<i>N</i>-Methyldihydroquinazolinone Derivatives of Retro-2 with Enhanced Efficacy against Shiga Toxin

Noël, Romain; Gupta, Neetu; Pons, Véronique; Goudet, Amélie; García-Castillo, María; Michau, Aurélien; Martinez, Jennifer; Buisson, David‐Alexandre; Johannes, Ludger; Gillet, Daniel; Barbier, Julien; Cintrat, Jean‐Christophe

doi:10.1021/jm4002346

Cited by 80 publications

(76 citation statements)

References 26 publications

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“…The following inhibitors were used in this study: brefeldin A (BFA), golgicide A, and bafilomycin A1 (all from Sigma-Aldrich) and Retro-2 (EMD Millipore). Retro-2c and Retro-2.1 were synthesized inhouse (41,42). The effect of various drugs on cell viability was measured using the CellTiter-Glo assay (Promega).…”

Section: Methodsmentioning

confidence: 99%

“…High-throughput screens for small-molecule inhibitors of ricin toxicity have recently identified Retro-2 to be a specific blocker of retrograde transport acting at the endosome-TGN interface (59), and it has been shown that Retro-2 and more potent derivatives (41,42) block retrograde transport of ricin and Shiga toxin by displacing STX5 from the TGN toward small peripheral vesicles (42,59). In agreement with our STX5 siRNA data, treatment of HeLa cells with the cyclized analog Retro-2 cycl significantly decreased AAV2-Luc transduction in a dose-dependent manner, showing a reduction of approximately 80% at the highest soluble concentration of 100 M (Fig.…”

Section: Inhibition Of Aav Transport By Compounds Affecting Stx5mentioning

confidence: 99%

“…Because Retro-2 can spontaneously cyclize to Retro-2 cycl, both are referred to as Retro-2 in this article. A novel, highly potent derivative of Retro-2, Retro-2.1 (42), showed an ϳ100-fold improvement of the 50% effective concentration against AAV2 transduction and achieved more than 90% inhibition of transduction at a concentration of 20 M, the maximal dose tested (Fig. 7A).…”

Section: Inhibition Of Aav Transport By Compounds Affecting Stx5mentioning

confidence: 99%

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Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

Nonnenmacher

Cintrat

Gillet

et al. 2015

J Virol

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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. Due to their intrinsically low immunogenicity, their ability to infect a variety of tissues in vivo, and their capacity to confer prolonged transgene expression in postmitotic tissues (1), vectors based on adeno-associated virus (AAV) are among the most promising gene therapy tools. Although these properties make AAV an attractive candidate for many clinical applications, some tissues or cell types are not efficiently transduced by AAV vectors, presumably due to the absence of viral receptors, inefficient intracellular trafficking, or viral uncoating (recently reviewed in reference 2).AAVs contain a single-stranded DNA genome, and the entire viral replication cycle-second-strand DNA synthesis, replication of viral genomes, and encapsidation-takes place in the nucleus. Therefore, correct trafficking of incoming virions from the plasma membrane toward the nuclear compartment is of crucial importance for viral or therapeutic gene expression. Following the initial attachment to a primary glycoprotein receptor (heparan sulfate proteoglycan for AAV serotype 2 [AAV2], AAV3, and AAV6; sialic acids for AAV1, AAV4, AAV5, and AAV6; and N-linked galactose for AAV9 [2]), viral particles undergo rapid endocytosis. Whereas more than one e...

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Section: Methodsmentioning

confidence: 99%

Section: Inhibition Of Aav Transport By Compounds Affecting Stx5mentioning

confidence: 99%

Section: Inhibition Of Aav Transport By Compounds Affecting Stx5mentioning

confidence: 99%

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Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

Nonnenmacher

Cintrat

Gillet

et al. 2015

J Virol

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“…3E). During the course of our study, structure-activity relationship (SAR) studies focusing on Retro-2 optimization lead to cyclic analogues with an ∼100-fold improvement in the EC 50 against Shiga toxin (Noel et al, 2013). We therefore also investigated whether inhibition of retrograde transport through an improved (i.e.…”

Section: Stxb-ss-saporin Translocates From An Endosomal Compartment Tmentioning

confidence: 99%

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

García-Castillo

Tran

Bobard

et al. 2015

Journal of Cell Science

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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 STxBsaporin 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 nonreducible 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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“…In addition, since only one enantiomer was active on previous dihydroquinazolinones synthesized as racemates, 13 a chiral phase HPLC separation of Retro-2.1 was performed on a ChiralPak IB column (see Experimental Section and Figure 1). No contamination of Retro-2.1a by Retro-2.1b or vice versa could be identified on the chromatograms (Figure 1).…”

mentioning

confidence: 99%

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

Gupta

Pons

Noël

et al. 2013

ACS Med. Chem. Lett.

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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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N-Methyldihydroquinazolinone Derivatives of Retro-2 with Enhanced Efficacy against Shiga Toxin

Cited by 80 publications

References 26 publications

Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

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

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

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