Gilles Ferry - Academia.edu (original) (raw)

Papers by Gilles Ferry

Quantitative Structure Activity Relationships, Dec 1, 2001

Analytical Biochemistry, 2019

 VHH are small antibody-like proteins. They are entering the therapeutic arsenal.  VHH purity, ... more  VHH are small antibody-like proteins. They are entering the therapeutic arsenal.  VHH purity, homogeneity, structure and affinity towards their targets are described.  An analytical workflow to qualify those proteins is presented to validate the molecules.

La surexpression des metalloproteases, et plus particulierement des gelatinases, est decrite dans... more La surexpression des metalloproteases, et plus particulierement des gelatinases, est decrite dans un grand nombre de pathologies ou la degradation de la matrice extracellulaire est un element determinant comme dans la progression tumorale et dans la pathologie d'insuffisance respiratoire aigue (ards). Cependant, la presence ou la surexpression de ces gelatinases ne prouve pas qu'elles participent effectivement a ces pathologies. De plus, les techniques conventionnelles utilisees ne permettent pas de montrer les gelatinases activees non inhibees in situ. Dans le modele tumoral murin de carcinome pulmonaire de lewis, comme dans le modele d'ards induit chez le hamster choisi, une surexpression de la gelatinase mmp-9 a ete detectee au cours de l'evolution des pathologies et une activite de degradation du collagene de type iv qui depend des mmps a ete mise en evidence ex vivo. Ceci suggere l'implication de la mmp-9 dans les deux modeles. En parallele, nous avons tente...

Toxicon: X, 2019

Peptidic toxins that target specifically mammalian channels and receptors can be found in the ven... more Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg 1 or Arg/Lys 27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.

Frontiers in Pharmacology

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase... more The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance ...

Molecular Biology Reports

Background Posttranslational modifications of proteins are catalyzed by a large family of enzymes... more Background Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. Methods and results In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N 3 -lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. Conclusions In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach—the Halo Tag—to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.

Protein Science

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a s... more VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2‐Fc‐VHH2. The structure of VHH2‐Fc‐VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2‐Fc‐VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti‐HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2‐Fc). This new type of molecules, VHH2‐Fc‐VHH2, could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Molecules

Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts... more Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts as a substrate for quinone-reductases that may be associated with its antimalarial properties. Following our exploration of reactive oxygen species-producing compounds such as indolones, as possible new approaches for the research of new ways to treat this parasitosis, we explored derivatives of this natural product and their possible antiplasmodial and antimalarial properties, in vitro and in vivo, respectively. Apart from one compound, all the products tested had weak to moderate antiplasmodial activities, the best IC50 value being equal to 0.58 µM. In vivo activities in the murine model were moderate (at a dose of 50 mg/kg/mice, five times higher than the dose of chloroquine). These results encourage further pharmacomodulation steps to improve the targeting of the parasitized red blood cells and antimalarial activities.

Protein Science

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we i... more In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.

International Journal of Molecular Sciences

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is... more Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystalli...

Free Radical Biology and Medicine

ABSTRACT There is increasing evidence that oxidative stress is involved in the etiology and patho... more ABSTRACT There is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol‐quinone and then into highly deleterious semiquinone radicals after 1‐electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol‐quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2‐mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co‐factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS‐induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders. HIGHLIGHTSWe evaluated the role of QR2 in the production of ROS during the reduction of quinones.ROS production was measured using K562, neuroblast cells, and primary neurons overexpressing QR2.A strong increase of ROS was observed with menadione and adrenochrome.The absence of QR2 co‐substrate or the addition of a specific QR2 inhibitor decreased ROS production.The neuronal toxicity of catechol‐quinones may originate in part from QR2 enzymatic activity.

Journal of natural products, Jan 27, 2018

In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systemati... more In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systematic in vitro evaluation using enzymatic and plasmonic resonance assays was undertaken on 11 317 plant extracts. The screening procedure led to the selection of the New Caledonian endemic species Meiogyne baillonii for a chemical investigation. Using a bioguided isolation procedure, three enediyne-γ-butyrolactones (1-3) and two enediyne-γ-butenolides (4 and 5), named sapranthins H-L, respectively, two enediyne carboxylic acid (6 and 7), two depsidones, stictic acid (8) and baillonic acid (9), aristolactams AIa and AIIa (10 and 11), and two aporphines, dehydroroemerine (12) and noraristolodione (13), were isolated from the ethyl acetate extract of the bark. The structures of the new compounds (1-6, 9, and 11) and their relative configurations were established by NMR spectroscopic analysis and by X-ray diffraction analysis for compound 9. Only stictic acid (8) exhibited a significant inhibiti...

Acta crystallographica. Section F, Structural biology communications, 2017

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystal... more A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Acta crystallographica. Section F, Structural biology communications, 2017

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystal... more A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Proceedings of the National Academy of Sciences of the United States of America, Apr 9, 2018

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to ol... more The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling...

