffolds are derived from microbial natural products, but these valuable molecules are not well annotated or organized, limiting the efficacy of modern informatic analyses. Here, we provide a comprehensive resource defining the targets, chemical origins and families of the natural antibacterial collective through a retrobiosynthetic algorithm. From this we also detail the directed mining of biosynthetic scaffolds and resistance determinants to reveal structures with a high likelihood of having previously unknown modes of action. Implementing this pipeline led to investigations of the telomycin family of natural products from Streptomyces canus, revealing that these bactericidal molecules possess a new antibacterial mode of action dependent on the bacterial phospholipid cardiolipin.
Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Cardiolipinas/biossíntese , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos/farmacologia , Streptomyces/metabolismo , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Vias Biossintéticas , Cardiolipinas/genética , Contagem de Colônia Microbiana , Bases de Dados Genéticas , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/metabolismo , Testes de Sensibilidade Microbiana , Família Multigênica , Peptídeos/genética , Peptídeos/isolamento & purificação , NavegadorRESUMO
The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics that are structurally related to the clinically approved antibiotic daptomycin. In this article, we describe the synthesis of a small library of glycinocin analogues that differ by variation in the exocyclic fatty acyl substituent. The glycinocin analogues were screened against a panel of Gram-positive bacteria (as well as Gram-negative P. aeruginosa). These analogues exhibited similar calcium-dependent activity to the parent natural products against Gram-positive bacteria but showed no activity against P. aeruginosa. The length of the fatty acid was shown to be important for optimal biological activity, while the hybridisation at the α,ß position and branching within the fatty acyl chain had only subtle effects on activity.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Peptídeos/síntese química , Peptídeos/farmacologia , Antibacterianos/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Polyketide or polyketide-like macrolides (pMLs) continue to serve as a source of inspiration for drug discovery. However, their inherent structural and stereochemical complexity challenges efforts to explore related regions of chemical space more broadly. Here, we report a strategy termed the Targeted Sampling of Natural Product space (TSNaP) that is designed to identify and assess regions of chemical space bounded by this important class of molecules. Using TSNaP, a family of tetrahydrofuran-containing pMLs are computationally assembled from pML inspired building blocks to provide a large collection of natural product-like virtual pMLs. By scoring functional group and volumetric overlap against their natural counterparts, a collection of compounds are prioritized for targeted synthesis. Using a modular and stereoselective synthetic approach, a library of polyketide-like macrolides are prepared to sample these unpopulated regions of pML chemical space. Validation of this TSNaP approach by screening this library against a panel of whole-cell biological assays, reveals hit rates exceeding those typically encountered in small molecule libraries. This study suggests that the TSNaP approach may be more broadly useful for the design of improved chemical libraries for drug discovery.
Assuntos
Produtos Biológicos , Policetídeos , Macrolídeos/farmacologia , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Descoberta de DrogasRESUMO
Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia. Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger, while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells.
RESUMO
Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. Marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way they exert this antibacterial activity has not been established. In this study, we explore their structure-activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds.
Assuntos
Produtos Biológicos , Hidrocarbonetos Clorados , Animais , Antibacterianos/farmacologia , Pirróis/farmacologia , Testes de Sensibilidade Microbiana , MamíferosRESUMO
The rapid emergence of antimicrobial resistance presents serious health challenges to the management of infectious diseases, a problem that is further exacerbated by slowing rates of antimicrobial drug discovery in recent years. The phenomenon of collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities to address both these challenges. Here, we present a high-throughput screening platform termed Collateral Sensitivity Profiling (CSP) to map the difference in bioactivity of large chemical libraries across 29 drug-resistant strains of E. coli. CSP screening of 80 commercial antimicrobials demonstrated multiple CS interactions. Further screening of a 6195-member natural product library revealed extensive CS relationships in nature. In particular, we report the isolation of known and new analogues of borrelidin A with potent CS activities against cephalosporin-resistant strains. Co-dosing ceftazidime with borrelidin A slows broader cephalosporin resistance with no recognizable resistance to borrelidin A itself.
Assuntos
Anti-Infecciosos , Produtos Biológicos , Infecções por Escherichia coli , Humanos , Escherichia coli , Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Sensibilidade Colateral a Medicamentos , Resistência às Cefalosporinas , Testes de Sensibilidade MicrobianaRESUMO
Appending of ferrocene (Fc) to biologically-active organic backbones can generate novel multi-functional species for targeting bacteria and cancer. In this work Fc was linked to coumarin and anthraquinone with the goal of harnessing the redox-active Fc centre to generate new compounds that exhibit cytoxicity through the generation of toxic reactive oxygen species (ROS). A Cu(I)-catalyzed azide-alkyne cycloaddition "click" reaction was used to connect the organic and Fc components via a triazole linker. Cyclic voltammetry shows that the Fc potentials are suitable for oxidation by biological hydrogen peroxide to give reactive ferrocenium (Fc+) species, which can then generate hydroxyl radicals. The ability of the compounds to generate hydroxyl radicals in the presence of hydrogen peroxide was shown directly using EPR spin-trapping experiments. Furthermore, in vitro studies in MCF-7 breast cancer cells show significant increases in ROS following incubation with the Fc-functionalized compounds. Screening for antibacterial activity produced negative results for all of the Fc compounds, consitent with low levels of hydrogen peroxide typically found in bacteria. By contrast, Fc-coumarin showed cytotoxicity against A549 lung cancer and SKOV3 ovarian cancer cell lines, whereas the parent compound was inactive. This is consistent both with the cytoxic potential of the Fc group and the elevated hydrogen peroxide levels found in many cancers. Interestingly, the anthraquinone compounds showed the opposite effect with the parent compounds showing modest activity against A549 cells, but the Fc compounds being inactive. This demonstrates other potential negative impacts of including Fc, such as significantly increased lipophilicity.
