Publications by authors named "Julien Barbier"

45 Publications

An adamantamine derivative as a drug candidate for the treatment of visceral leishmaniasis.

J Antimicrob Chemother 2021 Jul 2. Epub 2021 Jul 2.

Université Paris-Saclay, CNRS, BioCIS, 92290, Châtenay-Malabry, France.

Background: This study aimed to investigate compounds acting on the host cell machinery to impair parasite installation with the possible advantage of limiting drug resistance. The strategy therefore consisted of selecting compounds that are poorly active on the axenic parasite, but very active on the intramacrophage form of Leishmania.

Objectives: To identify a drug candidate from focused screening of adamantamine derivatives that can inhibit the development of Leishmania infantum in macrophages.

Methods: In vitro screening was performed on a library of 142 adamantamine derivatives with axenic and intramacrophage forms of L. infantum, as well as cytotoxicity assays, allowing selection of the most promising compound. Absorption, distribution, metabolism and excretion (ADME) experiments, including pharmacokinetics and microsomal stability, were performed and finally the physicochemical stability of the compound was investigated to assess its suitability for further drug development.

Results: VP343 was identified first in vitro, with a CC50 value of 63.7 μM and an IC50 value of 0.32 μM for L. infantum intramacrophage amastigotes and then in vivo, with a 59% reduction of the liver parasite burden after oral administration at 10 mg/kg/day for 5 days. In addition, the ADME data were compatible with moving this compound further through the antileishmanial drug candidate pipeline.

Conclusions: VP343 has the properties of a good drug candidate and merits further investigations.
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http://dx.doi.org/10.1093/jac/dkab226DOI Listing
July 2021

Broad spectrum compounds targeting early stages of rabies virus (RABV) infection.

Antiviral Res 2021 04 11;188:105016. Epub 2021 Jan 11.

Unit Antiviral Strategies, Institut Pasteur, 75724, Paris, France; Institut Pasteur de Guinée, BP 4416, Conakry, Guinea. Electronic address:

ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies.
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http://dx.doi.org/10.1016/j.antiviral.2021.105016DOI Listing
April 2021

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Toxins (Basel) 2020 07 10;12(7). Epub 2020 Jul 10.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.
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http://dx.doi.org/10.3390/toxins12070449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404996PMC
July 2020

Structure-Activity Relationship Studies of Retro-1 Analogues against Shiga Toxin.

J Med Chem 2020 08 27;63(15):8114-8133. Epub 2020 Jul 27.

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, 91191 Gif-sur-Yvette, France.

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00298DOI Listing
August 2020

Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

Nat Chem Biol 2020 03 17;16(3):327-336. Epub 2020 Feb 17.

Institut Curie, PSL Research University, Cellular and Chemical Biology unit, U1143 INSERM, UMR3666 CNRS, Endocytic Trafficking and Intracellular Delivery team, Paris, France.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.
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http://dx.doi.org/10.1038/s41589-020-0474-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039708PMC
March 2020

Revisiting Old Ionophore Lasalocid as a Novel Inhibitor of Multiple Toxins.

Toxins (Basel) 2020 01 1;12(1). Epub 2020 Jan 1.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France.

The ionophore lasalocid is widely used as a veterinary drug against coccidiosis. We found recently that lasalocid protects cells from two unrelated bacterial toxins, the cytotoxic necrotizing factor-1 (CNF1) from and diphtheria toxin. We evaluated lasalocid's capacity to protect cells against other toxins of medical interest comprising toxin B from , Shiga-like toxin 1 from enterohemorrhagic and exotoxin A from . We further characterized the impact of lasalocid on the endolysosomal and the retrograde pathways and organelle integrity, especially the Golgi apparatus. We found that lasalocid protects cells from all toxins tested and impairs the drop of vesicular pH along the trafficking pathways that are required for toxin sorting and translocation to the cytoplasm. Lasalocid also has an impact on the cellular distribution of GOLPH4 and GOLPH2 Golgi markers. Other intracellular trafficking compartments positive for EEA1 and Rab9A display a modified cellular pattern. In conclusion, lasalocid protects cells from multiple deadly bacterial toxins by corrupting vesicular trafficking and Golgi stack homeostasis.
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http://dx.doi.org/10.3390/toxins12010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020423PMC
January 2020

Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.

