Biodereplication of antiplasmodial extracts: application of the amazonian medicinal plant piper coruscans kunth

Improved methodological tools to hasten antimalarial drug discovery remain of interest, especially when considering natural products as a source of drug candidates. We propose a biodereplication method combining the classical dereplication approach with the early detection of potential antiplasmodial compounds in crude extracts. Heme binding is used as a surrogate of the antiplasmodial activity and is monitored by mass spectrometry in a biomimetic assay. Molecular networking and automated annotation of targeted mass through data mining were followed by mass-guided compound isolation by taking advantage of the versatility and finely tunable selectivity offered by centrifugal partition chromatography. This biodereplication workflow was applied to an ethanolic extract of the Amazonian medicinal plant Piper coruscans Kunth (Piperaceae) showing an IC50 of 1.36 ug/mL on the 3D7 Plasmodium falciparum strain. It resulted in the isolation of twelve compounds designated as potential antiplasmodial compounds by the biodereplication workflow. Two chalcones, aurentiacin (1) and cardamonin (3), with IC50 values of 2.25 and 5.5 uM, respectively, can be considered to bear the antiplasmodial activity of the extract, with the latter not relying on a heme-binding mechanism. This biodereplication method constitutes a rapid, efficient, and robust technique to identify potential antimalarial compounds in complex extracts such as plant extracts

Caractérisation de toxines animales innovantes pour l'étude fonctionnelle des récepteurs aux angiotensines

At the pathophysiological level, the renin-angiotensin system (RAS) is one of the systems responsible for the regulation of cardiovascular function. The main mediator of the RAS is angiotensin II (AngII), which targets the angiotensin type 1 (AT1) receptor, which, when activated, is responsible for, among other things, an increase in blood pressure. There are other effectors of the RAS that are thought to have opposite effects to those of the AngII/AT1 axis. This is the case in particular for the angiotensin 1-7 or alamandine peptides which target the Mas and MrgD receptors respectively, or the AT2 receptor, which once activated by AngII leads to cardiobeneficial effects. These angiotensin receptors are G protein-coupled receptors (GPCRs) with multiple intracellular signalling pathways that are still poorly studied and whose biological roles are not fully understood. The idea is therefore to better understand the signal transduction mechanisms involving these angiotensin receptors through the discovery of new tools allowing their functional study. To enable the study of these receptors, we need to find novel ligands that bind to the receptor in a specific and selective manner. One promising and understudied source of ligands that would meet these criteria is toxins from animal venoms. These venoms constitute a vast library of peptidic toxins with specific pharmacological properties, high affinity and selectivity for their targets in their prey. There are few toxins targeting GPCRs, and to date none identified as targeting angiotensin receptors.We therefore screened a library of 900 synthetic toxins on the AT2 receptor. 