Elsevier

Biochemical Pharmacology

Volume 76, Issue 10, 15 November 2008, Pages 1288-1297
Biochemical Pharmacology

Discovery of substituted sulfonamides and thiazolidin-4-one derivatives as agonists of human constitutive androstane receptor

https://doi.org/10.1016/j.bcp.2008.08.014Get rights and content

Abstract

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor responsible for the recognition of potentially toxic endo- and exogenous compounds whose elimination from the body is accelerated by the CAR-mediated inducible expression of metabolizing enzymes and transporters. Despite the importance of CAR, few human agonists are known so far. Following a sequential virtual screening procedure using a 3D pharmacophore and molecular docking approach, we identified 17 novel agonists that could activate human CAR in vitro and enhance its association with the nuclear receptor co-activator SRC1. Selected agonists also increased the expression of the human CAR target CYP2B6 mRNA in primary hepatocytes. Composed of substituted sulfonamides and thiazolidin-4-one derivatives, these agonists represent two novel chemotypes capable of human CAR activation, thus broadening the agonist spectrum of CAR.

Introduction

The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor (NR) superfamily, a group of ligand-regulated transcription factors which are involved in many physiological processes including cellular development, homeostasis and metabolism [1]. Together with the pregnane X receptor (PXR, NR1I2), CAR governs the drug-inducible expression of cytochrome P450s (CYPs), conjugating enzymes and transporter proteins [2]. As xenobiotic sensors, CAR and PXR recognize and are activated by many chemically diverse compounds including synthetic drugs, natural products and xenobiotics [3], and both receptors display wide species differences in their spectrum of activators [2], [3]. For example, endogenous substances including bilirubin, bile acids and testosterone metabolites have been identified as CAR ligands and/or activators [4]. The pesticide contaminant 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and the synthetic drugs chlorpromazine, meclizine and 17α-ethynyl-3,17β-estradiol (EE2) are efficient activators of mouse CAR [5], [6], [7]. However, these agonists do not activate human CAR at all; in contrast, meclizine and EE2 even inhibit it [8], [9], [10].

In unexposed liver, CAR is located in the cytoplasm where it forms a complex with the cytoplasmic CAR retention protein and heat shock protein 90 which help retain CAR in the cytoplasm [11], [12]. CYP inducers dissociate the cytoplasmic complex in a yet unknown manner, trigger translocation of CAR into the nucleus and lead to the transcriptional activation of CAR target genes. Recently, several protein kinases [13], [14], protein phosphatases [15], [16] and other factors [17] have been implicated in modulation of the CAR translocation process.

The common activation mechanism for NRs involves the direct binding of the ligand to a pocket that is harbored by the ligand-binding domain (LBD). Agonist binding modulates the orientation of the activation helix 12 (H12) so as to allow the LBD to interact with NR co-activators such as the steroid receptor coactivator-1 (SRC1) [1], [18]. However, only a few agonists which activate human CAR via direct binding to the ligand-binding pocket (LBP) are known. Among these, the steroid 5β-pregnane-3,20-dione and the synthetic compound 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) have been co-crystallized with the human CAR LBD [18]. Tri-p-methylphenyl phosphate (TMPP) and clotrimazole have been shown to bind to the LBD and to activate human CAR [8], [19], [20]. In addition to the direct CAR agonism, an indirect activation mechanism has been postulated for CAR. In this case, certain chemicals including bilirubin, phenobarbital (PB), phenytoin and acetaminophen are able to translocate and activate CAR and exert their CYP-inducing effects despite the fact that no direct binding to the CAR LBP could be demonstrated for these compounds [3], [21], [22]. The details of this indirect action are not understood, and some interpretations may be based on problems in detecting CAR agonism in assays with high basal activity of CAR [20], [23], [24].

In order to identify novel bioactive molecules, virtual screening (VS) techniques are a well-established technology. Despite its limitations, in silico screening has emerged as increasingly important in the drug discovery process. While diverse G protein-coupled receptors, enzymes and ion channels have been targeted by different VS approaches, only a few success stories for NRs have been reported although NRs represent an equally important class of pharmaceutically relevant proteins [25]. So far, VS efforts have led to the identification of retinoic acid receptor and thyroid hormone receptor antagonists as well as estrogen receptor modulators [26], [27], [28], [29], [30], [31].

We aimed to discover novel human CAR agonists by virtually screening a drug-like compound library. The selection of CAR-interacting candidates was first guided by a ligand-based screening approach followed by a molecular docking procedure. Among 30 molecules tested in vitro, 17 emerged as agonists in a cell-based human CAR activation assay, and their agonism was confirmed by assays measuring co-activator recruitment. Selected agonists also activated the CAR target CYP2B6 gene expression. The sulfonamides and thiazolidin-4-one derivatives identified here represent two novel chemotypes as efficacious human CAR agonists.

Section snippets

Molecular modeling

All calculations were performed on Intel-based Linux workstations. The compounds of the LeadQuest® database (Tripos Inc., St. Louis, USA) were transformed into 3D molecular structures using the Concord module within SYBYL 7.0 (Tripos Inc., St. Louis, USA). All compounds were generated in the protonation state assumed to exist under physiological conditions.

Crystal structures of the CAR LBD were obtained from the Protein Data Bank (PDB) [32]. The volume of the CAR LBP was ascertained using the

Virtual screening and validation of CAR activation

Initially, we used a homology model of the human CAR LBD for virtual screening as no crystal structure was available at that time [20]. Structural comparison of the model and the recently published crystal structures revealed a good agreement of side chain conformations within the LBP [36]. Moreover, binding modes of the co-crystallized ligands could be reproduced quite well in the model. Based on docked agonists TMPP and clotrimazole, a 3D pharmacophore model was generated within the UNITY

Discussion

Here, we report the discovery of 17 novel human CAR agonists based on a two-step virtual screening approach. With the combination of a pharmacophore-based database screening and a molecular docking approach, we identified 30 potential CAR agonists. We obtained a high hit rate of 56.6% among these compounds as judged by our cell-based CAR activation assay. Of these 17 confirmed agonists, three ligands (3, 9 and 14) were highly active (more than fivefold CAR induction). With nearly eightfold

Acknowledgments

This work was supported by grants from the Academy of Finland and the Finnish Funding Agency for Technology and Innovation.

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