Publications by authors named "Jean-Christophe Cintrat"

49 Publications

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

Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity.

Nucleic Acids Res 2020 11;48(20):11615-11625

Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette cedex, France.

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.
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http://dx.doi.org/10.1093/nar/gkaa903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672478PMC
November 2020

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

NMR Characterization of the Influence of Zinc(II) Ions on the Structural and Dynamic Behavior of the New Delhi Metallo-β-Lactamase-1 and on the Binding with Flavonols as Inhibitors.

ACS Omega 2020 May 28;5(18):10466-10480. Epub 2020 Apr 28.

Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Sud, Université Paris-Saclay, LabEx LERMIT, 1 avenue de la Terrasse, 91190 Gif-sur-Yvette, France.

New Delhi metallo-β-lactamase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to all common β-lactam antibiotics. Understanding the molecular basis of β-lactam hydrolysis by NDM is crucial for designing NDM inhibitors or β-lactams resistant to their hydrolysis. In this study, for the first time, NMR was used to study the influence of Zn(II) ions on the dynamic behavior of NDM-1. Our results highlighted that the binding of Zn(II) in the NDM-1 active site induced several structural and dynamic changes on active site loop 2 (ASL2) and L9 loops and on helix α2. We subsequently studied the interaction of several flavonols: morin, quercetin, and myricetin were identified as natural and specific inhibitors of NDM-1. Quercetin conjugates were also synthesized in an attempt to increase the solubility and bioavailability. Our NMR investigations on NDM-1/flavonol interactions highlighted that both Zn(II) ions and the residues of the NDM-1 ASL1, ASL2, and ASL4 loops are involved in the binding of flavonols. This is the first NMR interaction study of NDM-1/inhibitors, and the models generated using HADDOCK will be useful for the rational design of more active inhibitors, directed against NDM-1.
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http://dx.doi.org/10.1021/acsomega.0c00590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226869PMC
May 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

Photochemical Strain-Release-Driven Cyclobutylation of C(sp )-Centered Radicals.

Angew Chem Int Ed Engl 2020 02 6;59(7):2618-2622. Epub 2019 Nov 6.

Service de Chimie Bio-organique et Marquage (SCBM), CEA, Université Paris-Saclay, 91191, Gif-sur-Yvette, France.

A new photoredox-catalyzed decarboxylative radical addition approach to functionalized cyclobutanes is described. The reaction involves an unprecedented formal Giese-type addition of C(sp )-centered radicals to highly strained bicyclo[1.1.0]butanes. The mild photoredox conditions, which make use of a readily available and bench stable phenyl sulfonyl bicyclo[1.1.0]butane, proved to be amenable to a diverse range of α-amino and α-oxy carboxylic acids, providing a concise route to 1,3-disubstituted cyclobutanes. Furthermore, kinetic studies and DFT calculations unveiled mechanistic details on bicyclo[1.1.0]butane reactivity relative to the corresponding olefin system.
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http://dx.doi.org/10.1002/anie.201908951DOI Listing
February 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

Glucocorticoids delay RAF-induced senescence promoted by EGR1.

J Cell Sci 2019 08 22;132(16). Epub 2019 Aug 22.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France

Expression of hyperactive RAF kinases, such as the oncogenic B-RAF-V600E mutant, in normal human cells triggers a proliferative arrest that blocks tumor formation. We discovered that glucocorticoids delayed the entry into senescence induced by B-RAF-V600E in human fibroblasts, and allowed senescence bypass when the cells were regularly passaged, but that they did not allow proliferation of cells that were already senescent. Transcriptome and siRNA analyses revealed that the EGR1 gene is one target of glucocorticoid action. Transcription of the gene is activated by the RAF-MEK-ERK MAPK pathway and acts as a sensor of hyper-mitogenic pathway activity. The EGR1 transcription factor regulates the expression of p15 and p21 (encoded by and , respectively) that are redundantly required for the proliferative arrest of BJ fibroblasts upon expression of B-RAF-V600E. Our results highlight the need to evaluate the action of glucocorticoid on cancer progression in melanoma, thyroid and colon carcinoma in which B-RAF-V600E is a frequent oncogene, and cancers in which evasion from senescence has been shown.
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http://dx.doi.org/10.1242/jcs.230748DOI Listing
August 2019

Spermine-NBD as fluorescent probe for studies of the polyamine transport system in Leishmania donovani.

