Etude de l'activité antivirale d'extraits d'Euphorbia de Corse : recherche de nouveaux diterpènes d'intérêt biologique

Chikungunya fever is caused by an arthropod-borne virus that is associated with massive epidemics and severe morbidity (virus-induced arthralgia, fever, myalgia and rashes). Worldwide expansion of the mosquito vectors, such as Aedes albopictus ("Tiger moquito) is responsible for the spread of Chikungunya virus (CHIKV) throughout the world. A. albopictus has spread throughout Mediterranean areas, which could lead to epidemics outbreaks. Currently, no antiviral drugs or vaccines are available for the treatment or prevention of CHIKV infection. Since ten years, however, recent results showed that diterpene esters from Trignostemon (Euphorbiaceae) possess inhibiting activity of CHIKV replication.With the objective to discover new compounds with antiviral activities, 45 extracts from various plant parts of 11 Euphorbiaceae species native to Corsica were evaluated for selective inhibition of CHIKV replication. In collaboration with Dr. Leyssen (KU Leuven, Belgium), several extracts made from 10 Euphorbia species exhibited significant and selective anti-CHIKV activity in a virus-cell-based assay. The antiviral activities of 29 commercially available phorboïds were studied. Some phorboïds were potent inhibitors of CHIKV and human immunodeficiency virus (VIH) replication. Results allowed drawing new structure-activity relationships, which supported the hypothesis that PKC may be an important target in CHIKV replication. In order to confirm or infirm the presence of phorboïds with anti-CHIKV activity in Euphorbia extracts, a liquid chromatography (LC) coupled to linear ion trap mass spectrometry (MSn) method was developed using standard compounds. Application of this methodology indicated that none anti-CHIKV phorboïds was present in Euphorbia extracts. A second LC-MSn procedure was developed to profile untargeted phorboïdes. Results suggested that numerous other diterpene esters were present in the Euphorbia extracts. The species Euphorbia amygdaloides ssp. semiperfoliata was selected to perform a bioassay-guided purification procedure, which led to the isolation and identification of 14 jatrophane esters, including eight new components. Among them, antiviral evaluation indicated that one jatrophane ester was possessing anti-CHIKV and anti-HIV activities. Furthermore, the structure of an atypical jatrophane ester derivative, jatrohemiketal, was determined unambiguously through an original strategy combining NMR spectroscopy and molecular modeling. Finally, an original tandem mass spectrometry (MS/MS)-targeted supercritical fluid chromatography (SFC) method was developed and used to study bioactive fractions of E. amygdaloïdes ssp. semiperfoliata. The MS/MS data were analyzed by molecular networking. Thanks to this approach, four 4-deoxyphorbol esters and two new jatrophane esters were targeted, isolated and identified. Collaboration with the group of Pr. Alcami (CNM, Espagne) was started to explore the anti-HIV properties of the isolated diterpene esters. Thus, one compound derived from 4-deoxyphorbol esters proved to possess a potent inhibiting activity of HIV-1 replication (IC50 = 8 nM, and selectivity index > 6250). The investigation of the mechanism of this component indicated that it acted like prostratin, but with antiviral effect more than 28-fold. Furthermore, the evaluation of the anti-CHIKV activity indicated that another 4-deoxyphorbol derivative was one of the strongest inhibitor of CHIKV replication isolated up to date (EC50 = 0.34 ± 0.12 µM and selectivity index > 638).Le chikungunya est une maladie transmise par des moustiques du genre Aedes (dont A. albopictus, dit "moustique tigre"). Cette maladie provoque d'intenses fièvres et des douleurs articulaires chroniques fortement invalidantes. Les moustiques potentiellement vecteurs du virus du chikungunya (CHIKV) sont des espèces invasives qui, à la faveur du réchauffement climatique, se sont récemment implantés dans plusieurs régions du monde, dont la région méditerranéenne. Sa présence constitue un terreau favorable à la survenue d'épidémie. A l'heure actuelle, il n'existe ni vaccin, ni traitement médicamenteux efficace. Toutefois, des articles scientifiques ont récemment rapporté que des esters de diterpène isolés du genre Trigonostemon (Euphorbiaceae), avaient une activité inhibitrice de la réplication du CHIKV.Dans le cadre de ces travaux de thèse, des extraits de plantes du genre Euphorbia de Corse ont été étudiés dans le but d'isoler de nouvelles molécules douées d'activité antivirale sur la réplication du CHIKV. En collaboration avec le Dr. P. Leyssen (KU Leuven, Belgique), l'évaluation de l'activité anti-CHIKV de 45 extraits, obtenus à partir de 11 Euphorbiaceae de Corse, a permis de mettre en évidence la forte activité inhibitrice et sélective de des extraits d'espèces du genre Euphorbia in cellulo. L'activité antivirale d'une série de 27 diterpènes de type phorboïde, disponibles commercialement, a également été étudiée. Les résultats ont montré que certains dérivés avaient une forte activité inhibitrice de la réplication du CHIKV, mais aussi sur celle du virus de l'immunodéficience humaine (VIH). Ces études ont permis d'une part, de déduire des relations structure-activité inédites et d'autre part, de soutenir l'hypothèse d'un mécanisme d'action anti-CHIKV impliquant la modulation des protéines kinases C (PKCs) par les phorboïdes. Dans le but de confirmer ou d'infirmer la présence des phorboïdes dans les extraits d'Euphorbia, une première méthode utilisant la chromatographie liquide (LC) haute performance couplée à un spectromètre de masse à trappe d'ions (MSn), a été développée à partir des composés standards. L'application de cette méthodologie a révélé qu'aucun des phorboïdes ciblés n'était présent dans les extraits d'Euphorbia. Ainsi, une seconde procédure LC-MSn a été mise en œuvre afin de détecter - de manière non ciblée - différents types d’esters diterpéniques. L'utilisation de cette approche a révélé que de nombreux diterpènes, non-apparentés aux phorboïdes, étaient présents dans les extraits. Un extrait de l'espèce Euphorbia amygdaloides subsp. semiperfoliata a été sélectionné pour réaliser un fractionnement bio-guidé, aboutissant à l'isolement et l'identification de 14 esters de jatrophane, dont neuf nouveaux composés. Parmi eux, l'un s'est avéré inhiber la réplication du CHIKV et du VIH. Par ailleurs, la structure d'un ester de jatrophane atypique, le jatrohémicétal, a été élucidée grâce à une approche originale combinant modélisation moléculaire et spectroscopie par résonance magnétique nucléaire (RMN). Enfin, une nouvelle procédure de purification ciblée par spectrométrie de masse tandem (MS/MS) en chromatographie en phase fluide supercritique (SFC) a été développée et appliquée sur des fractions bioactives d'E. amygdaloïdes subsp. semiperfoliata. L’interprétation des données MS/MS s’est appuyée sur la génération de réseaux moléculaires. Par cette méthodologie, quatre nouvelles molécules ont pu être détectées, purifiées et identifiées ; il s’agit de deux nouveaux esters de jatrophane et de quatre esters dérivés du 4-déoxyphorbol. L'activité anti-VIH des constituants isolés a également pu être explorée dans le cadre d’une collaboration avec l'équipe du Pr. Alcami (CNM, Espagne). Ainsi, l’un des esters de 4-deoxyphorbol s’est révélé être doué d'un exceptionnel pouvoir inhibiteur de la réplication du VIH-1 (IC50 = 8 nM et index de sélectivité > 6250). Son mécanisme d'action semble s'apparenter à celui de la prostratine (molécule antivirale de référence) mais avec des propriétés antivirales environ 28 fois supérieur. Un deuxième ester de 4-déoxyphorbol s’est avéré être un des plus puissants inhibiteurs du CHIKV isolé à ce jour (EC50 = 0,34 ± 0,12 µM and SI > 638). Mots clés : Euphorbia, activité antivirale, chikungunya, diterpène, spectrométrie de mass

MEMO: mass spectrometry-based sample vectorization to explore chemodiverse datasets

In natural products research, chemodiverse extracts coming from multiple organisms are explored for novel bioactive molecules, sometimes over extended periods. Samples are usually analyzed by liquid chromatography coupled with fragmentation mass spectrometry to acquire informative mass spectral ensembles. Such data is then exploited to establish relationships among analytes or samples (e.g., via molecular networking) and annotate metabolites. However, the comparison of samples profiled in different batches is challenging with current metabolomics methods since the experimental variation-changes in chromatographical or mass spectrometric conditions - hinders the direct comparison of the profiled samples. Here we introduce MEMO-MS2 BasEd SaMple VectOrization-a method allowing to cluster large amounts of chemodiverse samples based on their LC-MS/MS profiles in a retention time agnostic manner. This method is particularly suited for heterogeneous and chemodiverse sample sets. MEMO demonstrated similar clustering performance as state-of-the-art metrics considering fragmentation spectra. More importantly, such performance was achieved without the requirement of a prior feature alignment step and in a significantly shorter computational time. MEMO thus allows the comparison of vast ensembles of samples, even when analyzed over long periods of time, and on different chromatographic or mass spectrometry platforms. This new addition to the computational metabolomics toolbox should drastically expand the scope of large-scale comparative analysis

4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4β-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4β-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4β-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4β-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4β-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4β-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4β-dPE A. Furthermore, NF-κb transcription factor is involved in 4β-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4β-dPE A in combination with other LRAs. When 4β-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4β-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4β-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.This work was supported by Ministry of Education of the Peruvian government (PRONABEC), the Universidad Complutense de Madrid (UCM-Santander PR87/19), the Instituto de Salud Carlos III (ISCIII-FIS PI16CIII/00034) and the Spanish AIDS Research Network RD12/0017/0015 that is included in the Spanish I D I Plan and is co-financed by ISCIII-Subdirección General de Evaluación and European Funding for Regional Development (FEDER). This work has also benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ANR- 10-LABX-25-01).S

Africa in the click stream: audience metrics and foreign correspondents in africa

Digital technologies have transformed the relationship between news outlets, journalists and their audiences. Notably, editors can now monitor their websites and discern the exact news preferences of their readers. Research suggests that some editors are using this data to help them produce more popular, ‘click friendly’ content. To date, research on this phenomenon has focused on journalists working within newsrooms. This article adds to the literature by exploring the relationship of foreign correspondents in Africa with their audiences, and asks whether readership metrics are influencing the journalists’ selection and development of news stories. Drawing on 67 interviews with foreign correspondents in East and West Africa, the article identifies three different approaches to audience metrics: correspondents who are 1) data-driven; 2) data informed; and 3) data denialists. The article discusses the implications of these approaches for the media image of Africa that is distributed around the globe

Open Access Repository-Scale Propagated Nearest Neighbor Suspect Spectral Library for Untargeted Metabolomics

Abstract Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of public MS/MS spectra. Annotations were propagated based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer’s brain phenotype. The nearest neighbor suspect spectral library is openly available through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data

Massive multi-informative molecular networks : a map to navigate through the chemical space of bioactive Euphorbiaceae

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