RESUMEN
The macrocyclic tumonolide (1) with enamide functionality and the linear tumonolide aldehyde (2) are new interconverting natural products from a marine cyanobacterium with a peptide-polyketide skeleton, representing a hybrid of apratoxins and palmyrolides or laingolides. The planar structures were established by NMR and mass spectrometry. The relative configuration of the stereogenically-rich apratoxin-like polyketide portion was determined using J-based configuration analysis. The absolute configuration of tumonolide (1) was determined by chiral analysis of the amino acid units and computational methods, followed by NMR chemical shift and ECD spectrum prediction, indicating all-R configuration for the polyketide portion, as in palmyrolide A and contrary to the all-S configuration in apratoxins. Functional screening against a panel of 168 GPCR targets revealed tumonolide (1) as a selective antagonist of TACR2 with an IC50 of 7.0â µM, closely correlating with binding affinity. Molecular docking studies established the binding mode and rationalized the selectivity for TACR2 over TACR1 and TACR3. RNA sequencing upon treatment of HCT116 colorectal cancer cells demonstrated activation of the pulmonary fibrosis idiopathic signaling pathway and the insulin secretion signaling pathway at 20â µM, indicating its potential to modulate these pathways.
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Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.
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Antineoplásicos/síntesis química , Proteínas Bacterianas/síntesis química , Productos Biológicos/síntesis química , Depsipéptidos/síntesis química , Microtúbulos/efectos de los fármacos , Moduladores de Tubulina/síntesis química , Tubulina (Proteína)/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Sitios de Unión , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Línea Celular Tumoral , Colchicina/química , Colchicina/farmacología , Cristalografía por Rayos X , Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Descubrimiento de Drogas , Células HCT116 , Humanos , Maitansina/química , Maitansina/farmacología , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Pironas/química , Pironas/farmacología , Taxoides/química , Taxoides/farmacología , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/aislamiento & purificación , Moduladores de Tubulina/farmacología , Alcaloides de la Vinca/química , Alcaloides de la Vinca/farmacologíaRESUMEN
Lyngbyastatins (Lbns) 1 (1) and 3 (2) belong to a group of cyclic depsipeptides that inhibit cancer cell proliferation. These compounds have been isolated from different marine cyanobacterial collections, while further development of these compounds relies on their lengthy total synthesis. Biosynthetic studies of these compounds can provide viable strategies to access these compounds and develop new analogs. In this study, we report the identification and characterization of one Lbn biosynthetic gene cluster (BGC) from the marine cyanobacterium Okeania sp. VPG18-21. We initially identified 1 and 2 in the organic extract by mass spectrometry and performed the targeted isolation of these compounds, which feature a (2S,3R)-3-amino-2-methylpentanoic acid (MAP) and a (2S,3R)-3-amino-2-methylhexanoic acid (Amha) moiety, respectively. Parallel metagenomic sequencing of VPG18-21 led to the identification of a putative Lbn BGC that encodes six megaenzymes (LbnA-F), including one polyketide synthase (PKS, LbnE), four nonribosomal peptide synthetases (NRPSs, LbnB-D and -F), and one PKS-NRPS hybrid (LbnA). Bioinformatic analysis of these enzymes suggested that the BGC produces 1 and 2. Furthermore, our biochemical studies of three recombinant adenylation domains uncovered their substrate specificities, supporting the identity of the BGC. Finally, we identified near-complete Lbn-like BGCs in the genomes of two other marine cyanobacteria.
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Antineoplásicos , Cianobacterias , Depsipéptidos , Neoplasias , Humanos , Antineoplásicos/farmacología , Cianobacterias/química , Depsipéptidos/química , Sintasas Poliquetidas/genética , Péptido Sintasas/genética , Familia de MultigenesRESUMEN
Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside (1) and its C-31 O-demethyl analogue, iezoside B (2), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC50 of 1.5 and 3.0 µΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 µΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC50 values of 0.3 and 0.6 µΜ in A549 cells and 0.1 and 0.5 µΜ, respectively, in HeLa cells, near the IC50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.