Bioorg Med Chem Lett 2019 07 17;29(14):1710-1713. Epub 2019 May 17.

Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), CNRS, Univ Paris Sud, Université Paris-Saclay, 15 rue Georges Clemenceau, 91405 Orsay Cedex, France. Electronic address:

This study describes the synthesis of fluorescent probes as potential substrates for the polyamine transport system (PTS) of Leishmania donovani. A competitive radioassay was used to determine the most efficient probe. We observed that the conjugate spermine-nitrobenzofurazan (Spm-NBD) was able to compete with [H]-spermidine in L. donovani at a potent IC of 60 µM.
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http://dx.doi.org/10.1016/j.bmcl.2019.05.030DOI Listing
July 2019

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

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

Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis.

J Virol 2016 Nov 28;90(22):10120-10132. Epub 2016 Oct 28.

The Pirbright Institute, Pirbright, Surrey, United Kingdom

Poxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growth and reduced weight loss and clinical signs in an murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication. Cellular retrograde transport pathways traffic cargo from endosomes to the -Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.
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http://dx.doi.org/10.1128/JVI.01464-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105650PMC
November 2016

Palladium-Catalyzed Chemoselective and Biocompatible Functionalization of Cysteine-Containing Molecules at Room Temperature.

Chemistry 2016 Aug 30;22(32):11365-70. Epub 2016 Jun 30.

BioCIS, Equipe Labellisée Ligue Contre le Cancer, Univ. Paris-Sud, CNRS, University Paris-Saclay, 92296, Châtenay-Malabry, France.

The third generation of aminobiphenyl palladacycle pre-catalyst "G3-Xantphos" enables functionalization of peptides containing cysteine in high yields. The conjugation (bioconjugation) occurs chemoselectively at room temperature under biocompatible conditions. Extension of the method to protein functionalization allows selective bioconjugation of the trastuzumab antibody.
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http://dx.doi.org/10.1002/chem.201602277DOI Listing
August 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

Regioselective Halogenation of 1,4-Benzodiazepinones via CH Activation.

Sci Rep 2015 Jul 16;5:12131. Epub 2015 Jul 16.

CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage, LabEx LERMIT. 91191 Gif-sur-Yvette (France).

This article reports an efficient CH activation process for regioselective halogenation of 1,4-benzodiazepinones. Direct halogenation with NXS (X = Br, I) affords halogenated benzodiazepinones on the central aromatic ring whereas catalyst (Pd(OAc)2) controlled CH activation furnishes regioselectively ortho halogenated benzodiazepinones on the phenyl side chain.
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http://dx.doi.org/10.1038/srep12131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503988PMC
July 2015

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

Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.

J Cell Sci 2015 Jul 14;128(13):2373-87. Epub 2015 May 14.

Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France

Antigen-presenting cells have the remarkable capacity to transfer exogenous antigens to the cytosol for processing by proteasomes and subsequent presentation on major histocompatibility complex class-I (MHC-I) molecules, a process termed cross-presentation. This is the target of biomedical approaches that aim to trigger a therapeutic immune response. The receptor-binding B-subunit of Shiga toxin (STxB) has been developed as an antigen delivery tool for such immunotherapy applications. In this study, we have analyzed pathways and trafficking factors that are involved in this process. A covalent conjugate between STxB and saporin was generated to quantitatively sample the membrane translocation step to the cytosol in differentiated monocyte-derived THP-1 cells. We have found that retrograde trafficking to the Golgi complex was not required for STxB-saporin translocation to the cytosol or for STxB-dependent antigen cross-presentation. Depletion of endosomal Rab7 inhibited, and lowering membrane cholesterol levels favored STxB-saporin translocation. Interestingly, experiments with reducible and non-reducible linker-arm-STxB conjugates led to the conclusion that after translocation, STxB remains associated with the cytosolic membrane leaflet. In summary, we report new facets of the endosomal escape process bearing relevance to antigen cross-presentation.
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http://dx.doi.org/10.1242/jcs.169383DOI Listing
July 2015

Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction.