FEBS J 2020 08 21;287(15):3184-3199. Epub 2020 Jan 21.

Université Paris-Saclay, CEA, INRAE, Médicaments et Technologies pour la Santé (MTS), SIMoS, Gif-sur-Yvette, 91191, France.

The endo-lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo-lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high-throughput screening against the cytotoxicity of ricin toxin, exhibits a broad-spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo-lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad-spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo-lysosome system-related cellular physiology and mechanisms of pathogenesis.
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http://dx.doi.org/10.1111/febs.15201DOI Listing
August 2020

DABMA: A Derivative of ABMA with Improved Broad-Spectrum Inhibitory Activity of Toxins and Viruses.

ACS Med Chem Lett 2019 Aug 2;10(8):1140-1147. Epub 2019 Jul 2.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette, France.

The small molecule ABMA has been previously shown to protect cells against multiple toxins and pathogens including virus, intracellular bacteria, and parasite. Its mechanism of action is directly associated with host endolysosomal pathway rather than targeting toxin or pathogen itself. However, the relationship of its broad-spectrum anti-infection activity and chemical structure is not yet resolved. Here, we synthesized a series of derivatives and compared their activities against diphtheria toxin (DT). Dimethyl-ABMA (DABMA), one of the most potent analogs with about 20-fold improvement in protection efficacy against DT, was identified with a similar mechanism of action to ABMA. Moreover, DABMA exhibited enhanced efficacy against toxin B (TcdB), lethal toxin (TcsL), Exotoxin A (PE) as well as Rabies and Ebola viruses. The results revealed a structure-activity relationship of ABMA, which is a starting point for its clinical development as broad-spectrum drug against existing and emerging infectious diseases.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691562PMC
August 2019

23rd and 24th Meetings of the French society of toxinology (SFET): Special issue on "Toxins: Immunity, inflammation and pain".

Toxicon 2018 07 25;149:1-5. Epub 2018 Apr 25.

CEA, Institut des Sciences du vivant Frédéric Joliot, Service d'Ingénierie Moléculaire des Protéines, Université Paris-Saclay, France; Institut des Neurosciences Paris-Saclay, UMR 9197 CNRS/Université Paris-Sud, Gif-sur-Yvette, France. Electronic address:

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http://dx.doi.org/10.1016/j.toxicon.2018.04.020DOI Listing
July 2018

Antiviral Effect of Retro-2.1 against Herpes Simplex Virus Type 2 In Vitro.

J Microbiol Biotechnol 2018 Jun;28(6):849-859

National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, P.R. China.

Herpes simplex virus type 2 (HSV-2) infection has been a public health concern worldwide. It is the leading cause of genital herpes and a contributing factor to cervical cancer and human immunodeficiency virus (HIV) infection. No vaccine is available yet for the treatment of HSV-2 infection, and routinely used synthetic nucleoside analogs have led to the emergence of drug resistance. The small molecule Retro-2 has been reported to be active against several pathogens by acting on intracellular vesicle transport, which also participates in the HSV-2 lifecycle. Here, we showed that Retro-2.1, which is an optimized, more potent derivative of Retro-2, could inhibit HSV-2 infection, with 50% inhibitory concentrations of 5.58 μM and 6.35 μM in cytopathic effect inhibition and plaque reduction assays, respectively. The cytotoxicity of Retro-2.1 was relatively low, with a 50% cytotoxicity concentration of 116.5 μM. We also preliminarily identified that Retro-2.1 exerted the antiviral effect against HSV-2 by a dual mechanism of action on virus entry and late stages of infection. Therefore, our study for the first time demonstrated Retro-2.1 as an effective antiviral agent against HSV-2 in vitro with targets distinct from those of nucleoside analogs.
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http://dx.doi.org/10.4014/jmb.1712.12052DOI Listing
June 2018

Ribosome Inactivating Proteins: From Plant Defense to Treatments against Human Misuse or Diseases.