3 toxins were identified with affinities in the µM range. These toxins were produced by solid phase peptide synthesis and underwent an oxidation step to form the disulphide bridges necessary to obtain the final functional form of the toxin. These toxins were then characterised by binding studies on AT2 and then AT1 receptors. One toxin, A-CTX-cMila, has a 100-fold higher affinity for AT1 than for AT2 (Ki of 300 nM). It is a cyclic toxin of 7 residues from Conus miliaris. Pharmacological characterisation focused on this toxin and on AT1 receptor. Using FRET second messenger assays and BRET biosensors, we showed that the toxin is a competitive antagonist of the AT1 receptor. It blocks the Gq, Gi, Go pathways, as well as β-arrestin recruitment and ERK1/2 activation.Au niveau physiopathologique, le système rénine-angiotensine (SRA) est un des systèmes responsables de la régulation de la fonction cardiovasculaire. Le principal médiateur du SRA est l'angiotensine II (AngII) ayant pour cible le récepteur à l'angiotensine de type 1 (AT1), qui, une fois activé, est responsable entre autres d'une augmentation de la pression artérielle. Il existe d'autres effecteurs du SRA qui auraient des effets opposés à ceux de l'axe AngII/AT1. C'est le cas notamment des peptides angiotensine 1-7 ou alamandine qui cibleraient respectivement les récepteurs Mas et MrgD, ou encore du récepteur AT2, qui une fois activé par AngII entraine des effets cardiobénéfiques. Ces récepteurs aux angiotensines sont des récepteurs couplés aux protéines G (RCPG), aux voies de signalisation intracellulaires multiples qui sont encore peu étudiés et dont les rôles biologiques ne sont pas parfaitement élucidés. L'idée est donc de mieux comprendre quels sont les mécanismes de transduction du signal impliquant ces récepteurs aux angiotensines par la découverte de nouveaux outils permettant leur étude fonctionnelle. Pour permettre l'étude de ces récepteurs, nous devons trouver des ligands innovants qui se lient au récepteur, de façon spécifique et sélective. Il existe une source de ligands prometteurs encore sous-étudiée qui répondrait à ces critères : les toxines issues de venins d'animaux. Ces venins constituent une vaste bibliothèque de toxines peptidiques aux propriétés pharmacologiques particulières, avec une affinité et une sélectivité élevées pour leurs cibles chez leurs proies. Il existe peu de toxines ciblant les RCPG, et à ce jour, aucune n'est identifiée comme ciblant les récepteurs aux angiotensines.Nous avons donc criblé une banque de 900 toxines synthétiques sur le récepteur AT2. 3 toxines ont été mises en évidence avec des affinités de l'ordre du µM. Ces toxines ont été produites par synthèse peptidique sur phase solide et ont subi une étape d'oxydation pour former les ponts disulfure nécessaires à l'obtention de la forme finale fonctionnelle de la toxine. Ces toxines ont ensuite été caractérisées par des études de liaison sur les récepteurs AT2 puis AT1. Une toxine, A-CTX-cMila, présente une affinité 100 fois plus intéressante pour AT1 que pour AT2 (Ki de 300 nM). C'est une toxine cyclique de 7 résidus issue de Conus miliaris. La caractérisation pharmacologique s'est concentrée sur cette toxine et sur le récepteur AT1. Par des méthodes de dosages de seconds messagers par FRET et par utilisation de biosenseurs de BRET, nous avons montré que la toxine est un antagoniste compétitif du récepteur AT1. Elle bloque les voies Gq, Gi, Go, ainsi que le recrutement de la β-arrestine et l'activation de ERK1/2

