ABC-Transmembrantransporter sind an der Aufnahme, Verteilung und Ausscheidung vieler Arznei- und Fremdstoffe beteiligt. Sie spielen eine Schlüsselrolle in der Pharmakokinetik und in der Ausscheidung toxischer endogener oder exogener Substanzen. Das Ziel der hier präsentierten Untersuchungen war deshalb, den Einfluss genetischer Polymorphismen des bekanntesten Vertreters dieser Proteinfamilie, MDR1 (ABCB1) zu untersuchen. Darüberhinaus sollte der ebenfalls zur ABC-Transporterfamilie gehörende hepatozelluläre Exporter für monoanionische Gallensäuren identifiziert und charakterisiert werden. MDR1 erwies sich als ein hochpolymorphes Gen mit zahlreichen Einzelbasenaustauschen (SNPs). Die meisten SNPs waren intronisch oder stumm. Für den nichtkodierenden SNP im Exon 26 3435C>T ergab sich bei homozygoten Trägern des T-Allels eine im Vergleich zum Wildtyp geringere intestinale P-Glykoprotein Expression mit einer entsprechend höheren und schnelleren Absorption von Digoxin. Die Auswertung pharmakokinetischer Profile von Digoxin in Individuen mit MDR1-Haplotypen der miteinander verbundenen SNPs in Exon 21 2677 und Exon 26 3435 untermauerte die beobachteten pharmakogenetischen Effekte. Nach oraler Einzelgabe von 1 mg Digoxin konnten wahrscheinlich aufgrund der Überschreitung der P-Glykoprotein Transportkapazität keine genotypischen Unterschiede beobachtet werden. Der biliäre Exporter für monoanionische Gallensäuren (Bsep) konnte als ein 160 kDa Glykoprotein aus einer Rattenleber cDNA-Bibliothek identifiziert werden und gehört ebenfalls zur ABC Transporter-Familie. Die transkriptionelle Regulation und Möglichkeiten der Modulation der Expression des Bsep-Gens wurden in vitro und in Tiermodellen der Cholestase untersucht. Dabei zeigte sich, dass Gallensäuren über ein proximales FXRE-Motiv die Bsep Promotoraktivität stimulierten. Arzneistoffe hatten ebenfalls einen Einfluss auf die Transkription des Bsep-Gens. Die adaptive Regulation hepatozellulärer Transporter während der Cholestase ergab eine verminderte Expression der meisten basolateralen Aufnahmetransporter und eine unveränderte oder heraufregulierte Proteinmasse kanalikulärer (apikaler) Efflux-Transporter. Dieses Regulationsmuster dient dem Schutz der Leberzelle, indem eine intrazelluläre Anreicherung toxischer Gallensäuren vermindert und der Gallefluss für eine intakte biliäre Clearance aufrechterhalten wird. / ABC transmembrane transporters are involved in absorption, distribution and excretion of diverse drugs and xenobiotics. They are key factors in pharmacokinetics and in the elimination of toxic endogenous or exogenous compounds. Therefore, the aim of the present study was to investigate the influence of genetic polymorphisms of the best known member of this protein family, MDR1 (ABCB1). In addition, the identity of another ABC transporter assumed to be the major hepatocellular export pump for monoanionic bile acids should be revealed and characterized. MDR1 turned out as a highly polymorphic gene with many single nucleotide polymorphisms (SNPs). Most of the SNPs were intronic or silent. Homozygous carriers of the non-coding SNP in exon 26 3435C>T had lower intestinal P-glycoprotein expression rates and thus enhanced absorption of the model compound digoxin as compared to wildtype controls. The analysis of pharmacokinetic profiles in different MDR1-haplotypes of the linked SNPs in exon 21 2677 and exon 26 3435 supported the above data. An oral single dose of 1 mg digoxin did not result in genotypic differences of tested genotypes, probably because this dose was above the maximal transport capacity of P-glycoprotein. The biliary export pump for monoanionic bile acids (Bsep) was identified as an 160 kDa glycoprotein of the ABC transporter family by screening a rat liver cDNA library. The transcriptional regulation and modulatory factors of Bsep (Abcb11) gene expression were analyzed in vitro and in animal models of cholestasis. The promoter activity of Bsep was stimulated by bile acids via a proximal FXRE motif. Drugs were also able to modify Bsep gene transcription. Adaptive regulation of hepatocellular transporters during cholestasis followed a pattern of diminished expression of most basolateral uptake carrier systems and maintained or even upregulated protein mass of canalicular (apical) exporters. This pattern serves as a protective mechanism of the liver cells preventing intracellular accumulation of toxic bile acids and providing unimpaired biliary flow and clearance.

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.

ABCB11/BSEP est le transporteur des acides biliaires, localisé au niveau du pôle canaliculaire des hépatocytes. Les mutations de ce gène sont responsables de la cholestase familiale intrahépatique progressive de type 2.Au cours de ma thèse, j’ai évalué la capacité des aminoglycosides et du PTC124 à induire la translecture de codons stop prématurés, l’adressage et la fonction de mutants non-sens et faux sens de Bsep ainsi que l’effet d’une bithérapie (translecture+chaperone).Dans nos modèles cellulaires, la gentamicine était capable d’induire la translecture du codon-stop prématuré du mutant non-sens BsepR1090X dans les lignées NIH3T3, HEK293 et Can 10. La protéine entière générée était partiellement détectée aux membranes plasmiques des cellules HEK293 et canaliculaires des cellules Can 10 et était partiellement fonctionnelle puisqu’elle était responsable d’une augmentation de l’activité de transport de 3H-taurocholate (3H-TC) dans les clones MDCK. Ces effets étaient potentialisés par l’addition de drogues chaperones telles que le 4-phenylbutyrate (4-PB).J’ai également mis en évidence la capacité de nouveaux composés dérivés du 4-PB (MHMPB, OTNC et HMPB) à corriger l’adressage et à augmenter le transport de 3H-TC du mutant faux sens BsepR1128C à des concentrations plus faibles que le 4-PB. Enfin, j’ai pu montrer que d'autres drogues chaperones (GPB, PA, SAHA et C18), pouvaient corriger l’adressage canaliculaire de BsepR1128C et augmenter son activité de transport de 3H-TC dans les clones MDCK. / ABCB11/BSEP is the main bile acids transporter located at the canalicular pole of hepatocytes. Mutations of ABCB11 are responsible for progressive familial intrahepatic cholestasis type 2.During my phD, I evaluated the ability of aminoglycosides and PTC124 to induce readthrough of premature termination codons, targeting and function of nonsense and missense mutants of Bsep and also the effect of combined therapy (readthrough + chaperone).In our expermental models, gentamicin increased readthrough of p.R1090X mutation NIH3T3, HEK293 and Can 10 lines. The resulting full-length protein was detected at the plasma membrane of HEK293 and at the canalicular membrane of Can 10 cells; and was partially functional since it was responsible for increasing the transport activity of 3H-taurocholate (3H-TC) in MDCK clones. These effects were potentiated by the addition of chaperone drugs such as 4-phenylbutyrate (4-PB).I have also demonstrated the ability of new 4-PB derived compounds (MHMPB, OTNC and HMPB) to correct mistrafficking and to increase 3H-TC transport of BsepR1128C missense mutant at lower concentrations than 4-PB. Finally, I showed that other chaperone drugs (GPB, PA, SAHA, and C18) were able to correct mistrafiking of BsepR1128C and to increase its 3H-TC transport activity in MDCK clones.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.