RESUMEN
To cope with environmental challenges, plants produce a wide diversity of phytochemicals, which are also the source of numerous medicines. Despite decades of research in chemical ecology, we still lack an understanding of the organization of plant chemical diversity across species and ecosystems. To address this challenge, we hypothesized that molecular diversity is not only related to species diversity, but also constrained by trophic, climatic, and topographical factors. We screened the metabolome of 416 vascular plant species encompassing the entire alpine elevation range and four alpine bioclimatic regions in order to characterize their phytochemical diversity. We show that by coupling phylogenetic information, topographic, edaphic, and climatic variables, we predict phytochemical diversity, and its inherent composition, of plant communities throughout landscape. Spatial mapping of phytochemical diversity further revealed that plant assemblages found in low to midelevation habitats, with more alkaline soils, possessed greater phytochemical diversity, whereas alpine habitats possessed higher phytochemical endemism. Altogether, we present a general tool that can be used for predicting hotspots of phytochemical diversity in the landscape, independently of plant species taxonomic identity. Such an approach offers promising perspectives in both drug discovery programs and conservation efforts worldwide.
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Metaboloma , Fitoquímicos/clasificación , Plantas/química , Plantas/clasificación , Altitud , Biodiversidad , Clima , Conservación de los Recursos Naturales/métodos , Descubrimiento de Drogas/métodos , Ecosistema , Europa (Continente) , Concentración de Iones de Hidrógeno , Filogenia , Fitoquímicos/biosíntesis , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Plantas/genética , Plantas/metabolismo , Suelo/química , TemperaturaRESUMEN
Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.
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Productos Biológicos/química , Espectrometría de Masas , Biología Computacional/métodos , Bases de Datos Factuales , Metabolómica/métodos , Programas InformáticosRESUMEN
Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8).
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Alcaloides , Antihelmínticos , Productos Biológicos , Poríferos , Alcaloides/química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Estructura Molecular , Poríferos/química , Pez CebraRESUMEN
Drug powder composition analysis is of particular interest in forensic investigations to identify illicit substance content, cutting agents and impurities. Powder profiling is difficult to implement due to multiple analytical methods requirement and remains a challenge for forensic toxicology laboratories. Furthermore, visualization tools allowing seizure products identification appear to be under-used to date. The aim of this study is to present the utility of molecular networking for the composition establishment of natural origin drugs. A powder suspected to contain heroin and three powders suspected to contain cocaine obtained from law enforcement agency seizures were analyzed using untargeted screening by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). Molecular networking and metabolite annotation applied to suspected heroin sample allowed rapid confirmation of its illicit content (heroin), the identification of structurally related major impurities (6-monoacetylmorphine, 6-monoacetylcodeine, noscapine, and papaverine), as well as cutting agents (acetaminophen and caffeine). The cocaine powder profiling allowed the comparison of its constituents in a semi-quantitative manner (cocaine, benzoylecgonine, trans/cis-cinnamoylcocaine, trimethoxycocaine, hexanoylecgonine methylester, caffeine, hydroxyzine, levamisole, and phenacetin), bringing additional information for their identification, including geographically sourcing of natural product and their putative place in the supply chain. Although this approach does not replace the profiling techniques used by forensic laboratories, the use of molecular networks provides a visual overview of structurally related constituents which aids the comparison and investigation of seizure powders. Molecular networks offers here an ideal way to depict structurally related and unrelated compounds in these often complex mixtures of chemicals.
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Drogas Ilícitas , Acetaminofén , Cafeína , Heroína/análisis , Heroína/química , Humanos , Drogas Ilícitas/análisis , Drogas Ilícitas/química , ConvulsionesRESUMEN
Metabolomics is playing an increasingly prominent role in chemical ecology and in the discovery of bioactive natural products (NPs). The identification of metabolites is a common/central objective in both research fields. NPs have significant biological properties and play roles in multiple chemical-ecological interactions. Classically, in pharmacognosy, their chemical structure is determined after a complex process of isolating and interpreting spectroscopic data. With the advent of powerful analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) the annotation process of the specialised metabolome of plants and microorganisms has improved considerably. In this article, we summarise the possibilities opened by these advances and illustrate how we harnessed them in our own research to automate annotations of NPs and target the isolation of key compounds. In addition, we are also discussing the analytical and computational challenges associated with these emerging approaches and their perspective.