J Virol 2015 Feb 19;89(3):1673-87. Epub 2014 Nov 19.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA

Unlabelled: Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.

Importance: Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.
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http://dx.doi.org/10.1128/JVI.02520-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300741PMC
February 2015

Directing peptide crystallization through curvature control of nanotubes.

J Pept Sci 2014 Jul 11;20(7):508-16. Epub 2014 Jun 11.

iBiTec-S, SB2SM, UMR 8221, CEA/CNRS, CEA-Saclay, 91191, Gif-sur-Yvette, France; Institut de Physique de Rennes, UMR 6251 CNRS/Université Rennes 1, Campus Beaulieu, 35042, Rennes Cedex, France; Laboratoire Interdisciplinaire sur l'Organisation Nanométrique et Supramoléculaire (LIONS), IRAMIS, NIMBE, UMR 3299 CEA/CNRS CEA-Saclay, 91191, Gif-sur-Yvette Cedex, France.

In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact. Indeed, we show that it is possible to modify the curvature of the lanreotide nanotubes and hence their diameter. This fine tuning leads to crystallization because the radius of curvature of the initially bidimensional peptide wall can be increased up to a point where the wall is essentially flat and a crystal is allowed to grow along a third dimension. By comparing X-ray diffraction data and Fourier transform Raman spectra, we show that the nanotubes and the crystals share similar cell parameters and molecular conformations, proving that there is indeed a structural continuum between these two morphologies. These results illustrate a novel approach to crystallization and represent the first step towards the acquisition of an Å-resolution structure of the lanreotide nanotubes β-sheet assembly.
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http://dx.doi.org/10.1002/psc.2647DOI Listing
July 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

Discovery of compounds blocking the proliferation of Toxoplasma gondii and Plasmodium falciparum in a chemical space based on piperidinyl-benzimidazolone analogs.

Antimicrob Agents Chemother 2014 May 18;58(5):2586-97. Epub 2014 Feb 18.

Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Grenoble Alpes, Institut National de la Recherche Agronomique, Unité Mixte de Recherche 5168, Institut de Recherches en Technologies et Sciences pour le Vivant, Grenoble, France.

A piperidinyl-benzimidazolone scaffold has been found in the structure of different inhibitors of membrane glycerolipid metabolism, acting on enzymes manipulating diacylglycerol and phosphatidic acid. Screening a focus library of piperidinyl-benzimidazolone analogs might therefore identify compounds acting against infectious parasites. We first evaluated the in vitro effects of (S)-2-(dibenzylamino)-3-phenylpropyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)piperidine-1-carboxylate (compound 1) on Toxoplasma gondii and Plasmodium falciparum. In T. gondii, motility and apical complex integrity appeared to be unaffected, whereas cell division was inhibited at compound 1 concentrations in the micromolar range. In P. falciparum, the proliferation of erythrocytic stages was inhibited, without any delayed death phenotype. We then explored a library of 250 analogs in two steps. We selected 114 compounds with a 50% inhibitory concentration (IC50) cutoff of 2 μM for at least one species and determined in vitro selectivity indexes (SI) based on toxicity against K-562 human cells. We identified compounds with high gains in the IC50 (in the 100 nM range) and SI (up to 1,000 to 2,000) values. Isobole analyses of two of the most active compounds against P. falciparum indicated that their interactions with artemisinin were additive. Here, we propose the use of structure-activity relationship (SAR) models, which will be useful for designing probes to identify the target compound(s) and optimizations for monotherapy or combined-therapy strategies.
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http://dx.doi.org/10.1128/AAC.01445-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993238PMC
May 2014