Browsing by Author "Ghebreghiorghis, Luam"

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    Surface plasmon resonance-based investigation of the human constitutive androstane receptor with regard to co-activator binding and ligand-dependent activation
    (2011) Ghebreghiorghis, Luam; Hauer, Bernhard (Prof. Dr.)
    During detoxification, xenobiotics including pharmaceuticals are subject to biotransformation reactions which enable their excretion outside the cell and the human body. The cytochrome P450 monooxygenases (CYPs) are the most important group of enzymes in xenobiotic metabolism. CAR (constitutive androstane receptor) and PXR (pregnane X receptor) seem to be crucial for pharmaceutical metabolism, too. CAR belongs to the family of nuclear receptors and is mainly responsible for regulation of CYP2B6 in humans. The most outstanding property of the nuclear receptor CAR is its constitutive activity which results from ligand-independent recruitment of transcriptional co-activators unlike most classical nuclear receptors. Yet, it was also shown that CAR, due to exposure to Phenobarbital (PB), translocates from the cytoplasm into the nucleus of hepatocytes. The co-activators SRC-1 (steroid receptor co-activator 1) and SRC-2 (steroid receptor co-activator 2) belong to the p160 family of co-activators and co-activate many nuclear receptors among others CAR and ER (estrogen receptor). Though CAR recruits ligand-independently co-activators and, therefore, does not need agonist binding to be active, it has been shown that its activity can be further enhanced by interactions with agonists. In this study Biacore technology, which relies on the principle of surface plasmon resonance (SPR), was used to investigate and characterize the nuclear receptor CAR. Several drugs selected in the course of a screening performed at the IKP (Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie) were chosen to investigate the influence on CAR with regard to co-activator binding and ligand-dependent activation. For this purpose, the first goal was the soluble expression of both CAR and the co-activators SRC-1 and SRC-2 in E. coli cells, and the subsequent purification for binding experiments via SPR. The aim of this work was to investigate to what extent the selected drugs influence the constitutive association of CAR with SRC-1 or SRC-2 by means of SPR. Furthermore, the work aimed to characterize the kinetics of both the association and the dissociation of the receptor – co-activator complex in the presence and absence of ligands. Additionally, the examination of the ligand-free interaction aimed to characterize the constitutive binding of receptor and co-activator. Binding experiments of immobilized SRC-1 with CAR in the presence of the selected drugs revealed a distinctive ligand-dependent hierarchy in association of receptor and co-activator which allowed a discrimination of the drugs into non- or low, weak, and strong binders of the receptor. The association hierarchy included Arteether > CITCO > Triphenylphosphate to be the three most competent agonists. Thus, CAR – SRC-1 interaction appears to be more susceptible to regulation by the selected drugs, especially by the pharmaceutically relevant substances Arteether and Clofibrate. Since ligand-induced binding of CAR and SRC-1 proved to be significantly diminished by Clotrimazole, side effects including cross reactivity caused by the simultaneous taking of the inverse agonist and Arteether or Clofibrate may occur in vivo. Yet, Clotrimazole could only be partly confirmed as inverse agonist of CAR since it did not lead to co-activator release in the absence of ligands. Yet, the co-activator SRC-1, unlike SRC-2, revealed to be a powerful tool of identification and characterization of putative agonists which might distinctively influence the constitutive binding with CAR. Kinetic binding assays demonstrated that the constitutive binding of CAR with SRC-1 occurred nine times faster than with SRC-2 whereas the stability of both receptor - co-activator complexes revealed to be low but displayed no distinctive differences. Thus, both ligand-induced binding experiments and ligand-free kinetic assays strongly indicate that SRC-1 is the prime co-activator of interest for CAR regardless of expression levels and tissue-specific expression profiles. Surprisingly, equilibrium dissociation constants of ligand-induced kinetic binding assays of CAR and SRC-1 revealed weaker affinities when interactions took place with all agonists, indicating that no ligand was able to accelerate recognition of SRC-1. Furthermore, two classes of CAR ligands were revealed. The first class of ligands including Artemether, Triphenylphosphate, and Fenofibrate led to the formation of more complexes, as demonstrated by ligand-induced increase in binding, but could not enhance the stability of the complex. The second class of ligands which comprised CITCO, Clofibrate, Arteether, and Artemisinin furthermore enhanced the stability of the complex but also caused distinctively slower association rates which might be assigned to a two-step association. Unlike the HMG-CoA reductase inhibitor Atorvastatin and its metabolites, Fenofibrate and Clofibrate, were identified as CAR agonists.