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Resistance in clear cell renal cell carcinoma (ccRCC) against sunitinib is a multifaceted process encompassing numerous molecular aberrations. This induces clinical complications, reducing the treatment success. Understanding these aberrations helps us to select an adapted treatment strategy that surpasses resistance mechanisms, reverting the treatment insensitivity. In this regard, we investigated the dominant mechanisms of resistance to sunitinib and validated an optimized multidrug combination to overcome this resistance. Human ccRCC cells were exposed to single or chronic treatment with sunitinib to obtain three resistant clones. Upon manifestation of sunitinib resistance, morphometric changes in the cells were observed. At the molecular level, the production of cell membrane and extracellular matrix components, chemotaxis, and cell cycle progression were dysregulated. Molecules enforcing the cell cycle progression, i.e., cyclin A, B1, and E, were upregulated. Mass spectrometry analysis revealed the intra- and extracellular presence of N-desethyl sunitinib, the active metabolite. Lysosomal sequestration of sunitinib was confirmed. After treatment with a synergistic optimized drug combination, the cell metabolic activity in Caki-1-sunitinib-resistant cells and 3D heterotypic co-cultures was reduced by >80%, remaining inactive in non-cancerous cells. These results demonstrate geno- and phenotypic changes in response to sunitinib treatment upon resistance induction. Mimicking resistance in the laboratory served as a platform to study drug responses.
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Antineoplásicos/farmacología , Carcinoma de Células Renales/genética , Resistencia a Antineoplásicos/genética , Neoplasias Renales/genética , Inhibidores de Proteínas Quinasas/farmacología , Sunitinib/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Modelos Biológicos , Inhibidores de Proteínas Quinasas/uso terapéutico , Sunitinib/uso terapéuticoRESUMEN
Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.
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Antibacterianos/metabolismo , Antibiosis/fisiología , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , GMP Cíclico/análogos & derivados , GMP Cíclico/análisis , Glucolípidos/análisis , Oligopéptidos/análisis , Fenoles/análisis , Pseudomonas aeruginosa/genética , Quinolinas/análisis , Percepción de Quorum/genética , Sideróforos/análisis , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tiazoles/análisisRESUMEN
Acebutolol is a ß1-selective adrenergic receptor antagonist with moderate membrane-stabilizing activity and intrinsic sympathomimetic activity; accordingly, the drug is indicated in hypertension, angina pectoris, and arrhythmia. However, acebutolol's beta-blocking properties also extend the QRS and QTc intervals, and may predispose the patient to ventricular tachydysrhythmia. Here, we report autopsy and toxicological findings on a fatal case of acebutolol self-poisoning in a 70-year-old woman. Toxicological analyses of post-mortem samples (using a liquid chromatography high-resolution mass spectrometry (LC-HR-MS) method) highlighted high concentrations of acebutolol and its metabolite diacetolol in femoral blood (92.8 mg/L and 21.2 mg/L, respectively) and other matrices (cardiac blood, urine, bile, and gastric contents). A molecular networking approach provided useful information on acebutolol's metabolism and revealed the existence of an unknown phase II metabolite of acebutolol. Molecular networking also facilitated visualization of the complex LC-HR-MS/MS datasets and the sample-to-sample comparisons that confirmed massive acebutolol intoxication by ingestion.
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Acebutolol/análogos & derivados , Acebutolol/sangre , Acebutolol/envenenamiento , Anciano , Autopsia , Cromatografía Liquida , Femenino , Humanos , Imagen Molecular , Suicidio , Espectrometría de Masas en TándemRESUMEN
The published version of this article unfortunately contained a mistake. In Figure 1 on the molecular network of acebutololol, two molecular structures are not displayed ("acebutolol glucuronide "and "impurity J"). The Figure is corrected here.
