LC-HRMS-Database Screening Metrics for Rapid Prioritization of Samples to Accelerate the Discovery of Structurally New Natural Products

In order to accelerate the isolation and characterization of structurally new or novel secondary metabolites, it is crucial to develop efficient strategies that prioritize samples with greatest promise early in the workflow so that resources can be utilized in a more efficient and cost-effective manner. We have developed a metrics-based prioritization approach using exact LC-HRMS, which uses data for 24 618 marine natural products held in the PharmaSea database. Each sample was evaluated and allocated a metric score by a software algorithm based on the ratio of new masses over the total (sample novelty), ratio of known masses over the total (chemical novelty), number of peaks above a defined peak area threshold (sample complexity), and peak area (sample diversity). Samples were then ranked and prioritized based on these metric scores. To validate the approach, eight marine sponges and six tunicate samples collected from the Fiji Islands were analyzed, metric scores calculated, and samples targeted for isolation and characterization of new compounds. Structures of new compounds were elucidated by spectroscopic techniques, including 1D and 2D NMR, MS, and MS/MS. Structures were confirmed by computer-assisted structure elucidation methods (CASE) using the ACD/Structure Elucidator Suite

Validation d'une méthode de déreplication de substances naturelles marines

Rising incidence of cancers and ever-growing microbial infections are the main concerns of modern medicine, coevolving with the respective drug targeting resistances. This “growing health crisis”, as defined by the World Health Organization in 2014, prompts researchers to find new resources of drug leads. Despite the large number of terrestrial natural products derivatives approved as pharmaceutics, industry has reduced its reliance on it, mostly due to the high rate of rediscovery of known compounds. Therefore, academicians and private industry have turned their efforts toward exploring the marine world as new sources of bioactive molecules. We contributed to this effort in participating in a research project named PharmaSea, based on the bio discovery, identification and development of new substances from marine organisms. The key step to successful drug bio discovery from marine sources is the rapid identification of the known compounds before isolating them. Called dereplication, this approach is essential to assess chemical novelty of crude extracts and their fractions. The profusion of data sets and the lack of confirmatory data make this task arduous. Different strategies of dereplication procedures involving Liquid chromatography coupled with High Resolution Mass Spectrometry are thus emerging. In this regard, we investigated the PharmaSea dereplication method on 21 tunicates extracts. This method combines the ACDLabs IntelliXtract software with the MarinLit database from the Royal Society of Chemistry to dereplicate mass data. Nuclear Magnetic Resonance was also used to streamline the prioritization of samples to study to validate the method. The most interesting sample according to these strategies was selected for further purification leading to the isolation of seven compounds of which five are new as evidence of the success of the method.L’émergence de résistance aux antibiotiques et aux anticancéreux est aujourd’hui définie par l’Organisation Mondiale de la Santé comme une crise de santé mondiale, incitant les chercheurs à développer des agents thérapeutiques innovants et plus spécifiques. L’industrie a restreint son recours aux substances naturelles végétales, notamment en raison de la fréquente redécouverte de composés connus. Les scientifiques tournent alors désormais leurs efforts vers le milieu marin, encore très peu exploité. Nous avons contribué à cet effort en participant au projet de recherche Européen PharmaSea axé sur la découverte, la caractérisation, et le développement de nouvelles substances pharmaceutiques tirées d'organismes marins. Une étape clé pour réussir dans la découverte de « médicaments de la mer » est la déréplication : l'identification rapide de composés déjà connus avant l’isolement. L’abondance de données et le manque d’informations confirmatives compliquent cette tâche. Différentes stratégies de déréplication comme la Chromatographie Liquide couplée à la Spectrométrie de Masse Haute Résolution émergent actuellement. Nous avons investigué la méthode de déréplication des données de masse de PharmaSea sur 21 extraits de tuniciers. Cette méthode est basée sur une combinaison du logiciel ACDLabs IntelliXtract et de la base de données MarinLit de la Royal Society of Chemistry. Grâce à l’ajout d’une analyse par Résonnance Magnétique Nucléaire, nous avons pu classer les extraits à étudier pour valider la méthode. Les plus prometteurs de nouveauté ont été purifiés, conduisant à l'isolement de sept composés dont cinq nouveaux, preuve de l'efficacité du mode de déréplication utilisé

Comprehensive Study of the Fungal Endophyte Community of the Long-lived Amazonian Palm <i>Astrocaryum Sciophilum</i>: A Model for Deciphering Plant-microbe Interactions at Chemical and Bioactive Levels