Toxins (Basel) 2018 04 18;10(4). Epub 2018 Apr 18.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette, France.

Ribosome inactivating proteins (RIPs) form a vast family of hundreds of toxins from plants, fungi, algae, and bacteria. RIP activities have also been detected in animal tissues. They exert an N-glycosydase catalytic activity that is targeted to a single adenine of a ribosomal RNA, thereby blocking protein synthesis and leading intoxicated cells to apoptosis. In many cases, they have additional depurinating activities that act against other nucleic acids, such as viral RNA and DNA, or genomic DNA. Although their role remains only partially understood, their functions may be related to plant defense against predators and viruses, plant senescence, or bacterial pathogenesis.
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http://dx.doi.org/10.3390/toxins10040160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923326PMC
April 2018

Antiviral Effects of ABMA against Herpes Simplex Virus Type 2 In Vitro and In Vivo.

Viruses 2018 03 9;10(3). Epub 2018 Mar 9.

National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, Jilin, China.

Herpes simplex virus type 2 (HSV-2) is the causative pathogen of genital herpes and is closely associated with the occurrence of cervical cancer and human immunodeficiency virus (HIV) infection. The absence of an effective vaccine and the emergence of drug resistance to commonly used nucleoside analogs emphasize the urgent need for alternative antivirals against HSV-2. Recently, ABMA [1-adamantyl (5-bromo-2-methoxybenzyl) amine] has been demonstrated to be an inhibitor of several pathogens exploiting host-vesicle transport, which also participates in the HSV-2 lifecycle. Here, we showed that ABMA inhibited HSV-2-induced cytopathic effects and plaque formation with 50% effective concentrations of 1.66 and 1.08 μM, respectively. We also preliminarily demonstrated in a time of compound addition assay that ABMA exerted a dual antiviral mechanism by impairing virus entry, as well as the late stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that ABMA protected BALB/c mice from intravaginal HSV-2 challenge with an improved survival rate of 50% at 5 mg/kg (8.33% for the untreated virus infected control). Consequently, our study has identified ABMA as an effective inhibitor of HSV-2, both in vitro and in vivo, for the first time and presents an alternative to nucleoside analogs for HSV-2 infection treatment.
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http://dx.doi.org/10.3390/v10030119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869512PMC
March 2018

Screening of a Drug Library Identifies Inhibitors of Cell Intoxication by CNF1.

ChemMedChem 2018 04 27;13(7):754-761. Epub 2018 Feb 27.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, 91191, Gif/Yvette, France.

Cytotoxic necrotizing factor 1 (CNF1) is a toxin produced by pathogenic strains of Escherichia coli responsible for extra-intestinal infections. CNF1 deamidates Rac1, thereby triggering its permanent activation and worsening inflammatory reactions. Activated Rac1 is prone to proteasomal degradation. There is no targeted therapy against CNF1, despite its clinical relevance. In this work we developed a fluorescent cell-based immunoassay to screen for inhibitors of CNF1-induced Rac1 degradation among 1120 mostly approved drugs. Eleven compounds were found to prevent CNF1-induced Rac1 degradation, and five also showed a protective effect against CNF1-induced multinucleation. Finally, lasalocid, monensin, bepridil, and amodiaquine protected cells from both diphtheria toxin and CNF1 challenges. These data highlight the potential for drug repurposing to fight several bacterial infections and Rac1-based diseases.
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http://dx.doi.org/10.1002/cmdc.201700631DOI Listing
April 2018

Retro-2 and its dihydroquinazolinone derivatives inhibit filovirus infection.

Antiviral Res 2018 01 22;149:154-163. Epub 2017 Nov 22.