Assuntos
Citotoxinas , Peróxido de Hidrogênio , Antraquinonas/farmacologia , Cumarínicos/farmacologia , Humanos , Metalocenos , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Few tools exist in natural products discovery to integrate biological screening and untargeted mass spectrometry data at the library scale. Previously, we reported Compound Activity Mapping as a strategy for predicting compound bioactivity profiles directly from primary screening results on extract libraries. We now present NP Analyst, an open online platform for Compound Activity Mapping that accepts bioassay data of almost any type, and is compatible with mass spectrometry data from major instrument manufacturers via the mzML format. In addition, NP Analyst will accept processed mass spectrometry data from the MZmine 2 and GNPS open-source platforms, making it a versatile tool for integration with existing discovery workflows. We demonstrate the utility of this new tool for both the dereplication of known compounds and the discovery of novel bioactive natural products using a challenging low-resolution antimicrobial bioassay data set. This new platform is available at www.npanalyst.org.
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Ohmyungsamycin A and ecumicin are structurally related cyclic depsipeptide natural products that possess activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Herein, we describe the design and synthesis of a library of analogues of these two natural products using an efficient solid-phase synthesis and late-stage macrolactamization strategy. Lead analogues possessed potent activity against Mtb in vitro (minimum inhibitory concentration 125-500 nM) and were shown to inhibit protein degradation by the mycobacterial ClpC1-ClpP1P2 protease with an associated enhancement of ClpC1 ATPase activity. The most promising analogue from the series exhibited rapid bactericidal killing activity against Mtb, capable of sterilizing cultures after 7 days, and retained bactericidal activity against hypoxic non-replicating Mtb. This natural product analogue was also active in an in vivo zebrafish model of infection.
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Produtos Biológicos , Depsipeptídeos , Mycobacterium tuberculosis , Animais , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Produtos Biológicos/farmacologia , Depsipeptídeos/farmacologia , Chaperonas Moleculares , Mycobacterium tuberculosis/metabolismo , Peptídeos Cíclicos , Peixe-Zebra/metabolismoRESUMO
The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for anti-trypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.
Assuntos
Alanina-tRNA Ligase/antagonistas & inibidores , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Treonina-tRNA Ligase/antagonistas & inibidores , Trypanosoma/efeitos dos fármacos , Alanina-tRNA Ligase/genética , Alanina-tRNA Ligase/metabolismo , Antiprotozoários/química , Inibidores Enzimáticos/química , Humanos , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Trypanosoma/enzimologia , Trypanosoma/genética , Tripanossomíase/parasitologiaRESUMO
Despite rapid evolution in the area of microbial natural products chemistry, there is currently no open access database containing all microbially produced natural product structures. Lack of availability of these data is preventing the implementation of new technologies in natural products science. Specifically, development of new computational strategies for compound characterization and identification are being hampered by the lack of a comprehensive database of known compounds against which to compare experimental data. The creation of an open access, community-maintained database of microbial natural product structures would enable the development of new technologies in natural products discovery and improve the interoperability of existing natural products data resources. However, these data are spread unevenly throughout the historical scientific literature, including both journal articles and international patents. These documents have no standard format, are often not digitized as machine readable text, and are not publicly available. Further, none of these documents have associated structure files (e.g., MOL, InChI, or SMILES), instead containing images of structures. This makes extraction and formatting of relevant natural products data a formidable challenge. Using a combination of manual curation and automated data mining approaches we have created a database of microbial natural products (The Natural Products Atlas, www.npatlas.org) that includes 24â¯594 compounds and contains referenced data for structure, compound names, source organisms, isolation references, total syntheses, and instances of structural reassignment. This database is accompanied by an interactive web portal that permits searching by structure, substructure, and physical properties. The Web site also provides mechanisms for visualizing natural products chemical space and dashboards for displaying author and discovery timeline data. These interactive tools offer a powerful knowledge base for natural products discovery with a central interface for structure and property-based searching and presents new viewpoints on structural diversity in natural products. The Natural Products Atlas has been developed under FAIR principles (Findable, Accessible, Interoperable, and Reusable) and is integrated with other emerging natural product databases, including the Minimum Information About a Biosynthetic Gene Cluster (MIBiG) repository, and the Global Natural Products Social Molecular Networking (GNPS) platform. It is designed as a community-supported resource to provide a central repository for known natural product structures from microorganisms and is the first comprehensive, open access resource of this type. It is expected that the Natural Products Atlas will enable the development of new natural products discovery modalities and accelerate the process of structural characterization for complex natural products libraries.
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We present here the genome sequence ofStreptomyces canusATCC 12647, a producer of the antibiotic telomycin, noted for its unique antibacterial activity against cardiolipin. Genomic analysis using the bioinformatics tool PRISM revealed the presence of multiple biosynthetic gene clusters, including those for telomycin and other natural products of potential pharmacological interest.