RESUMEN
Environmental conditions influence specialized plant metabolism. However, many studies aiming to understand these modulations have been conducted with model plants and/or under controlled conditions, thus not reflecting the complex interaction between plants and environment. To fully grasp these interactions, we investigated the specialized metabolism and genetic diversity of a native plant in its natural environment. We chose Myrcia bella due to its medicinal interest and occurrence in Brazilian savanna regions with diverse climate and soil conditions. An LC-HRMS-based metabolomics approach was applied to analyze 271 samples harvested across seven regions during the dry and rainy season. Genetic diversity was assessed in a subset of 40 samples using amplified fragment length polymorphism. Meteorological factors including rainfall, temperature, radiation, humidity, and soil nutrient and mineral composition were recorded in each region and correlated with chemical variation through multivariate analysis (MVDA). Marker compounds were selected using a statistically informed molecular network and annotated by dereplication against an in silico database of natural products. The integrated results evidenced different chemotypes, with variation in flavonoid and tannin content mainly linked to soil conditions. Different levels of genetic diversity and distance of populations were found to be correlated with the identified chemotypes. These observations and the proposed analytical workflow contribute to the global understanding of the impact of abiotic factors and genotype on the accumulation of given metabolites and, therefore, could be valuable to guide further medicinal exploration of native species.
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Flavonoides/química , Metabolómica/métodos , Myrtaceae/química , Taninos/química , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Cromatografía Liquida , ADN de Plantas/genética , Variación Genética , Pradera , Myrtaceae/genética , Extractos Vegetales/químicaRESUMEN
INTRODUCTION: Bitter melon (Momordica charantia, Cucurbitaceae) is a popular edible medicinal plant, which has been used as a botanical dietary supplement for the treatment of diabetes and obesity in Chinese folk medicine. Previously, our team has proved that cucurbitanes triterpenoid were involved in bitter melon's anti-diabetic effects as well as on increasing energy expenditure. The triterpenoids composition can however be influenced by changes of varieties or habitats. OBJECTIVES: To clarify the significance of bioactive metabolites diversity among different bitter melons and to provide a guideline for selection of bitter melon varieties, an exploratory study was carried out using a UHPLC-HRMS based metabolomic study to identify chemotypes. METHODS: Metabolites of 55 seed samples of bitter melon collected in different parts of China were profiled by UHPLC-HRMS. The profiling data were analysed with multivariate (MVA) statistical methods. Principle component analysis (PCA) and hierarchical cluster analysis (HCA) were applied for sample differentiation. Marker compounds were identified by comparing spectroscopic data with isolated compounds, and additional triterpenes were putatively identified by propagating annotations through a molecular network (MN) generated from UHPLC-HRMS & MS/MS metabolite profiling. RESULTS: PCA and HCA provided a good discrimination between bitter melon samples from various origins in China. This study revealed for the first time the existence of two chemotypes of bitter melon. Marker compounds of those two chemotypes were identified at different MSI levels. The combined results of MN and MVA demonstrated that the two chemotypes mainly differ in their richness in cucurbitane versus oleanane triterpenoid glycosides (CTGs vs. OTGs). CONCLUSION: Our finding revealed a clear chemotype distribution of bioactive components across bitter melon varieties. While bioactivities of individual CTGs and OTGs still need to be investigated in more depth, our results could help in future the selection of bitter melon varieties with optimised metabolites profile for an improved management of diabetes with this popular edible Chinese folk medicine.