Texas Biomedical Research Institute, San Antonio, TX, USA. Electronic address:

Members of the family Filoviridae cause severe, often fatal disease in humans, for which there are no approved vaccines and only a few experimental drugs tested in animal models. Retro-2, a small molecule that inhibits retrograde trafficking of bacterial and plant toxins inside host cells, has been demonstrated to be effective against a range of bacterial and virus pathogens, both in vitro and in animal models. Here, we demonstrated that Retro-2 and its derivatives, Retro-2.1 and compound 25, blocked infection by Ebola virus and Marburg virus in vitro. We show that the derivatives were more potent inhibitors of infection as compared to the parent compound. Pseudotyped virus assays indicated that the compounds affected virus entry into cells while virus particle localization to Niemann-Pick C1-positive compartments showed that they acted at a late step in virus entry. Our work demonstrates a potential for Retro-type drugs to be developed into anti-filoviral therapeutics.
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http://dx.doi.org/10.1016/j.antiviral.2017.11.016DOI Listing
January 2018

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Sci Rep 2017 Nov 14;7(1):15567. Epub 2017 Nov 14.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, LabEx LERMIT, 91191, Gif-sur-Yvette, France.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
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http://dx.doi.org/10.1038/s41598-017-15466-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686106PMC
November 2017

Antiviral effects of Retro-2 and Retro-2.1 against Enterovirus 71 in vitro and in vivo.

Antiviral Res 2017 08 5;144:311-321. Epub 2017 Jul 5.

National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, China. Electronic address:

Enterovirus 71 (EV71) is one of the causative pathogens of hand, foot and mouth disease (HFMD), especially the form associated with fatal neurological disorders. Sustained outbreaks of EV71 infections remain a serious health threat worldwide. However, no antiviral agent against EV71 for clinical therapy has been approved. Retro-2 and Retro-2.1 are inhibitors of several pathogens specifically targeting the intracellular vesicle transport, which also participates in the EV71 lifecycle processes including progeny virus release. Here, we reported that Retro-2 and Retro-2.1, respectively, could inhibit EV71 infection with 50% effective concentrations of 12.56 μM and 0.05 μM in a cytopathic effect inhibition assay and showed relatively low cytotoxicity with 50% cytotoxicity concentrations of more than 500 μM and 267.80 μM. Preliminary mechanism studies revealed that Retro-2 and Retro-2.1 did not inhibit EV71 protein synthesis or RNA replication but could block progeny EV71 release specifically. Furthermore, administration of Retro-2 at the dose of 10 mg/kg significantly protected 90% of newborn mice from lethal EV71 challenge. Consequently, our results for the first time identified Retro-2 and Retro-2.1 as effective inhibitors of EV71 as well as lead compounds, which would contribute to anti-EV71 drug development. We also identified progeny virus release and the intracellular vesicle transport as antiviral targets for EV71.
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http://dx.doi.org/10.1016/j.antiviral.2017.07.001DOI Listing
August 2017

Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

ChemMedChem 2016 11 25;11(22):2506-2510. Epub 2016 Oct 25.

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, USA.

Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct.
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http://dx.doi.org/10.1002/cmdc.201600463DOI Listing
November 2016

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Chem Biol Interact 2017 Apr 3;267:96-103. Epub 2016 Oct 3.

Institute of Biology and Technology of Saclay (IBITECS), CEA, LabEx LERMIT, Université Paris-Saclay, F-91191, Gif Sur Yvette, France.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.
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http://dx.doi.org/10.1016/j.cbi.2016.10.005DOI Listing
April 2017

The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

PLoS One 2016 12;11(4):e0153401. Epub 2016 Apr 12.

CEA, iBiTec-S/SIMOPRO, CEA-Saclay, Paris Saclay University, LabEx LERMIT, F-91191 Gif-sur-Yvette, France.

Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153401PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829238PMC
August 2016

Special Issue on "Toxins: New targets and new functions".

Toxicon 2016 Jun 17;116:1-3. Epub 2016 Mar 17.

CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif-sur-Yvette, France.

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http://dx.doi.org/10.1016/j.toxicon.2016.03.012DOI Listing
June 2016

Intracellular pathogens convert macrophages from death traps into hospitable homes.

