Studies on nuclear receptors involved in drug metabolism

Abstract: The molecular basis for drug metabolism is starting to emerge as a result of cloning and characterization of a group of nuclear receptors that respond to drugs by transcriptional regulation of key genes encoding enzymes involved in metabolism of commonly used drugs. This thesis describes the identification and functional, as well as the structural, characterization of one of these receptors, the human pregnenolone activated receptor (hPAR, NR1I2) that also has been referred to as the pregnane X receptor (PXR) or the steroid and xenobiotic receptor (SXR) by others. Functional characterization of this, and the closely related, constitutively active receptor CAR (NR1I3) has shown that both PXR and CAR are important for cytochrome P450 gene induction and thus are likely to be important mediators of drug metabolism. PXR was identified by homology searching of human EST databases and two differentially spliced mRNAs were cloned. The expression was high in liver and intestine, and PXR was activated by a large and chemically diverse group of compounds including drugs metabolized by CYP3A4. A transient transfection study using an expression plasmid for PXR together with a luciferase reporter construct, containing a part of the CYP3A4 promoter (- 176 to - 146), showed that PXR induced the expression of this reporter. Based on this study we suggested that PXR was a novel sensor for sterol and xenobiotic metabolism. To further investigate the regulatory role of PXR in CYP3A induction we cloned approximately 10 kb of both the CYP3A7 and the CYP3A4 gene promoters. These two promoters exhibited 90 % sequence identity up to -8,8 kb, indicating a close evolutionary distance between the two genes. A combination of promoter deletion analyses and transient transfections suggests that PYCR and CAR are important for CYP3A4 and CYP3A7 gene induction through a functionally conserved distally located xenobiotic responsive enhancer module. Taken together, we propose that PXR and CAR play important roles in xenobiotic regulation, not only for CYP3A4 regulation in adults, but also for CYP3A7 to protect the embryo against endogenous and exogenous toxins. The chemically broad activation profile of PXR suggested that this receptor could be structurally related to the PPARs (NR1C1-3), another subgroup of orphan nuclear receptors with promiscuous ligand binding proper-ties. We found from the crystal structure of the PPAR gamma ligand binding domain that this receptor has a larger and more accessible ligand binding pocket than other nuclear receptors binding to a structurally more limited set of ligands. We also made a model of the ligand binding domain of PXR based on the available co-crystal structure of VDR (NR1I1), and 1alpha 25-dihydroxyvitamin D3. The model indicated important residues of the ligand binding pocket of PXR. We mutated polar residues from human to mouse and tested them in transient transfection in combination with species specific compounds. The results have increased our understanding for species specific ligand binding to PXR. In addition, we also made a single point mutation in PYR that yielded a constitutive active form of the receptor. PPARgamma agonists belonging to the thiazolidinedione (TZD) class of compounds are currently used for treatment of diabetes type 2. Troglitazone, a TZD, is known to induce CYP3A4 activity. The TZI)s troglitazone, pioglitazone and rosiglitazone were all found to activate PXR on a CYP3A4 promoter. Furthermore an insulin sensitizing non-TZD ligand, JTT-501, did not activate the nuclear receptor PXR but dually activated PPAR alpha and gamma. Therefore JTT is less likely to mediate drug- interactions due to PXR mediated CYP3A4 induction. In conclusion, screening against PXR activation is predicted to be a valuable tool in pre-clinical drug development to obtain better and safer drugs.