Caractérisation de toxines animales innovantes pour l'étude fonctionnelle des récepteurs aux angiotensines

At the pathophysiological level, the renin-angiotensin system (RAS) is one of the systems responsible for the regulation of cardiovascular function. The main mediator of the RAS is angiotensin II (AngII), which targets the angiotensin type 1 (AT1) receptor, which, when activated, is responsible for, among other things, an increase in blood pressure. There are other effectors of the RAS that are thought to have opposite effects to those of the AngII/AT1 axis. This is the case in particular for the angiotensin 1-7 or alamandine peptides which target the Mas and MrgD receptors respectively, or the AT2 receptor, which once activated by AngII leads to cardiobeneficial effects. These angiotensin receptors are G protein-coupled receptors (GPCRs) with multiple intracellular signalling pathways that are still poorly studied and whose biological roles are not fully understood. The idea is therefore to better understand the signal transduction mechanisms involving these angiotensin receptors through the discovery of new tools allowing their functional study. To enable the study of these receptors, we need to find novel ligands that bind to the receptor in a specific and selective manner. One promising and understudied source of ligands that would meet these criteria is toxins from animal venoms. These venoms constitute a vast library of peptidic toxins with specific pharmacological properties, high affinity and selectivity for their targets in their prey. There are few toxins targeting GPCRs, and to date none identified as targeting angiotensin receptors.We therefore screened a library of 900 synthetic toxins on the AT2 receptor. 3 toxins were identified with affinities in the µM range. These toxins were produced by solid phase peptide synthesis and underwent an oxidation step to form the disulphide bridges necessary to obtain the final functional form of the toxin. These toxins were then characterised by binding studies on AT2 and then AT1 receptors. One toxin, A-CTX-cMila, has a 100-fold higher affinity for AT1 than for AT2 (Ki of 300 nM). It is a cyclic toxin of 7 residues from Conus miliaris. Pharmacological characterisation focused on this toxin and on AT1 receptor. Using FRET second messenger assays and BRET biosensors, we showed that the toxin is a competitive antagonist of the AT1 receptor. It blocks the Gq, Gi, Go pathways, as well as β-arrestin recruitment and ERK1/2 activation.Au niveau physiopathologique, le système rénine-angiotensine (SRA) est un des systèmes responsables de la régulation de la fonction cardiovasculaire. Le principal médiateur du SRA est l'angiotensine II (AngII) ayant pour cible le récepteur à l'angiotensine de type 1 (AT1), qui, une fois activé, est responsable entre autres d'une augmentation de la pression artérielle. Il existe d'autres effecteurs du SRA qui auraient des effets opposés à ceux de l'axe AngII/AT1. C'est le cas notamment des peptides angiotensine 1-7 ou alamandine qui cibleraient respectivement les récepteurs Mas et MrgD, ou encore du récepteur AT2, qui une fois activé par AngII entraine des effets cardiobénéfiques. Ces récepteurs aux angiotensines sont des récepteurs couplés aux protéines G (RCPG), aux voies de signalisation intracellulaires multiples qui sont encore peu étudiés et dont les rôles biologiques ne sont pas parfaitement élucidés. L'idée est donc de mieux comprendre quels sont les mécanismes de transduction du signal impliquant ces récepteurs aux angiotensines par la découverte de nouveaux outils permettant leur étude fonctionnelle. Pour permettre l'étude de ces récepteurs, nous devons trouver des ligands innovants qui se lient au récepteur, de façon spécifique et sélective. Il existe une source de ligands prometteurs encore sous-étudiée qui répondrait à ces critères : les toxines issues de venins d'animaux. Ces venins constituent une vaste bibliothèque de toxines peptidiques aux propriétés pharmacologiques particulières, avec une affinité et une sélectivité élevées pour leurs cibles chez leurs proies. Il existe peu de toxines ciblant les RCPG, et à ce jour, aucune n'est identifiée comme ciblant les récepteurs aux angiotensines.Nous avons donc criblé une banque de 900 toxines synthétiques sur le récepteur AT2. 3 toxines ont été mises en évidence avec des affinités de l'ordre du µM. Ces toxines ont été produites par synthèse peptidique sur phase solide et ont subi une étape d'oxydation pour former les ponts disulfure nécessaires à l'obtention de la forme finale fonctionnelle de la toxine. Ces toxines ont ensuite été caractérisées par des études de liaison sur les récepteurs AT2 puis AT1. Une toxine, A-CTX-cMila, présente une affinité 100 fois plus intéressante pour AT1 que pour AT2 (Ki de 300 nM). C'est une toxine cyclique de 7 résidus issue de Conus miliaris. La caractérisation pharmacologique s'est concentrée sur cette toxine et sur le récepteur AT1. Par des méthodes de dosages de seconds messagers par FRET et par utilisation de biosenseurs de BRET, nous avons montré que la toxine est un antagoniste compétitif du récepteur AT1. Elle bloque les voies Gq, Gi, Go, ainsi que le recrutement de la β-arrestine et l'activation de ERK1/2

Characterization of innovative animal toxins for the functional study of angiotensin receptors

Au niveau physiopathologique, le système rénine-angiotensine (SRA) est un des systèmes responsables de la régulation de la fonction cardiovasculaire. Le principal médiateur du SRA est l'angiotensine II (AngII) ayant pour cible le récepteur à l'angiotensine de type 1 (AT1), qui, une fois activé, est responsable entre autres d'une augmentation de la pression artérielle. Il existe d'autres effecteurs du SRA qui auraient des effets opposés à ceux de l'axe AngII/AT1. C'est le cas notamment des peptides angiotensine 1-7 ou alamandine qui cibleraient respectivement les récepteurs Mas et MrgD, ou encore du récepteur AT2, qui une fois activé par AngII entraine des effets cardiobénéfiques. Ces récepteurs aux angiotensines sont des récepteurs couplés aux protéines G (RCPG), aux voies de signalisation intracellulaires multiples qui sont encore peu étudiés et dont les rôles biologiques ne sont pas parfaitement élucidés. L'idée est donc de mieux comprendre quels sont les mécanismes de transduction du signal impliquant ces récepteurs aux angiotensines par la découverte de nouveaux outils permettant leur étude fonctionnelle. Pour permettre l'étude de ces récepteurs, nous devons trouver des ligands innovants qui se lient au récepteur, de façon spécifique et sélective. Il existe une source de ligands prometteurs encore sous-étudiée qui répondrait à ces critères : les toxines issues de venins d'animaux. Ces venins constituent une vaste bibliothèque de toxines peptidiques aux propriétés pharmacologiques particulières, avec une affinité et une sélectivité élevées pour leurs cibles chez leurs proies. Il existe peu de toxines ciblant les RCPG, et à ce jour, aucune n'est identifiée comme ciblant les récepteurs aux angiotensines.Nous avons donc criblé une banque de 900 toxines synthétiques sur le récepteur AT2. 3 toxines ont été mises en évidence avec des affinités de l'ordre du µM. Ces toxines ont été produites par synthèse peptidique sur phase solide et ont subi une étape d'oxydation pour former les ponts disulfure nécessaires à l'obtention de la forme finale fonctionnelle de la toxine. Ces toxines ont ensuite été caractérisées par des études de liaison sur les récepteurs AT2 puis AT1. Une toxine, A-CTX-cMila, présente une affinité 100 fois plus intéressante pour AT1 que pour AT2 (Ki de 300 nM). C'est une toxine cyclique de 7 résidus issue de Conus miliaris. La caractérisation pharmacologique s'est concentrée sur cette toxine et sur le récepteur AT1. Par des méthodes de dosages de seconds messagers par FRET et par utilisation de biosenseurs de BRET, nous avons montré que la toxine est un antagoniste compétitif du récepteur AT1. Elle bloque les voies Gq, Gi, Go, ainsi que le recrutement de la β-arrestine et l'activation de ERK1/2.At the pathophysiological level, the renin-angiotensin system (RAS) is one of the systems responsible for the regulation of cardiovascular function. The main mediator of the RAS is angiotensin II (AngII), which targets the angiotensin type 1 (AT1) receptor, which, when activated, is responsible for, among other things, an increase in blood pressure. There are other effectors of the RAS that are thought to have opposite effects to those of the AngII/AT1 axis. This is the case in particular for the angiotensin 1-7 or alamandine peptides which target the Mas and MrgD receptors respectively, or the AT2 receptor, which once activated by AngII leads to cardiobeneficial effects. These angiotensin receptors are G protein-coupled receptors (GPCRs) with multiple intracellular signalling pathways that are still poorly studied and whose biological roles are not fully understood. The idea is therefore to better understand the signal transduction mechanisms involving these angiotensin receptors through the discovery of new tools allowing their functional study. To enable the study of these receptors, we need to find novel ligands that bind to the receptor in a specific and selective manner. One promising and understudied source of ligands that would meet these criteria is toxins from animal venoms. These venoms constitute a vast library of peptidic toxins with specific pharmacological properties, high affinity and selectivity for their targets in their prey. There are few toxins targeting GPCRs, and to date none identified as targeting angiotensin receptors.We therefore screened a library of 900 synthetic toxins on the AT2 receptor. 3 toxins were identified with affinities in the µM range. These toxins were produced by solid phase peptide synthesis and underwent an oxidation step to form the disulphide bridges necessary to obtain the final functional form of the toxin. These toxins were then characterised by binding studies on AT2 and then AT1 receptors. One toxin, A-CTX-cMila, has a 100-fold higher affinity for AT1 than for AT2 (Ki of 300 nM). It is a cyclic toxin of 7 residues from Conus miliaris. Pharmacological characterisation focused on this toxin and on AT1 receptor. Using FRET second messenger assays and BRET biosensors, we showed that the toxin is a competitive antagonist of the AT1 receptor. It blocks the Gq, Gi, Go pathways, as well as β-arrestin recruitment and ERK1/2 activation

The Positive Side of the Alzheimer’s Disease Amyloid Cross-Interactions: The Case of the Aβ 1-42 Peptide with Tau, TTR, CysC, and ApoA1

International audienceAlzheimer's disease (AD) represents a progressive amyloidogenic disorder whose advancement is widely recognized to be connected to amyloid-β peptides and Tau aggregation. However, several other processes likely contribute to the development of AD and some of them might be related to protein-protein interactions. Amyloid aggregates usually contain not only single type of amyloid protein, but also other type of proteins and this phenomenon can be rationally explained by the process of protein cross-seeding and co-assembly. Amyloid cross-interaction is ubiquitous in amyloid fibril formation and so a better knowledge of the amyloid interactome could help to further understand the mechanisms of amyloid related diseases. In this review, we discuss about the cross-interactions of amyloid-β peptides, and in particular Aβ1-42, with other amyloids, which have been presented either as integrated part of Aβ neurotoxicity process (such as Tau) or conversely with a preventive role in AD pathogenesis by directly binding to Aβ (such as transthyretin, cystatin C and apolipoprotein A1). Particularly, we will focus on all the possible therapeutic strategies aiming to rescue the Aβ toxicity by taking inspiration from these protein-protein interactions

Structural and Functional Diversity of Animal Toxins Interacting With GPCRs

International audiencePeptide toxins from venoms have undergone a long evolutionary process allowing host defense or prey capture and making them highly selective and potent for their target. This has resulted in the emergence of a large panel of toxins from a wide diversity of species, with varied structures and multiple associated biological functions. In this way, animal toxins constitute an inexhaustible reservoir of druggable molecules due to their interesting pharmacological properties. One of the most interesting classes of therapeutic targets is the G-protein coupled receptors (GPCRs). GPCRs represent the largest family of membrane receptors in mammals with approximately 800 different members. They are involved in almost all biological functions and are the target of almost 30% of drugs currently on the market. Given the interest of GPCRs in the therapeutic field, the study of toxins that can interact with and modulate their activity with the purpose of drug development is of particular importance. The present review focuses on toxins targeting GPCRs, including peptide-interacting receptors or aminergic receptors, with a particular focus on structural aspects and, when relevant, on potential medical applications. The toxins described here exhibit a great diversity in size, from 10 to 80 amino acids long, in disulfide bridges, from none to five, and belong to a large panel of structural scaffolds. Particular toxin structures developed here include inhibitory cystine knot (ICK), three-finger fold, and Kunitz-type toxins. We summarize current knowledge on the structural and functional diversity of toxins interacting with GPCRs, concerning first the agonist-mimicking toxins that act as endogenous agonists targeting the corresponding receptor, and second the toxins that differ structurally from natural agonists and which display agonist, antagonist, or allosteric properties

Characterization of the First Animal Toxin Acting as an Antagonist on AT1 Receptor

The renin-angiotensin system (RAS) is one of the main regulatory systems of cardiovascular homeostasis. It is mainly composed of angiotensin-converting enzyme (ACE) and angiotensin II receptors AT1 and AT2. ACE and AT1 are targets of choice for the treatment of hypertension, whereas the AT2 receptor is still not exploited due to the lack of knowledge of its physiological properties. Peptide toxins from venoms display multiple biological functions associated with varied chemical and structural properties. If Brazilian viper toxins have been described to inhibit ACE, no animal toxin is known to act on AT1/AT2 receptors. We screened a library of toxins on angiotensin II receptors with a radioligand competition binding assay. Functional characterization of the selected toxin was conducted by measuring second messenger production, G-protein activation and β-arrestin 2 recruitment using bioluminescence resonance energy transfer (BRET) based biosensors. We identified one original toxin, A-CTX-cMila, which is a 7-residues cyclic peptide from Conus miliaris with no homology sequence with known angiotensin peptides nor identified toxins, displaying a 100-fold selectivity for AT1 over AT2. This toxin shows a competitive antagonism mode of action on AT1, blocking Gαq, Gαi3, GαoA, β-arrestin 2 pathways and ERK1/2 activation. These results describe the first animal toxin active on angiotensin II receptors

Characterization of the First Animal Toxin Acting as an Antagonist on AT1 Receptor

The renin-angiotensin system (RAS) is one of the main regulatory systems of cardiovascular homeostasis. It is mainly composed of angiotensin-converting enzyme (ACE) and angiotensin II receptors AT1 and AT2. ACE and AT1 are targets of choice for the treatment of hypertension, whereas the AT2 receptor is still not exploited due to the lack of knowledge of its physiological properties. Peptide toxins from venoms display multiple biological functions associated with varied chemical and structural properties. If Brazilian viper toxins have been described to inhibit ACE, no animal toxin is known to act on AT1/AT2 receptors. We screened a library of toxins on angiotensin II receptors with a radioligand competition binding assay. Functional characterization of the selected toxin was conducted by measuring second messenger production, G-protein activation and β-arrestin 2 recruitment using bioluminescence resonance energy transfer (BRET) based biosensors. We identified one original toxin, A-CTX-cMila, which is a 7-residues cyclic peptide from Conus miliaris with no homology sequence with known angiotensin peptides nor identified toxins, displaying a 100-fold selectivity for AT1 over AT2. This toxin shows a competitive antagonism mode of action on AT1, blocking Gαq, Gαi3, GαoA, β-arrestin 2 pathways and ERK1/2 activation. These results describe the first animal toxin active on angiotensin II receptors

