RESUMEN
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
Asunto(s)
Bacterias , Nucleotidiltransferasas , ARN Viral , Fagos de Staphylococcus , Animales , 2',5'-Oligoadenilato Sintetasa/metabolismo , Bacterias/enzimología , Bacterias/inmunología , Evolución Molecular , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Oligonucleótidos/inmunología , Oligonucleótidos/metabolismo , ARN Viral/inmunología , ARN Viral/metabolismo , Transducción de Señal/inmunología , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/inmunologíaRESUMEN
DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomeraseâ I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29â cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomeraseâ I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.
Asunto(s)
Antineoplásicos , Productos Biológicos , Humanos , Ratones , Animales , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Productos Biológicos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Biología Computacional , Suelo , Tiazoles , Línea Celular TumoralRESUMEN
Dinoflagellate-derived polyketides are typically large molecules (>1000 Da) with complex structures, potent bioactivities, and high toxicities. Their discovery suffers three major bottlenecks: insufficient bioavailability, low-yield cultivation of producer organisms, and production of multiple highly related analogues by a single strain. Consequently, the biotechnological production of therapeutics or toxicological standards of dinoflagellate-derived polyketides is also hampered. Strategies based on sensitive and selective techniques for chemical prospection of dinoflagellate extracts could aid in overcoming these limitations, as it allows selecting the most interesting candidates for discovery and exploitation programs according to the biosynthetic potential. In this work, we assess the combination of data-dependent liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS2) and molecular networking to screen polyol polyketides. To demonstrate the power of this approach, we selected dinoflagellate Amphidinium carterae since it is commonly used as a biotechnological model and produces amphidinols, a family of polyol-polyene compounds with antifungal and antimycoplasmal activity. First, we screened families of compounds with multiple hydroxyl groups by examining MS2 profiles that contain sequential neutral losses of water. Then, we clustered MS2 spectra by molecular networking to facilitate the dereplication and discovery of amphidinols. Finally, we used the MS2 fragmentation behavior of well-characterized luteophanol D as a model to propose a structural hypothesis of nine novel amphidinols. We envision that this strategy is a valuable approach to rapidly monitoring toxin production of known and unknown polyol polyketides in dinoflagellates, even in small culture volumes, and distinguishing strains according to their toxin profiles.
Asunto(s)
Dinoflagelados , Policétidos , Antifúngicos/química , Dinoflagelados/química , Polienos , Policétidos/química , Polímeros , AguaRESUMEN
The demand for valuable products from dinoflagellate biotechnology has increased remarkably in recent years due to their many prospective applications. However, there remain many challenges that need to be addressed in order to make dinoflagellate bioactives a commercial reality. In this article, we describe the technical feasibility of producing and recovering amphidinol analogues (AMs) excreted into a culture broth of Amphidinium carterae ACRN03, successfully cultured in an LED-illuminated pilot-scale (80 L) bubble column photobioreactor operated in fed-batch mode with a pulse feeding strategy. We report on the isolation of new structurally related AMs, amphidinol 24 (1, AM24), amphidinol 25 (2, AM25) and amphidinol 26 (3, AM26), from a singular fraction resulting from the downstream processing. Their planar structures were elucidated by extensive NMR and HRMS analysis, whereas the relative configuration of the C-32âC-47 bis-tetrahydropyran core was confirmed to be antipodal in accord with the recently revised configuration of AM3. The hemolytic activities of the new metabolites and other related derivatives were evaluated, and structure-activity conclusions were established. Their isolation was based on a straightforward and high-performance bioprocess that could be suitable for the commercial development of AMs or other high-value compounds from shear sensitive dinoflagellates.
Asunto(s)
Organismos Acuáticos/química , Dinoflagelados/química , Animales , Fotobiorreactores , Proyectos Piloto , Relación Estructura-ActividadRESUMEN
Red algae of Laurencia continue to provide wide structural diversity and complexity of halogenated C15 acetogenin medium-ring ethers. Here, we described the isolation of three new C15 acetogenins (3-5), and one truncated derivative (6) from Laurencia viridis collected on the Canary Islands. These compounds are interesting variations on the pinnatifidenyne structure that included the first examples of ethynyl oxirane derivatives (3-4). The structures were elucidated by extensive study of NMR (Nuclear Magnetic Resonance) data, J-based configuration analysis and DFT (Density Functional Theory) calculations. Their antiproliferative activity against six human solid tumor cell lines was evaluated.
Asunto(s)
Acetogeninas/química , Éteres Cíclicos/química , Óxido de Etileno/química , Laurencia/química , Acetogeninas/aislamiento & purificación , Acetogeninas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Óxido de Etileno/aislamiento & purificación , Óxido de Etileno/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura MolecularRESUMEN
Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens. Here, we manipulated the acyl tail and the peptide core of cilagicin to identify an optimized collection of structural features that maintain potent antibiotic activity against a wide range of pathogens in the presence of serum. This led to the identification of the optimized antibiotic dodecacilagicin, which contains an N-terminal dodecanoic acid. Dodecacilagicin exhibits low MICs against clinically relevant pathogens in the presence of serum, retains polyprenyl phosphate binding, and evades resistance development even after long-term antibiotic exposure, making dodecacilagicin an appealing candidate for further therapeutic development.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Humanos , Farmacorresistencia Bacteriana/efectos de los fármacos , Depsipéptidos/farmacología , Depsipéptidos/química , Bacterias Grampositivas/efectos de los fármacosRESUMEN
Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids. Here we increase the bioinformatic and synthetic complexity of the synBNP approach by targeting BGCs that encode N-cinnamoyl lipids. This led to our synthesis of cinnamosyn, a 10-mer N-cinnamoyl-containing peptide that is cytotoxic to human cells.
Asunto(s)
Productos Biológicos , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/síntesis química , Humanos , Estructura Molecular , Biología Computacional , Familia de Multigenes , Lipopéptidos/química , Lipopéptidos/farmacología , Lipopéptidos/síntesis química , Cinamatos/química , Cinamatos/farmacología , Cinamatos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis químicaRESUMEN
Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.