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Antineoplásicos , Cianobacterias , Humanos , Células HeLa , Calcio/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Cianobacterias/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , AzúcaresRESUMEN
NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides. We isolated three new compounds of this class, taveuniamides L-N (1-3), along with the previously reported taveuniamide F (4), which was the most abundant analog. The planar structures of the new compounds were established using 1D and 2D NMR as well as mass spectrometry. We established the configuration of this chemical class to be R at C-8 via Mosher's analysis of 4 after reduction of the carboxamide group. Our biological investigations with 4 revealed that the compound binds to the cannabinoid receptor CNR1, acting as an antagonist/inverse agonist in the canonical G-protein signaling pathways. In selectivity profiling against 168 GPCR targets using the ß-arrestin functional assay, we found that 4 antagonizes GPR119, NPSR1b, CCR9, CHRM4, GPR120, HTR2A, and GPR103, in addition to CNR1. Interestingly, 4 showed a 6.8-fold selectivity for CNR1 over CNR2. The binding mode of 4 to CNR1 was investigated using docking and molecular dynamics simulations with both natural and unnatural stereoisomers, revealing important CNR1 residues for the interaction and also providing a possible reasoning for the observed CNR1/CNR2 selectivity.
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Cianobacterias , Agonismo Inverso de Drogas , Espectrometría de Masas en Tándem , Amidas/farmacología , Ácidos GrasosRESUMEN
The monounsaturated fatty acid 7(E)-9-keto-hexadec-7-enoic acid (1) and three structurally related analogues with different oxidation states and degrees of unsaturation (2-4) were discovered from a marine benthic cyanobacterial mat collected from Delta Shoal, Florida Keys. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The structure of 1 contained an α,ß-unsaturated carbonyl system, a key motif required for the activation of the Keap1/Nrf2-ARE pathway that is involved in the activation of antioxidant and phase II detoxification enzymes. Compounds 1-4 were screened in ARE-luciferase reporter gene assay using stably transfected HEK293 cells, and only 1 significantly induced Nrf2 activity at 32 and 10 µM, whereas 2-4 were inactive. As there is crosstalk between inflammation and oxidative stress, subsequent biological studies were focused on 1 to investigate its anti-inflammatory potential. Compound 1 induced Nqo1, a well-known target gene of Nrf2, and suppressed iNos transcript levels, which translated into reduced levels of nitric oxide in LPS-activated mouse macrophage RAW264.7 cells, a more relevant model for inflammation. RNA sequencing was performed to capture the effects of 1 on a global level and identified additional canonical pathways and upstream regulators involved in inflammation and immune response, particularly those related to multiple sclerosis. A targeted survey of marine cyanobacterial samples from other geographic locations, including Guam, suggested the widespread occurrence of 1. Furthermore, the previous isolation of 1 from marine diatoms and green algae implied a potentially important ecological role across marine algal eukaryotes and prokaryotes. The previous isolation from sea lettuce raises the possibility of dietary intervention to attenuate inflammation and related disease progression.