Il est désormais établi que les plantes ne sont pas des entités distinctes, mais qu'elles ont évolué en association avec des assemblages microbiens très complexes et diversifiés. Ces communautés de symbiotes microbiens des plantes (microbiote) et leur matériel génétique collectif constituent le microbiome végétal. Le microbiome semble jouer des rôles clés dans l'écologie et la physiologie des plantes, y compris leur croissance et survie. Notre intérêt se porte sur certains des habitants fongiques du microbiome: les champignons endophytes. Dans cette thèse, réalisée dans le cadre du projet franco-suisse SECIL (Study of Endophytes Communities In a Leaf, SECIL_ANR), nous avons voulu étudier le crosstalk microbien dans les endophytes fongiques des feuilles de plantes au niveau chimique et bioactif, en recherchant des métabolites biologiquement pertinents. Le palmier typique de la forêt primaire de Guyane française, Astrocaryum sciophilum (Miq.) Pulle a été choisi pour une étude approfondie en tant que modèle d'hôte, en raison de son cycle de vie particulièrement long et de la diversité des communautés endophytes trouvées dans ses feuilles. Son long âge de maturation suggère que ce palmier pourrait maintenir une association stable avec les communautés microbiennes sur une période de temps substantielle. Cela implique que les endophytes potentiels sont capables de survivre et de résister à l'environnement de la plante, probablement en développant des anti- pathogènes et des métabolites cytotoxiques impliqués dans la longévité des pousses et des feuilles du palmier. De plus, l'écosystème hautement compétitif de la forêt tropicale rend la pression de sélection intense, incitant probablement les microbes associés à cet hôte tropical à produire des entités chimiques intéressantes. Nous avons également émis l'hypothèse que des interactions chimiques réciproques se produisent entre la plante et les champignons, via une chimie spécialisée potentiellement exprimée dans les tissus de l'hôte. Nous avons donc développé une stratégie pour étudier, à un moment donné, cette interaction plante-champignon d'un point de vue moléculaire, de la manière la plus complète possible, ainsi que le potentiel bioactif qui résulte de cette interaction. Le workflow que nous avons développé dans cette thèse vise à prendre un apercu métabolomique de la feuille hôte et de sa communauté fongique endophyte et à étudier les interactions moléculaires et les interactions chimiques réciproques entre eux. Nous avons ensuite exploité, lorsque c'était possible, les métabolites spécialisés de la communauté fongique pour leur bioactivité antivirulentes ou anticancéreuses. Il a permis plus particulièrement de caractériser le métabolome de la communauté fongique et celui de la plante hôte. L’analyse poussée de ces données révèle des interactions jamais analysées auparavant de manière aussi détaillée et non ciblée au niveau d'un modèle hôte-feuille. Notre stratégie a également conduit à l'identification de 24 nouveaux métabolites secondaires d'une souche de Lasiodiplodia venezuelensis. Certains d'entre eux ont montré une activité intéressante d'inhibition de quorum sensing, avec un effet sur les gènes régulés par le quorum sensing et certains facteurs de virulence, et un composé mineur a montré une inhibition significative et spécifique de l'activité Wnt et de la prolifération de lignées cellulaires de cancer du sein triple négatif.It is now established that plants are not distinct entities, but have evolved in association with highly complex and diverse microbial assemblages. These plant microbial symbiont communities (microbiota) and their collective genetic material constitute the plant microbiome, thought to play key roles in plant ecology and physiology, including plant fitness (growth and survival). Our interest is turned towards some of the fungal inhabitants of the microbiome: endophytic fungi. Endophytes colonize the healthy internal tissues of plants in an asymptomatic way and have been extensively studied in the last decades for their ability to produce bioactive molecules as part of a defense response against pathogens for the host or for the fungi themselves. In this thesis, carried out in the scope of the Franco-Swiss project SECIL (Study of Endophyte Communities In a Leaf, SECIL_ANR), we aimed to study microbial crosstalk in plant leaf fungal endophytes at the chemical and bioactive level, searching for biologically relevant metabolites. The typical palm tree from the primary forest in French Guiana, Astrocaryum sciophilum (Miq.) Pulle was chosen for deep investigations as a host model, due to its remarkable long-life cycle and the diversity of endophytic communities found in its leaves. Its long maturation age suggests that this palm might maintain a stable association with microbial communities over a substantial period of time. This implies that potential endophytes are able to survive and resist the environment of the plant, likely by developing antipathogens and cytotoxic metabolites involved in the longevity of the shoots and leaves of the palm. Moreover, the highly competitive rainforest ecosystem makes the selection pressure intense, probably prompting the microbes associated with this tropical host to produce interesting chemical entities. We also hypothesized that reciprocal chemical interactions occur between plant and fungi, via specialised chemistry potentially expressed in host tissues. We thus developed a strategy to study at a given time, this plant-fungi interaction from a molecular point-of-view, in the most comprehensive way possible, along with the bioactive potential which results from the interaction. The workflow we developed in this thesis aims to take a metabolomic snapshot of the host leaf and its fungal endophytic community and study molecular interplay and reciprocal chemical interactions between them. We further exploited when possible specialised metabolites of the fungal community for their bioactivity (antivirulence or anticancer). It allowed more particularly to characterise the metabolome of the fungal community and that of the host plant. The in-depth analysis of these data revealed interactions never before analysed in such a detailed and untargeted manner at the level of a host-leaf model. Our workflow also led to the identification of 24 new secondary metabolites of a Lasiodiplodia venezuelensis strain. Some of which showed interesting quorum sensing inhibition activity, with effect on the quorum sensing- regulated genes and some virulence factors, a minor compound showed significant and specific inhibition of Wnt activity and of the proliferation of Triple Negative Breast Cancer cell lines.</p

