Study of drug/receptor interaction by mass spectrometry : development and validation of new tests of molecular screening

La découverte de nouveaux médicaments par le criblage biomoléculaire est au centre de la recherche pharmaceutique actuelle. La spectrométrie de masse, en tant que technique d’analyse fiable, reproductible, sensible, spécifique, compatible avec de nombreux types d’échantillons et permettant un débit d’analyse conséquent, trouve ainsi sa place dans les stratégies de recherche et développement de nouveaux médicaments. Le but de ce travail était de mettre en place et de valider une stratégie originale, impliquant la désorption/ionisation laser assistée par matrice couplée à la spectrométrie de masse (MALDI-MS) comme technique de détection, en vue du criblage de la liaison de composés à des cibles moléculaires applicable directement à des extraits de plantes. Le protocole que nous avons développé s’articule en trois étapes successives qui sont l’incubation des molécules à tester avec la cible moléculaire choisie (la tubuline ou la DHFR), l’élimination des composés non liés et enfin l’analyse par MALDI-TOFMS des composés liés. Notre démarche a fait l’objet d’une démarche de validation et les résultats pouvant être obtenus ont été discutés. Le débit pouvant être évalué à 60 échantillons en 1h50 à 3h30 soit de 18 à 32 échantillons à l’heure. Enfin, une démarche innovante nous a permis de prouver que notre approche pouvait être utile également en criblage secondaire. L’application de notre approche à des extraits bruts de plantes (Colchique d’Automne, Pervenche de Madagascar et Thé vert) à permis de mettre en évidence 20 molécules actives se liant à l’une ou l’autre des cibles moléculaires utilisées et d’évaluer l’affinité relative de l’une d’entre ellesDiscovering new drugs by biomolecular screening is a central task of pharmaceutical research. Mass spectrometry, as a reliable, reproducible, sensitive and specific technique, compatible with a wide range of samples and offering an excellent throughput, shows its potential in different strategies of research and development. The aim of this work was to develop and validate a new strategy, involving matrix assisted laser desorption/ionization coupled to time of flight mass spectrometry (MALDI-TOFMS) to screen the ability of different compounds, including plant extracts, to bind to two biological targets (tubulin and DHFR). The protocol is therefore divided into three main steps : an incubation of the compounds to be tested with target, an elimination of all unbound compounds and the MALDI-TOFMS detection of target-bound compounds. Our protocol was validated and the results that can be obtained were discussed. The throughput offered by this technique was evaluated as 60 samples in 1h50 to 3h30, or 18 to 32 samples per hour. Finally, we developed a new approach to perform a secondary screening of active compounds. The protocol was applied to screen crude plants extracts (colchicum autumnale, catharanthus roseus and green tea) and allowed to find 20 tubulin-binding or DHFR-binding molecules, and the relative affinity of one of these was also evaluate

Etude de l'interaction médicament/récepteur par spectrométrie de masse : mise en place et validation de nouveaux protocoles de criblage moléculaire

Discovering new drugs by biomolecular screening is a central task of pharmaceutical research. Mass spectrometry, as a reliable, reproducible, sensitive and specific technique, compatible with a wide range of samples and offering an excellent throughput, shows its potential in different strategies of research and development. The aim of this work was to develop and validate a new strategy, involving matrix assisted laser desorption/ionization coupled to time of flight mass spectrometry (MALDI-TOFMS) to screen the ability of different compounds, including plant extracts, to bind to two biological targets (tubulin and DHFR). The protocol is therefore divided into three main steps : an incubation of the compounds to be tested with target, an elimination of all unbound compounds and the MALDI-TOFMS detection of target-bound compounds. Our protocol was validated and the results that can be obtained were discussed. The throughput offered by this technique was evaluated as 60 samples in 1h50 to 3h30, or 18 to 32 samples per hour. Finally, we developed a new approach to perform a secondary screening of active compounds. The protocol was applied to screen crude plants extracts (colchicum autumnale, catharanthus roseus and green tea) and allowed to find 20 tubulin-binding or DHFR-binding molecules, and the relative affinity of one of these was also evaluatedLa découverte de nouveaux médicaments par le criblage biomoléculaire est au centre de la recherche pharmaceutique actuelle. La spectrométrie de masse, en tant que technique d?analyse fiable, reproductible, sensible, spécifique, compatible avec de nombreux types d?échantillons et permettant un débit d?analyse conséquent, trouve ainsi sa place dans les stratégies de recherche et développement de nouveaux médicaments. Le but de ce travail était de mettre en place et de valider une stratégie originale, impliquant la désorption/ionisation laser assistée par matrice couplée à la spectrométrie de masse (MALDI-MS) comme technique de détection, en vue du criblage de la liaison de composés à des cibles moléculaires applicable directement à des extraits de plantes. Le protocole que nous avons développé s?articule en trois étapes successives qui sont l?incubation des molécules à tester avec la cible moléculaire choisie (la tubuline ou la DHFR), l?élimination des composés non liés et enfin l?analyse par MALDI-TOFMS des composés liés. Notre démarche a fait l?objet d?une démarche de validation et les résultats pouvant être obtenus ont été discutés. Le débit pouvant être évalué à 60 échantillons en 1h50 à 3h30 soit de 18 à 32 échantillons à l?heure. Enfin, une démarche innovante nous a permis de prouver que notre approche pouvait être utile également en criblage secondaire. L?application de notre approche à des extraits bruts de plantes (Colchique d?Automne, Pervenche de Madagascar et Thé vert) à permis de mettre en évidence 20 molécules actives se liant à l?une ou l?autre des cibles moléculaires utilisées et d?évaluer l?affinité relative de l?une d?entre elle

Substances naturelles se fixant sur la tubuline (mise en oeuvre d'un criblage par spectométrie de masse)

La découverte de nouveaux médicaments par le criblage biomoléculaire est au centre de la recherche pharmaceutique actuelle. La spectrométrie de masse, en tant que technique d analyse fiable, reproductible, sensible, spécifique, compatible avec de nombreux types d échantillons et permettant un débit d analyse conséquent, trouve ainsi sa place dans les stratégies de recherche et développement de nouveaux médicaments. Le but de ce travail était de caractériser finement des extraits de plantes à activité anti-tubuline et de mettre en place et de valider une stratégie de criblage originale, impliquant la spectrométrie de masse comme technique de détection. Son potentiel devait être mis en évidence en l appliquant à différents échantillons complexes tels que des extraits de plantes. Le protocole que nous avons développé s articule en trois étapes successives qui sont l incubation des molécules à tester avec la cible moléculaire choisie (la tubuline), l élimination des composés non liés et enfin l analyse par MALDI-TOFMS des composés liés. Notre protocole a fait l objet d une démarche de validation et les résultats pouvant être obtenus ont été discutés. L application de notre approche à des extraits bruts de plantes (Colchique d Automne et Pervenche de Madagascar) à permis de mettre en évidence 19 molécules actives se liant à la tubuline.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

A Rapid and Efficient Method for Isolating High Quality DNA from Leaves of Carnivorous Plants from the Drosera Genus

International audienceDrosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A₂₆₀/A₂₈₀ ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h

A simple SDS-Page protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae)

International audiencePremise of the study - Carnivorous plants have always fascinated scientists because these plants are able to attract, capture and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. Methods - Here, we propose a new method to easily distinguish Nepenthes species based on a 1D SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed between specimens growing in different environmental conditions to ascertain the robustness of this method. Key results - Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. Conclusions - The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph informatio

Plant Milking Technology—An Innovative and Sustainable Process to Produce Highly Active Extracts from Plant Roots

We have used an original technology (Plant Milking Technology) based on aeroponic cultivation of plants associated with the gentle recovery of active ingredients from roots. Extraction of bioactive molecules was achieved by soaking the roots, still attached to the living plants, into a nontoxic solvent for a 2 h period. This nondestructive recovery process allows using the same root biomass for successive harvesting dates, in a recyclable way. We have applied this technology to Morus alba L. (mulberry tree), an emblematic tree of the Traditional Chinese Medicine (TCM). Trees were aeroponically grown in large-scale devices (100 m2) and were submitted to nitrogen deprivation to increase the content in active molecules (prenylated flavonoids). The Plant Milking technology applied to Morus alba L. allowed to produce an extract enriched in prenylated compounds (18-fold increase when compared to commercial root extract). Prenylated flavonoids (moracenin A and B, kuwanon C, wittiorumin F, morusin) presented a high affinity for the aged-associated collagenase enzyme, which was confirmed by activity inhibition. In accordance, M. alba extract presents efficient properties to regulate the skin matrisome, which is critical during skin aging. The benefits have been especially confirmed in vivo on wrinkle reduction, in a clinical study that involved aged women. Plant Milking technology is an optimal solution to produce active ingredients from plant roots, including trees, that meet both customer expectations around sustainability, as well as the need for an efficient production system for biotechnologists

EURECA – setting the scene for scintillators

voir fichier complet des proceedingsInternational audienceEURECA (European Underground Rare Event Calorimeter Array) will be an astro-particle physics facility aiming to directly detect galactic dark matter. TheLaboratoire Souterrain de Modane has been selected as host laboratory. TheEURECA collaboration concentrates effort on cryogenic detector research inEurope into a single facility by bringing together colleagues from CRESST,EDELWEISS, ROSEBUD and additional new member institutes. EURECA will use atarget mass of up to one ton for exploring WIMP-nucleon scalar scattering crosssections in the region of 10 −9 – 10 −10 picobarn. A major advantage of EURECAis the planned use of more than just one target material (multi targetexperiment for WIMP identification)