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Medicamentos Herbarios Chinos/metabolismo , Glicósidos/metabolismo , Metabolómica , Momordica charantia/metabolismo , Ácido Oleanólico/metabolismo , Triterpenos/metabolismo , Animales , China , Cromatografía Líquida de Alta Presión , Diabetes Mellitus/tratamiento farmacológico , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/uso terapéutico , Glicósidos/química , Glicósidos/uso terapéutico , Humanos , Espectrometría de Masas , Medicina Tradicional China , Conformación Molecular , Momordica charantia/química , Ácido Oleanólico/química , Ácido Oleanólico/uso terapéutico , Triterpenos/química , Triterpenos/uso terapéuticoRESUMEN
A biological evaluation of a library of extracts from entomopathogen strains showed that Pantoea sp. extract has significant antimicrobial and insecticidal activities. Three hydroxyacyl-phenylalanine derivatives were isolated from this strain. Their structures were elucidated by a comprehensive analysis of their NMR and MS spectroscopic data. The antimicrobial and insecticidal potencies of these compounds were evaluated, and compound 3 showed 67% mortality against Aedes aegypti larvae at a concentration of 100 ppm, and a minimum inhibitory concentration (MIC) of 16 µg/mL against methicillin-resistant Staphylococcus aureus. Subsequently, hydroxyacyl-phenylalanine analogues were synthesized to better understand the structure-activity relationships within this class of compounds. Bioassays highlighted the antimicrobial potential of analogues containing saturated medium-chain fatty acids (12 or 14 carbons), whereas an unsaturated long-chain fatty acid (16 carbons) imparted larvicidal activity. Finally, using a molecular networking-based approach, several close analogues of the isolated and newly synthesized lipoamino acids were discovered in the Pantoea sp. extract.
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Antiinfecciosos/química , Ácidos Grasos/química , Insecticidas/química , Pantoea/química , Aedes/efectos de los fármacos , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Ácidos Grasos/síntesis química , Ácidos Grasos/farmacología , Insecticidas/síntesis química , Insecticidas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
A major goal in the discovery of bioactive natural products is to rapidly identify active compound(s) and dereplicate known molecules from complex biological extracts. The conventional bioassay-guided fractionation process can be time consuming and often requires multi-step procedures. Herein, we apply a metabolomic strategy merging multivariate data analysis and multi-informative molecular maps to rapidly prioritize bioactive molecules directly from crude plant extracts. The strategy was applied to 59 extracts of three Bacopa species (B. monnieri, B. caroliniana and B. floribunda), which were profiled by UHPLC-HRMS2 and screened for anti-lipid peroxidation activity. Using this approach, six lipid peroxidation inhibitors 1â6 of three Bacopa spp. were discovered, three of them being new compounds: monnieraside IV (4), monnieraside V (5) and monnieraside VI (6). The results demonstrate that this combined approach could efficiently guide the discovery of new bioactive natural products. Furthermore, the approach allowed to evidence that main semi-quantitative changes in composition linked to the anti-lipid peroxidation activity were also correlated to seasonal effects notably for B. monnieri.
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Bacopa/química , Productos Biológicos/química , Peroxidación de Lípido/efectos de los fármacos , Manósidos/química , Manósidos/farmacología , Animales , Encéfalo , Química Encefálica , Mezclas Complejas/química , Manósidos/aislamiento & purificación , Metabolómica/métodos , Análisis Multivariante , Extractos Vegetales/química , Análisis de Componente Principal , Ratas , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
Lung cancer is the most lethal form of cancer in the world. Its development often involves an overactivation of the nuclear factor kappa B (NF-κB) pathway, leading to increased cell proliferation, survival, mobility, and a decrease in apoptosis. Therefore, NF-κB inhibitors are actively sought after for both cancer chemoprevention and therapy, and fungi represent an interesting unexplored reservoir for such molecules. The aim of the present work was to find naturally occurring lung cancer chemopreventive compounds by investigating the metabolites of Penicillium vulpinum, a fungus that grows naturally on dung. Penicillium vulpinum was cultivated in Potato Dextrose Broth and extracted with ethyl acetate. Bioassay-guided fractionation of this extract was performed by measuring NF-κB activity using a HEK293 cell line transfected with an NF-κB-driven luciferase reporter gene. The mycotoxin patulin was identified as a nanomolar inhibitor of TNF-α-induced NF-κB activity. Immunocytochemistry and Western blot analyses revealed that its mechanism of action involved an inhibition of p65 nuclear translocation and was independent from the NF-κB inhibitor α (IκBα) degradation process. Enhancing its interest in lung cancer chemoprevention, patulin also exhibited antiproliferative, proapoptotic, and antimigration effects on human lung adenocarcinoma cells through inhibition of the Wnt pathway.