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Ácidos Grasos , Factor 2 Relacionado con NF-E2 , Humanos , Ratones , Animales , Factor 2 Relacionado con NF-E2/metabolismo , Ácidos Grasos/uso terapéutico , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Células HEK293 , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Inflamación/metabolismo , FN-kappa B/metabolismo , Lipopolisacáridos/farmacologíaRESUMEN
Black band disease is a globally distributed and easily recognizable coral disease. Despite years of study, the etiology of this coral disease, which impacts dozens of stony coral species, is not completely understood. Although black band disease mats are predominantly composed of the cyanobacterial species Roseofilum reptotaenium, other filamentous cyanobacterial strains and bacterial heterotrophs are readily detected. Through chemical ecology and metagenomic sequencing, we uncovered cryptic strains of Roseofilum species from Siderastrea siderea corals that differ from those on other corals in the Caribbean and Pacific. Isolation of metabolites from Siderastrea-derived Roseofilum revealed the prevalence of unique forms of looekeyolides, distinct from previously characterized Roseofilum reptotaenium strains. In addition, comparative genomics of Roseofilum strains showed that only Siderastrea-based Roseofilum strains have the genetic capacity to produce lasso peptides, a family of compounds with diverse biological activity. All nine Roseofilum strains examined here shared the genetic capacity to produce looekeyolides and malyngamides, suggesting these compounds support the ecology of this genus. Similar biosynthetic gene clusters are not found in other cyanobacterial genera associated with black band disease, which may suggest that looekeyolides and malyngamides contribute to disease etiology through yet unknown mechanisms.
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Antozoos , Cianobacterias , Animales , Antozoos/microbiología , Cianobacterias/metabolismo , Genómica , MetagenómicaRESUMEN
Emerging diseases can have devastating consequences for wildlife and require a rapid response. A critical first step towards developing appropriate management is identifying the etiology of the disease, which can be difficult to determine, particularly early in emergence. Gathering and synthesizing existing information about potential disease causes, by leveraging expert knowledge or relevant existing studies, provides a principled approach to quickly inform decision-making and management efforts. Additionally, updating the current state of knowledge as more information becomes available over time can reduce scientific uncertainty and lead to substantial improvement in the decision-making process and the application of management actions that incorporate and adapt to newly acquired scientific understanding. Here we present a rapid prototyping method for quantifying belief weights for competing hypotheses about the etiology of disease using a combination of formal expert elicitation and Bayesian hierarchical modeling. We illustrate the application of this approach for investigating the etiology of stony coral tissue loss disease (SCTLD) and discuss the opportunities and challenges of this approach for addressing emergent diseases. Lastly, we detail how our work may apply to other pressing management or conservation problems that require quick responses. We found the rapid prototyping methods to be an efficient and rapid means to narrow down the number of potential hypotheses, synthesize current understanding, and help prioritize future studies and experiments. This approach is rapid by providing a snapshot assessment of the current state of knowledge. It can also be updated periodically (e.g., annually) to assess changes in belief weights over time as scientific understanding increases. Synthesis and applications: The rapid prototyping approaches demonstrated here can be used to combine knowledge from multiple experts and/or studies to help with fast decision-making needed for urgent conservation issues including emerging diseases and other management problems that require rapid responses. These approaches can also be used to adjust belief weights over time as studies and expert knowledge accumulate and can be a helpful tool for adapting management decisions.
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Antozoos , Animales , Teorema de Bayes , IncertidumbreRESUMEN
Our ongoing efforts to explore the chemical space associated with marine cyanobacteria from coral reefs of Guam have yielded two new members of the anaenamide family of natural products, anaenamides C (3) and D (4). These compounds were isolated from a novel Hormoscilla sp. (VPG16-58). Our phylogenetic profiling (16S rDNA) of this cyanobacterium indicated that VPG16-58 is taxonomically distinct from the previously reported producer of the anaephenes, VPG16-59 (Hormoscilla sp.), and other previously documented species of the genus Hormoscilla. The planar structures of 3 and 4 were determined via spectroscopic methods, and absolute configurations of the α-hydroxy acids were assigned by enantioselective HPLC analysis. To address the requirement for sufficient material for testing, we first adapted our published linear synthetic approach for 1 and 2 to generate anaenoic acid (7), which served as a point for diversification, providing the primary amides 3 and 4 from synthetic intermediates 5 and 6, respectively. The compounds were then tested for effects on HCT116 colon cancer cell viability and in an ARE-luciferase reporter gene assay for Nrf2 modulation using HEK293 human embryonic kidney cells. Our findings indicate that, in contrast to cytotoxic methyl esters 1 and 2, the primary amides 3 and 4 activate the Nrf2 pathway at noncytotoxic concentrations. Overall, our data suggest that the anaenamide scaffold is tunable to produce differential biological outcomes.