Biodereplication of Antiplasmodial Extracts: Application of the Amazonian Medicinal Plant Piper coruscans Kunth

International audienceImproved methodological tools to hasten antimalarial drug discovery remain of interest, especially when considering natural products as a source of drug candidates. We propose a biodereplication method combining the classical dereplication approach with the early detection of potential antiplasmodial compounds in crude extracts. Heme binding is used as a surrogate of the antiplasmodial activity and is monitored by mass spectrometry in a biomimetic assay. Molecular networking and automated annotation of targeted mass through data mining were followed by mass-guided compound isolation by taking advantage of the versatility and finely tunable selectivity offered by centrifugal partition chromatography. This biodereplication workflow was applied to an ethanolic extract of the Amazonian medicinal plant Piper coruscans Kunth (Piperaceae) showing an IC50 of 1.36 µg/mL on the 3D7 Plasmodium falciparum strain. It resulted in the isolation of twelve compounds designated as potential antiplasmodial compounds by the biodereplication workflow. Two chalcones, aurentiacin (1) and cardamonin (3), with IC50 values of 2.25 and 5.5 µM, respectively, can be considered to bear the antiplasmodial activity of the extract, with the latter not relying on a heme-binding mechanism. This biodereplication method constitutes a rapid, efficient, and robust technique to identify potential antimalarial compounds in complex extracts such as plant extracts

Metabolomic approach of the antiprotozoal activity of medicinal Piper species used in Peruvian Amazon

International audienceEthnopharmacological relevance : In the Peruvian Amazon as in the tropical countries of South America, the use of medicinal Piper species (cordoncillos) is common practice, particularly against symptoms of infection by protozoal parasites. However, there is few documented information about the practical aspects of their use and few scientific validation. The starting point of this work was a set of interviews of people living in six rural communities from the Peruvian Amazon (Alto Amazonas Province) about their uses of plants from Piper genus: one community of Amerindian native people (Shawi community) and five communities of mestizos. Infections caused by parasitic protozoa take a huge toll on public health in the Amazonian communities, who partly fight it using traditional remedies. Validation of these traditional practices contributes to public health care efficiency and may help to identify new antiprotozoal compounds.Aims of study : To record and validate the use of medicinal Piper species by rural people of Alto Amazonas Province (Peru) and annotate active compounds using a correlation study and a data mining approach.Materials and methods : Rural communities were interviewed about traditional medication against parasite infections with medicinal Piper species. Ethnopharmacological surveys were undertaken in five mestizo villages, namely: Nueva Arica, Shucushuyacu, Parinari, Lagunas and Esperanza, and one Shawi community (Balsapuerto village). All communities belong to the Alto Amazonas Province (Loreto region, Peru). Seventeen Piper species were collected according to their traditional use for the treatment of parasitic diseases, 35 extracts (leaves or leaves and stems) were tested in vitro on P. falciparum (3D7 chloroquine-sensitive strain and W2 chloroquine-resistant strain), Leishmania donovani LV9 strain and Trypanosoma brucei gambiense. Assessments were performed on HUVEC cells and RAW 264.7 macrophages. The annotation of active compounds was realized by metabolomic analysis and molecular networking approach.Results : Nine extracts were active (IC50 ≤ 10 μg/mL) on 3D7 P. falciparum and only one on W2 P. falciparum, six on L. donovani (axenic and intramacrophagic amastigotes) and seven on Trypanosoma brucei gambiense. Only one extract was active on all three parasites (P. lineatum). After metabolomic analyses and annotation of compounds active on Leishmania, P. strigosum and P. pseudoarboreum were considered as potential sources of leishmanicidal compounds.Conclusions : This ethnopharmacological study and the associated in vitro bioassays corroborated the relevance of use of Piper species in the Amazonian traditional medicine, especially in Peru. A series of Piper species with few previously available phytochemical data have good antiprotozoal activity and could be a starting point for subsequent promising work. Metabolomic approach appears to be a smart, quick but still limited methodology to identify compounds with high probability of biological activity