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Antineoplásicos/farmacología , Neoplasias Pulmonares/metabolismo , Patulina/farmacología , Penicillium/química , Factor de Necrosis Tumoral alfa/metabolismo , Células A549 , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Patulina/química , Patulina/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
With the aim of discovering new cytotoxic prenylated stilbenes of the schweinfurthin series, Macaranga tanarius was selected for detailed phytochemical investigation among 21 Macaranga species examined by using a molecular networking approach. From an ethanol extract of the fruits, seven new prenylated stilbenes, schweinfurthins K-Q (7-13), were isolated, along with vedelianin (1), schwenfurthins E-G (2-4), mappain (5), and methyl-mappain (6). The structures of the new compounds were established by spectroscopic data analysis. The relative configurations of compounds 8, 12, and 13 were determined based on ROESY NMR spectroscopic analysis. The cytotoxic activities of compounds 1-13 were evaluated against the human glioblastoma (U87) and lung (A549) cancer cell lines.
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Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Euphorbiaceae/química , Estilbenos/aislamiento & purificación , Estilbenos/farmacología , Antineoplásicos Fitogénicos/química , Ensayos de Selección de Medicamentos Antitumorales , Flavonoides/química , Frutas/química , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Hojas de la Planta/química , Prenilación , Estilbenos/química , VietnamRESUMEN
Phytochemical investigation of the alkaloid extract of Palicourea sessilis by LC-HRMS/MS using molecular networking and an in silico MS/MS fragmentation approach suggested the presence of several new monoterpene indole alkaloids. These compounds were isolated by semipreparative HPLC, and their structures confirmed by means of HRMS, NMR, and ECD measurements as 4-N-methyllyaloside (3), 4-N-methyl-3,4-dehydrostrictosidine (4), 4ß-hydroxyisodolichantoside (6), and 4α-hydroxyisodolichantoside (7), as well as the known alkaloids alline (1), N-methyltryptamine (2), isodolichantoside (5), and 5-oxodolichantoside (8). In addition, the acetylcholinesterase inhibitory activity of the compounds was evaluated up to 50 µM.
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Inhibidores de la Colinesterasa/aislamiento & purificación , Rubiaceae/química , Alcaloides de Triptamina Secologanina/aislamiento & purificación , Acetilcolinesterasa/efectos de los fármacos , Brasil , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Hojas de la Planta/química , Alcaloides de Triptamina Secologanina/química , Triptaminas/químicaRESUMEN
INTRODUCTION: Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites. OBJECTIVE: To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains. METHODS: Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I isoform HDAC1 and the class II isoform HDAC6. RESULTS: Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in P. griseofulvum is described here for the first time. CONCLUSION: The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.
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Cromatografía Líquida de Alta Presión/métodos , Hongos/metabolismo , Histona Desacetilasas/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Dereplication represents a key step for rapidly identifying known secondary metabolites in complex biological matrices. In this context, liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is increasingly used and, via untargeted data-dependent MS/MS experiments, massive amounts of detailed information on the chemical composition of crude extracts can be generated. An efficient exploitation of such data sets requires automated data treatment and access to dedicated fragmentation databases. Various novel bioinformatics approaches such as molecular networking (MN) and in-silico fragmentation tools have emerged recently and provide new perspective for early metabolite identification in natural products (NPs) research. Here we propose an innovative dereplication strategy based on the combination of MN with an extensive in-silico MS/MS fragmentation database of NPs. Using two case studies, we demonstrate that this combined approach offers a powerful tool to navigate through the chemistry of complex NPs extracts, dereplicate metabolites, and annotate analogues of database entries.
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Productos Biológicos/química , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Simulación por ComputadorRESUMEN
A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8-methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography-high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS(2) spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19-22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.
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Alcaloides/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Productos Biológicos/aislamiento & purificación , Rutaceae/química , Alcaloides/química , Alcaloides/farmacología , Antifúngicos/química , Productos Biológicos/química , Productos Biológicos/farmacología , Brasil , Candida albicans/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Resonancia Magnética Nuclear Biomolecular , Corteza de la Planta/químicaRESUMEN
A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy.