Étude du transporteur de multiples drogues MRP1 (caractérisation des NBD, et étude de modulateurs conduisant à la mort des cellules surexprimant le transporteur)

L acquisition du phénotype de résistance des cancers est souvent corrélée à l expression de transporteurs membranaires appartenant à la superfamille des transporteurs ABC ( ATP-Binding Cassette ). Un de ces transporteurs, MRP1 ( Multidrug Resistance Protein 1 ), permet l efflux de nombreux substrats, de type anioniques, conjugués au GSH, glucuronates ou sulfates, ou en co-transport avec le GSH. Dans un premier temps, ce travail a porté sur l étude des domaines de fixation des nucléotides isolés. Une étude biochimique a montré leur caractère fonctionnel asymétrique concernant les nucléotides, prouvant que seul la séquence primaire de ces NBD est responsable de cette fonctionnalité différentielle. L étude de la fixation de substrats sur les NBD, a montré que ceux-ci pourraient, de part leur proximité avec les domaines transmembranaires, avoir un rôle dans la fixation des substrats. La deuxième étape de ce travail a concerné la caractérisation de l activité des énantiomères du vérapamil. Les résultats ont montré que le S-vérapamil est l isomère responsable de la stimulation du transport du GSH, conduisant à la mort des cellules surexprimant MRP1. Le R-vérapamil est caractérisé comme un inhibiteur de MRP1. Ces résultats ont des répercussions importantes en terme de thérapie. Deux études préliminaires de relation structure/fonction ont été menées en ce qui concerne des dérivés du vérapamil et des dérivés de flavonoïdes, afin de trouver des molécules plus efficaces contre MRP1The acquisition of cancer resistance phenotype is mostly correlated to the expression of membrane transporters belonging to the ABC transporters super family ( ATP-Binding Cassette ). One of these transporters, MRP1 ( Multidrug Resistance Protein1 ), is a transmembrane protein which has the ability to efflux many substrates. First, this work has focused on the characterisation of the isolated nucleotide binding domains. A biochemical study has shown their asymmetric behaviour towards nucleotides, and their ability to bind substrates, which could lead to the involvement of NBDs in the substrate binding site. Then, a characterisation of the verapamil enantiomers has been performed, showing that S-verapamil was responsible of glutathione efflux, whereas R-verapamil had an inhibitory effect on MRP1. These results may have great impact in term of therapy. Finally, structure/function relationships of verapamil and flavonoids derivatives has been accessed, in order to find more potent modulators for MRP1LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Etude du transporteur MRP1 humain responsable de la résistance de cellules cancéreuses à la chimiothérapie et recherche d'inhibiteurs spécifiques

L'émergence du phénotype MDR (" MultiDrug Resistance ") des cellules cancéreuses est souvent corrélée à la surexpression de deux ATPases membranaires, appartenant à la superfamille des transporteurs ABC (" ATP-Binding Cassette "), la glycoprotéine-P et MRP1 (" Multidrug Resistance Protein "), qui utilisent l'énergie issue de l'hydrolyse de l'ATP pour expulser les agents antitumoraux hors des cellules. Nos objectifs ont concerné d'une part l'étude fondamentale du mécanisme moléculaire d'efflux des drogues par le transporteur MRP1, dont la spécificité de substrat est différente de celle de la glycoprotéine-P, et d'autre part la recherche rationnelle de modulateurs spécifiques de MRP1, capables d'inhiber l'activité d'efflux des drogues. Les principaux résultats ont permis de mettre en évidence deux modulateurs; l'un de la famille des flavonoïdes qui abolit le phénotype MDR et l'autre, dérivé iodé du vérapamil, qui induit spécifiquement la mort par apoptose des cellules surexprimant MRP1LYON1-BU Santé (693882101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Molecular analysis of the massive GSH transport mechanism mediated by the human Multidrug Resistant Protein 1/ABCC1

International audienceThe transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1

Nucleotide-free Crystal Structure of Nucleotide-binding Domain 1 from Human ABCC1 Supports a ‘General-Base Catalysis’ Mechanism for ATP Hydrolysis

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Iodination of verapamil for a stronger induction of death, through GSH efflux, of cancer cells overexpressing MRP1

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Monoterpene indole alkaloid azine derivatives as MDR reversal agents

International audienceAiming at generating a library of bioactive indole alkaloid derivatives as multidrug resistance (MDR) reversers, two epimeric indole alkaloids (1 and 2) were submitted to chemical transformations, giving rise to twenty-four derivatives (5-28), bearing new aromatic or aliphatic azine moieties. The structure of the compounds was established by 1D and 2D NMR (COSY, HMBC, HMQC and NOESY) experiments. Two different strategies were employed for assessing their anti-MDR potential, namely through the evaluation of their activity as inhibitors of typical MDR ABC transporters overexpressed by cell transfection, such as ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP), or by evaluating their ability as collateral sensitivity (CS) agents in cells overexpressing MRP1. A considerable MDR reversing activity was observed for compounds bearing the aromatic azine moiety. The strongest and most selective P-gp inhibition was found for the epimeric azines 5 and 6, bearing a para-methylbenzylidene moiety. Instead, compounds 17 and 18 that possess a di-substituted benzylidene portion with methoxy and hydroxyl groups, selectively inhibited MRP1 drug-efflux. None of these compounds inhibited BCRP. Compounds 5, 6 and 18 were further investigated in drug combination experiments, which corroborated their anti-MDR potential. Moreover, it was observed that compound 12, with an aromatic azine moiety, and compounds 23-26, sharing a new aliphatic substituent, displayed a CS activity, selectively killing MRP1-overexpress-ing cells. Among these last compounds, it could be established that addition of 19, 23 and 25 to MRP1-overexpressing cells led to glutathione depletion triggering cell death through apoptosis

MRP1-dependent Collateral Sensitivity of Multidrug-resistant Cancer Cells: Identifying Selective Modulators Inducing Cellular Glutathione Depletion

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Optimizing the flavanone core toward new selective nitrogen-containing modulators of ABC transporters

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