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Cianobacterias , Factor 2 Relacionado con NF-E2 , Amidas/farmacología , Cianobacterias/química , Células HEK293 , Humanos , FilogeniaRESUMEN
Stony corals (Scleractinia) are invertebrates that form symbiotic relationships with eukaryotic algal endosymbionts and the prokaryotic microbiome. The microbiome has the potential to produce bioactive natural products providing defense and resilience to the coral host against pathogenic microorganisms, but this potential has not been extensively explored. Bacterial pathogens can pose a significant threat to corals, with some species implicated in primary and opportunistic infections of various corals. In response, probiotics have been proposed as a potential strategy to protect corals in the face of increased incidence of disease outbreaks. In this study, we screened bacterial isolates from healthy and diseased corals for antibacterial activity. The bioactive extracts were analyzed using untargeted metabolomics. Herein, an UpSet plot and hierarchical clustering analyses were performed to identify isolates with the largest number of unique metabolites. These isolates also displayed different antibacterial activities. Through application of in silico and experimental approaches coupled with genome analysis, we dereplicated natural products from these coral-derived bacteria from Florida's coral reef environments. The metabolomics approach highlighted in this study serves as a useful resource to select probiotic candidates and enables insights into natural product-mediated chemical ecology in holobiont symbiosis.
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Antozoos , Productos Biológicos , Animales , Antozoos/microbiología , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias/genética , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Metabolómica , SimbiosisRESUMEN
Dysidazirine carboxylic acid (1) was isolated from the lipophilic extract of a collection of the benthic marine cyanobacterium Caldora sp. from reefs near Fort Lauderdale, Florida. The planar structure of this new compound was determined by spectroscopic methods and comparisons between HRMS and NMR data with its reported methyl ester. The absolute configuration of the single chiral center was determined by the conversion of 1 to the methyl ester and the comparison of its specific rotation data with the two known methyl ester isomers, 2 and 3. Molecular sequencing with 16S rDNA indicated that this cyanobacterium differs from Caldora penicillata (Oscillatoriales) and represents a previously undocumented and novel Caldora species. Dysidazirine (2) showed weak cytotoxicity against HCT116 colorectal cancer cells (IC50 9.1 µM), while dysidazirine carboxylic acid (1) was non-cytotoxic. Similar cell viability patterns were observed in RAW264.7 cells with dysidazirine only (2), displaying cytotoxicity at the highest concentration tested (50 µM). The non-cytotoxic dysidazirine carboxylic acid (1) demonstrated anti-inflammatory activity in RAW264.7 cells stimulated with LPS. After 24 h, 1 inhibited the production of NO by almost 50% at 50 µM, without inducing cytotoxicity. Compound 1 rapidly decreased gene expression of the pro-inflammatory gene iNOS after 3 h post-LPS treatment and in a dose-dependent manner (IC50 ~1 µM); the downregulation of iNOS persisted at least until 12 h.
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Azirinas , Ácidos Carboxílicos , Antiinflamatorios/farmacología , Ácidos Carboxílicos/farmacología , Florida , Humanos , Estructura MolecularRESUMEN
Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.
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Antifúngicos/metabolismo , Membrana Celular/metabolismo , Macrólidos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Animales , Antifúngicos/química , Antifúngicos/farmacología , Membrana Celular/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Ergosterol/química , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemólisis/efectos de los fármacos , Liposomas/química , Liposomas/metabolismo , Macrólidos/química , Macrólidos/farmacología , Nistatina/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , OvinosRESUMEN
By 2004, Belize was exhibiting classic fishing down of the food web. Groupers (Serranidae) and snappers (Lutjanidae) were scarce and fisheries turned to parrotfishes (Scarinae), leading to a 41% decline in their biomass. Several policies were enacted in 2009-2010, including a moratorium on fishing parrotfish and a new marine park with no-take areas. Using a 20-year time series on reef fish and benthos, we evaluated the impact of these policies approximately 10 years after their implementation. Establishment of the Southwater Caye Marine Reserve led to a recovery of snapper at 2 out of 3 sites, but there was no evidence of recovery outside the reserve. Snapper populations in an older reserve continued to increase, implying that at least 9 years is required for their recovery. Despite concerns over the feasibility of banning parrotfish harvest once it has become a dominant fin fishery, parrotfishes returned and exceeded biomass levels prior to the fishery. The majority of these changes involved an increase in parrotfish density; species composition and adult body size generally exhibited little change. Recovery occurred equally well in reserves and areas open to other forms of fishing, implying strong compliance. Temporal trends in parrotfish grazing intensity were strongly negatively associated with the cover of macroalgae, which by 2018 had fallen to the lowest levels observed since measurements began in 1998. Coral populations remained resilient and continued to exhibit periods of net recovery after disturbance. We found that a moratorium on parrotfish harvesting is feasible and appears to help constrain macroalgae, which can otherwise impede coral resilience.
Reservas Marinas, Vedas Pesqueras y 20 Años de Cambios Positivos en un Ecosistema de Arrecife de Coral Resumen Para el año 2004, Belice estaba exhibiendo la clásica pesca de los niveles más bajos de las cadenas alimenticias marinas. Los meros (Serranidae) y los pargos (Lutjanidae) eran escasos y las pesquerías comenzaron a consumir a los peces loro (Scarinae), lo que resultó en una declinación del 41% de su biomasa. Entre el 2009 y el 2010 se promulgaron varias políticas, incluyendo una moratoria para la pesca del pez loro y un nuevo parque marino con zonas de no consumo. Mediante una serie temporal de 20 años para los peces de arrecifes y el bentos, evaluamos el impacto de estas políticas aproximadamente diez años después de su implementación. La creación de la Reserva Marina del Cayo Southwater resultó en la recuperación del pargo en dos de tres sitios, pero no hubo evidencias de la recuperación fuera de la reserva. Las poblaciones de pargos en una reserva más vieja continuaron su incremento, lo que implica que se requieren al menos nueve años para su recuperación. A pesar de la preocupación por la viabilidad de la veda para el pez loro una vez que se haya convertido en una pesquería dominante, los peces loro regresaron al sitio de pesca y excedieron los niveles de biomasa previos a la pesquería. La mayoría de estos cambios involucró un incremento en la densidad de los peces loro; la composición de especies y la talla corporal adulta generalmente exhibieron pocos cambios. La recuperación ocurrió equitativamente bien en las reservas y en las áreas abiertas a otras formas de pesca, lo que implica un estricto cumplimiento de las restricciones. Las tendencias temporales en la intensidad de pastoreo de los peces loro estuvieron fuertemente asociadas de manera negativa con la cobertura de macroalgas, la cual para el 2018 había caído a los niveles más bajos observados desde que se comenzó a medir en 1998. Las poblaciones coralinas permanecieron resilientes y continuaron exhibiendo periodos de recuperación neta después de la perturbación. Descubrimos que una moratoria para la pesca de pez loro es viable y parece ayudar a restringir las macroalgas, las cuales de otra forma pueden impedir la resiliencia del coral.
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Antozoos , Arrecifes de Coral , Animales , Conservación de los Recursos Naturales , Ecosistema , Explotaciones Pesqueras , PecesRESUMEN
New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa, characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi, indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.
