Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

Abstract

Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)–approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of PXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in PXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates PXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates PXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.

Tryptophan 299 is a conserved residue of human pregnane X receptor critical for the functional consequence of ligand binding

“…Detailed methods have been described previously [24]. Briefly, the cells were transfected with Flag-hPXR, CMV-Renilla (Promega, Madison, WI; as a transfection control and used for normalization), and CYP3A4-luc plasmids using FuGENE 6 (Roche Diagnostics).…”

“…The CheckMate mammalian two-hybrid system (Promega, Madison, WI) was performed as described previously [24]. It consists of VP16-hPXR, Gal4-SRC-1, and a luciferase reporter pG5-luc co-transfected into HepG2 cells.…”

“…The qRT-PCR experiment was performed as described previously [24]. Briefly, total RNA was isolated from HepG2 cells using Maxwell 16 LEV simplyRNA purification kits (Promega).…”

Targeting xenobiotic receptors PXR and CAR in human diseases

“…These xenobiotic receptors are known to bind various structurally diverse chemicals [7,8]. Because they regulate an overlapping set of target genes, it is difficult to dictate their specificity in gene regulation and associated biological functions [9].…”

Clinical Impacts of Cyp3a4 Isoenzyme on Drugs Used in Treatment of Cardiovascular Diseases: Drug-Drug Interactions

“…Moreover, thiazide diuretics are well known first choice diuretics used in hypertension and congestive heart failure treatment. Thiazide like diuretic metalozone, used in treatment of these illnesses, activated human PXR (hPXR)mediated CYP3A4 expression in human hepatocytes and intestine cells at both mRNA and protein levels and researchers defined that doses of drugs that are metabolized with CYP3A4 should be adjusted [13].…”