Using Porcine Jejunum Ex Vivo to Study Absorption and Biotransformation of Natural Products in Plant Extracts: Pueraria lobata as a Case Study

Herbal preparations (HPs) used in folk medicine are complex mixtures of natural products (NPs). Their efficacy in vivo after ingestion depends on the uptake of the active ingredient, and, in some cases, their metabolites, in the gastrointestinal tract. Thus, correlating bioactivities measured in vitro and efficacy in vivo is a challenge. An extract of Pueraria lobata rich in different types of isoflavones was used to evaluate the capacity of viable porcine small intestine ex vivo to elucidate the absorption of HP constituents, and, in some cases, their metabolites. The identification and transport of permeants across the jejunum was monitored by liquid chromatography-mass spectrometry (LC-MS), combining targeted and untargeted metabolite profiling approaches. It was observed that the C-glycoside isoflavones were stable and crossed the intestinal membrane, while various O-glycoside isoflavones were metabolized into their corresponding aglycones, which were then absorbed. These results are consistent with human data, highlighting the potential of using this approach. A thorough investigation of the impact of absorption and biotransformation was obtained without in vivo studies. The combination of qualitative untargeted and quantitative targeted LC-MS methods effectively monitored a large number of NPs and their metabolites, which is essential for research on HPs

Structural Identification of Antibacterial Lipids from Amazonian Palm Tree Endophytes through the Molecular Network Approach

International audienc

Characterization, Diversity, and Structure-Activity Relationship Study of Lipoamino Acids from Pantoea sp. and Synthetic Analogues

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 &micro;g/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

Chemo-diversification of plant extracts using a generic bromination reaction and monitoring by metabolite profiling

A generic procedure for direct bromination of polyphenol in crude plant extracts was developed to generate multiple "unnatural" halogenated natural products for further bioassay evaluation. To better control the halogenation procedure, the bromination was optimized with a flavonoid standard, and the reactions were monitored by high-performance liquid chromatography photometric diode array coupled to the evaporative light scattering detection (ELSD). ELSD detection was successfully used for a relative yield estimation of the compounds obtained. From the halogenation of hesperitin (11), five brominated compounds were obtained. After optimization, the reaction was successfully applied to the methanolic extract of Citrus sinensis peels, a typical waste biomass and also to the methanolic extract of the medicinal plant Curcuma longa. In both cases, the methanolic extracts were profiled by NMR for a rapid estimation of the polyphenol versus primary metabolite content. An enriched secondary metabolites extract was obtained using vacuum liquid chromatography and submitted to bromination. Metabolite profiling performed by ultrahigh purity liquid chromatography time-of-flight high-resolution mass spectrometry revealed the presence of various halogenated products. To isolate these compounds, the reactions were scaled up, and six halogenated analogues were isolated and fully characterized by NMR and high-resolution electrospray ionization mass spectrometry analyses. The antibacterial properties of these compounds were evaluated using in vitro bioassays against multiresistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Some of the halogenated derivatives obtained presented moderate antibacterial properties

Identification and dereplication of endophytic Colletotrichum strains by MALDI TOF mass spectrometry and molecular networking

International audienceThe chemical diversity of biologically active fungal strains from 42 Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software. From five Colletotrichum strains producing cytotoxic specialized metabolites, we predicted the occurrence of peptide and cytochalasin analogues in four of them by MN, including a similar ion clusters in the MN algorithm provided by MetGem software. Chemoinformatics predictions were fully confirmed after isolation of three pentacyclopeptides (cyclo(Phe-Leu-Leu-Leu-Val), cyclo(Phe-Leu-Leu-Leu-Leu) and cyclo(Phe-Leu-Leu-Leu-Ile)) and two cytochalasins (cytochalasin C and cytochalasin D) exhibiting cytotoxicity at the micromolar concentration. Finally, the chemical study of the last active cytotoxic strain BSNB-0583 led to the isolation of four colletamides bearing an identical decadienamide chain