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Apoptosis/efectos de los fármacos , Cianobacterias/química , Pirroles/farmacología , Percepción de Quorum/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF , Línea Celular Tumoral , Humanos , Estructura Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Pirroles/química , Pirroles/aislamiento & purificación , Relación Estructura-Actividad , Vibrio/efectos de los fármacosRESUMEN
Chemical investigation of a benthic marine cyanobacterium yielded the anticancer agent dolastatin 15, originally isolated from a mollusk. Dolastatin 15 is a microtubule-destabilizing agent with analogues undergoing clinical evaluation. Profiling against a panel of isogenic HCT116 colorectal cancer cells showed remarkable differential cytotoxicity against the parental cells over isogenic cells lacking HIF or other key players in the pathway, including oncogenic KRAS and VEGF. Dolastatin 15 displayed an antivascularization effect in human endothelial cells and in zebrafish vhl mutants with activated Hif, thus signifying its clinical potential as a treatment for solid tumors with an angiogenic component. Global transcriptome analysis with RNA sequencing suggested that dolastatin 15 could affect other major cancer pathways that might not directly involve tubulin or HIF. The identification of the true producer of a clinically relevant agent is important for sustainable supply, as is understanding the biosynthesis, and future genetic manipulation of the biosynthetic gene cluster for analogue production.
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Supervivencia Celular/efectos de los fármacos , Cianobacterias/química , Depsipéptidos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Depsipéptidos/uso terapéutico , Células HCT116 , HumanosRESUMEN
A new, cyclic carbonate eudesmane-type sesquiterpene, eudesmacarbonate (1), was isolated from marine filamentous cyanobacterial mats associated with apparent ingestion-related intoxications of captive bottlenose dolphins in the Florida Keys. Sequencing of 16S rDNA revealed that mats were composed of closely related Oscillatoriacean species including a previously undocumented species of Neolyngbya. The structure of 1 was elucidated by (+)-HRESIMS, 1D and 2D NMR, single-crystal X-ray diffraction, and vibrational circular dichroism data. Toxicity of 1 was assessed in the zebrafish embryo/larval model, and 1 was found to exhibit effects qualitatively similar to those observed for the known neurotoxin brevetoxin-2 and consistent with neurobehavioral impairment.
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Cianobacterias/química , Síndromes de Neurotoxicidad/psicología , Neurotoxinas/toxicidad , Sesquiterpenos de Eudesmano/toxicidad , Sesquiterpenos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Embrión no Mamífero , Florida , Larva , Espectroscopía de Resonancia Magnética , Estructura Molecular , Difracción de Rayos X , Pez CebraRESUMEN
BACKGROUND: The MinION Access Program (MAP, 2014-2016) allowed selected users to test the prospects of long nanopore reads for diverse organisms and applications through the rapid development of improving chemistries. In 2014, faced with a fragmented Illumina assembly for the chloroplast genome of the green algal holobiont Caulerpa ashmeadii, we applied to the MAP to test the prospects of nanopore reads to investigate such intricacies, as well as further explore the hologenome of this species with native and hybrid approaches. RESULTS: The chloroplast genome could only be resolved as a circular molecule in nanopore assemblies, which also revealed structural variants (i.e. chloroplast polymorphism or heteroplasmy). Signal and Illumina polishing of nanopore-assembled organelle genomes (chloroplast and mitochondrion) reflected the importance of coverage on final quality and current limitations. In hybrid assembly, our modest nanopore data sets showed encouraging results to improve assembly length, contiguity, repeat content, and binning of the larger nuclear and bacterial genomes. Profiling of the holobiont with nanopore or Illumina data unveiled a dominant Rhodospirillaceae (Alphaproteobacteria) species among six putative endosymbionts. While very fragmented, the cumulative hybrid assembly length of C. ashmeadii's nuclear genome reached 24.4 Mbp, including 2.1 Mbp in repeat, ranging closely with GenomeScope's estimate (> 26.3 Mbp, including 4.8 Mbp in repeat). CONCLUSION: Our findings relying on a very modest number of nanopore R9 reads as compared to current output with newer chemistries demonstrate the promising prospects of the technology for the assembly and profiling of an algal hologenome and resolution of structural variation. The discovery of polymorphic 'chlorotypes' in C. ashmeadii, most likely mediated by homing endonucleases and/or retrohoming by reverse transcriptases, represents the first report of chloroplast heteroplasmy in the siphonous green algae. Improving contiguity of C. ashmeadii's nuclear and bacterial genomes will require deeper nanopore sequencing to greatly increase the coverage of these larger genomic compartments.
Asunto(s)
Caulerpa/genética , Genoma del Cloroplasto , Secuenciación de Nanoporos/métodos , Análisis de Secuencia de ADN/métodos , Genoma Bacteriano , Genoma Mitocondrial , Genómica/métodos , Polimorfismo Genético , Polimorfismo de Nucleótido SimpleRESUMEN
The transition from larva to adult is a critical step in the life history strategy of most marine animals. However, the genetic basis of this life history change remains poorly understood in many taxa, including most coral species. Recent evidence suggests that coral planula larvae undergo significant changes at the physiological and molecular levels throughout the development. To investigate this, we characterized differential gene expression (DGE) during the transition from planula to adult polyp in the abundant Caribbean reef-building coral Porites astreoides, that is from nonprobing to actively substrate-probing larva, a stage required for colony initiation. This period is crucial for the coral, because it demonstrates preparedness to locate appropriate substrata for settlement based on vital environmental cues. Through RNA-Seq, we identified 860 differentially expressed holobiont genes between probing and nonprobing larvae (p ≤ .01), the majority of which were upregulated in probing larvae. Surprisingly, differentially expressed genes of endosymbiotic dinoflagellate origin greatly outnumbered coral genes, compared with a nearly 1:1 ratio of coral-to-dinoflagellate gene representation in the holobiont transcriptome. This unanticipated result suggests that dinoflagellate endosymbionts may play a significant role in the transition from nonprobing to probing behaviour in dinoflagellate-rich larvae. Putative holobiont genes were largely involved in protein and nucleotide binding, metabolism and transport. Genes were also linked to environmental sensing and response and integral signalling pathways. Our results thus provide detailed insight into molecular changes prior to larval settlement and highlight the complex physiological and biochemical changes that occur in early transition stages from pelagic to benthic stages in corals, and perhaps more importantly, in their endosymbionts.
Asunto(s)
Antozoos/genética , Expresión Génica/genética , Larva/genética , Animales , Región del Caribe , Arrecifes de Coral , Dinoflagelados/genética , Simbiosis/genética , Transcriptoma/genéticaRESUMEN
Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.
Asunto(s)
Productos Biológicos/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Éteres Difenilos Halogenados/metabolismo , Metagenómica , Poríferos/metabolismo , Animales , Productos Biológicos/química , Éteres Difenilos Halogenados/química , Estructura MolecularRESUMEN
Black band disease (BBD), a lethal, polymicrobial disease consortium dominated by the cyanobacterium Roseofilum reptotaenium, kills many species of corals worldwide. To uncover chemical signals or cytotoxins that could be important in proliferation of Roseofilum and the BBD layer, we examined the secondary metabolites present in geographically diverse collections of BBD from Caribbean and Pacific coral reefs. Looekeyolide A (1), a 20-membered macrocyclic compound formed by a 16-carbon polyketide chain, 2-deamino-2-hydroxymethionine, and d-leucine, and its autoxidation product looekeyolide B (2) were extracted as major compounds (â¼1 mg g-1 dry wt) from more than a dozen field-collected BBD samples. Looekeyolides A and B were also produced by a nonaxenic R. reptotaenium culture under laboratory conditions at similar concentrations. R. reptotaenium genomes that were constructed from four different metagenomic data sets contained a unique nonribosomal peptide/polyketide biosynthetic cluster that is likely responsible for the biosynthesis of the looekeyolides. Looekeyolide A, which readily oxidizes to looekeyolide B, may play a biological role in reducing H2O2 and other reactive oxygen species that could occur in the BBD layer as it overgrows and destroys coral tissue.