FEBS J 2016 Feb 2;283(4):595-7. Epub 2016 Feb 2.

CEA, Section of Molecular Engineering of Proteins (SIMOPRO), Institute of Biology and Biotechnology of Saclay (iBiTec-S), LERMIT LabEx, Paris Saclay University, Gif sur Yvette, France.

Intracellular pathogens - bacteria, parasites and fungi - frequently infect macrophages in addition to other cells. They turn these deadly cells into harmless hosts to multiply and paralyze immunity. Understanding the complex mechanisms underlying this phenomenon may have a strong impact to identify new targets belonging to the pathogens but also to the host, thereby allowing the design of new therapeutic strategies.
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http://dx.doi.org/10.1111/febs.13657DOI Listing
February 2016

Proteomic analysis of the Simkania-containing vacuole: the central role of retrograde transport.

Mol Microbiol 2016 Jan 20;99(1):151-71. Epub 2015 Oct 20.

Department of Microbiology, University of Würzburg, Biocenter, Am Hubland, D-97074, Würzburg, Germany.

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane-bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania-containing vacuole (SnCV) is a critical host-pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER-SnCV-membranes using label free LC-MS(2) to define the pathogen-containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome-to-trans-Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome-to-TGN transport for nutrient acquisition and growth.
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http://dx.doi.org/10.1111/mmi.13222DOI Listing
January 2016

Synthesis, Chiral Separation, Absolute Configuration Assignment, and Biological Activity of Enantiomers of Retro-1 as Potent Inhibitors of Shiga Toxin.

ChemMedChem 2015 Jul 1;10(7):1153-6. Epub 2015 Jun 1.

CEA, iBiTec-S/SCBM, CEA-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette (France).

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro-1, a compound active against Stx and other such protein toxins. Retro-1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X-ray diffraction data revealed (S)-Retro-1 to be slightly more active than (R)-Retro-1.
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http://dx.doi.org/10.1002/cmdc.201500139DOI Listing
July 2015

Special Issue on "freshwater and marine toxins".

Toxicon 2014 Dec 20;91:1-4. Epub 2014 Nov 20.

CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif-sur-Yvette, France.

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http://dx.doi.org/10.1016/j.toxicon.2014.11.217DOI Listing
December 2014

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins.

ACS Med Chem Lett 2014 Jan 4;5(1):94-7. Epub 2013 Dec 4.

CEA, iBiTec-S/SCBM, CEA-Saclay, LabEx LERMIT, F-91191 Gif-sur-Yvette, France.

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.
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http://dx.doi.org/10.1021/ml400457jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027625PMC
January 2014

Special issue on «Toxins: from threats to benefits».

Toxicon 2013 Dec 6;75:1-2. Epub 2013 Aug 6.

Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille, CNRS/AMU, Marseille, France.

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http://dx.doi.org/10.1016/j.toxicon.2013.08.001DOI Listing
December 2013

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

J Med Chem 2013 Apr 5;56(8):3404-13. Epub 2013 Apr 5.

CEA, iBiTec-S/SCBM, CEA-Saclay, LabEx LERMIT, F-91191 Gif-sur-Yvette, France.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.
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http://dx.doi.org/10.1021/jm4002346DOI Listing
April 2013

The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides.

Nucleic Acids Res 2013 Apr 8;41(6):3673-87. Epub 2013 Feb 8.

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.

The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.
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http://dx.doi.org/10.1093/nar/gkt066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616695PMC
April 2013

Inhibitors of the cellular trafficking of ricin.

Toxins (Basel) 2012 01 6;4(1):15-27. Epub 2012 Jan 6.

Laboratory of Molecular Toxinology and Biotechnology, Molecular engineering of proteins (SIMOPRO), Life Sciences Division (DSV), Institute of Biology and Technology Saclay, French Alternative Energies and Atomic Energy Commission (CEA), F-91191 Gif sur Yvette, France.

Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress.
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http://dx.doi.org/10.3390/toxins4010015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277095PMC
January 2012
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