Scientific reports, Nov 23, 2016

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase ... more Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

Quantitative Structure Activity Relationships, Dec 1, 2001

Analytical Biochemistry, 2019

 VHH are small antibody-like proteins. They are entering the therapeutic arsenal.  VHH purity, ... more  VHH are small antibody-like proteins. They are entering the therapeutic arsenal.  VHH purity, homogeneity, structure and affinity towards their targets are described.  An analytical workflow to qualify those proteins is presented to validate the molecules.

La surexpression des metalloproteases, et plus particulierement des gelatinases, est decrite dans... more La surexpression des metalloproteases, et plus particulierement des gelatinases, est decrite dans un grand nombre de pathologies ou la degradation de la matrice extracellulaire est un element determinant comme dans la progression tumorale et dans la pathologie d'insuffisance respiratoire aigue (ards). Cependant, la presence ou la surexpression de ces gelatinases ne prouve pas qu'elles participent effectivement a ces pathologies. De plus, les techniques conventionnelles utilisees ne permettent pas de montrer les gelatinases activees non inhibees in situ. Dans le modele tumoral murin de carcinome pulmonaire de lewis, comme dans le modele d'ards induit chez le hamster choisi, une surexpression de la gelatinase mmp-9 a ete detectee au cours de l'evolution des pathologies et une activite de degradation du collagene de type iv qui depend des mmps a ete mise en evidence ex vivo. Ceci suggere l'implication de la mmp-9 dans les deux modeles. En parallele, nous avons tente...

Toxicon: X, 2019

Peptidic toxins that target specifically mammalian channels and receptors can be found in the ven... more Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg 1 or Arg/Lys 27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.

Frontiers in Pharmacology

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase... more The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance ...

Molecular Biology Reports

Background Posttranslational modifications of proteins are catalyzed by a large family of enzymes... more Background Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. Methods and results In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N 3 -lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. Conclusions In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach—the Halo Tag—to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.

Protein Science

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a s... more VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2‐Fc‐VHH2. The structure of VHH2‐Fc‐VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2‐Fc‐VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti‐HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2‐Fc). This new type of molecules, VHH2‐Fc‐VHH2, could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Molecules

Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts... more Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts as a substrate for quinone-reductases that may be associated with its antimalarial properties. Following our exploration of reactive oxygen species-producing compounds such as indolones, as possible new approaches for the research of new ways to treat this parasitosis, we explored derivatives of this natural product and their possible antiplasmodial and antimalarial properties, in vitro and in vivo, respectively. Apart from one compound, all the products tested had weak to moderate antiplasmodial activities, the best IC50 value being equal to 0.58 µM. In vivo activities in the murine model were moderate (at a dose of 50 mg/kg/mice, five times higher than the dose of chloroquine). These results encourage further pharmacomodulation steps to improve the targeting of the parasitized red blood cells and antimalarial activities.

Protein Science

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we i... more In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.

International Journal of Molecular Sciences

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is... more Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystalli...

Free Radical Biology and Medicine

ABSTRACT There is increasing evidence that oxidative stress is involved in the etiology and patho... more ABSTRACT There is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol‐quinone and then into highly deleterious semiquinone radicals after 1‐electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol‐quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2‐mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co‐factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS‐induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders. HIGHLIGHTSWe evaluated the role of QR2 in the production of ROS during the reduction of quinones.ROS production was measured using K562, neuroblast cells, and primary neurons overexpressing QR2.A strong increase of ROS was observed with menadione and adrenochrome.The absence of QR2 co‐substrate or the addition of a specific QR2 inhibitor decreased ROS production.The neuronal toxicity of catechol‐quinones may originate in part from QR2 enzymatic activity.

Journal of natural products, Jan 27, 2018

In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systemati... more In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systematic in vitro evaluation using enzymatic and plasmonic resonance assays was undertaken on 11 317 plant extracts. The screening procedure led to the selection of the New Caledonian endemic species Meiogyne baillonii for a chemical investigation. Using a bioguided isolation procedure, three enediyne-γ-butyrolactones (1-3) and two enediyne-γ-butenolides (4 and 5), named sapranthins H-L, respectively, two enediyne carboxylic acid (6 and 7), two depsidones, stictic acid (8) and baillonic acid (9), aristolactams AIa and AIIa (10 and 11), and two aporphines, dehydroroemerine (12) and noraristolodione (13), were isolated from the ethyl acetate extract of the bark. The structures of the new compounds (1-6, 9, and 11) and their relative configurations were established by NMR spectroscopic analysis and by X-ray diffraction analysis for compound 9. Only stictic acid (8) exhibited a significant inhibiti...

Acta crystallographica. Section F, Structural biology communications, 2017

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystal... more A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Acta crystallographica. Section F, Structural biology communications, 2017

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystal... more A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Proceedings of the National Academy of Sciences of the United States of America, Apr 9, 2018

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to ol... more The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling...

Scientific reports, Nov 23, 2016

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase ... more Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.