CINPA1 binds directly to constitutive androstane receptor and inhibits its activity.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that regulate the expression of drug-metabolizing enzymes and efflux transporters. CAR activation promotes drug elimination, thereby reducing therapeutic effectiveness, or causes adverse drug effects via toxic metabolites. CAR inhibitors could be used to attenuate these adverse drug effects. CAR and PXR share ligands and target genes, confounding the understanding of the regulation of receptor-specific activity. We previously identified a small-molecule inhibitor, CINPA1, that inhibits CAR (without activating PXR at lower concentrations) by altering CAR-coregulator interactions and reducing CAR recruitment to DNA response elements of regulated genes. However, solid evidence was not presented for the direct binding of CINPA1 to CAR. In this study, we demonstrate direct interaction of CINPA1 with the CAR ligand-binding domain (CAR-LBD) and identify key residues involved in such interactions through a combination of biophysical and computational methods. We found that CINPA1 resides in the ligand-binding pocket to stabilize the CAR-LBD in a more rigid, less fluid state. Molecular dynamics simulations, together with our previously reported docking model, enabled us to predict which CAR residues were critical for interactions with CINPA1. The importance of these residues for CINPA1 binding were then validated by directed mutations and testing the mutant CAR proteins in transcription reporter and coregulatory interaction assays. We demonstrated strong hydrogen bonding of CINPA1 with N165 and H203 and identified other residues involved in hydrophobic contacts with CINPA1. Overall, our data confirm that CINPA1 directly binds to CAR.

Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor.

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the promoter regions of its target genes. In this study, we showed that CINPA1 is converted to two main metabolites in human liver microsomes. By using cell-based reporter gene and biochemical coregulator recruitment assays, we showed that although metabolite 1 was very weak in inhibiting CAR function and disrupting CAR-coactivator interaction, metabolite 2 was inactive in this regard. Docking studies using the CAR ligand-binding domain structure showed that although CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding. These results indicate that the metabolites of CINPA1 may not interfere with the action of CINPA1. We also used in vitro enzyme assays to identify the cytochrome P450 enzymes responsible for metabolizing CINPA1 in human liver microsomes and showed that CINPA1 was first converted to metabolite 1 by CYP3A4 and then further metabolized by CYP2D6 to metabolite 2. Identification and characterization of the metabolites of CINPA1 enabled structure-activity relationship studies of this family of small molecules and provided information to guide in vivo pharmacological studies.

The orphan nuclear receptor NR4A2 is part of a p53-microRNA-34 network.

Nuclear receptor subfamily 4 group A member 2 (NR4A2) is an orphan nuclear receptor that is over-expressed in cancer and promotes cell proliferation, migration, transformation, and chemoresistance. Increased expression and function of NR4A2 have been attributed to various signaling pathways, but little is known about microRNA (miRNA) regulation of NR4A2 in cancer. To investigate the posttranscriptional regulation of NR4A2, we used a 3' untranslated region (UTR) reporter screen and identified miR-34 as a putative regulator of NR4A2. By using computer predictions, we identified and confirmed an miRNA recognition element in the 3' UTR of NR4A2 that was responsible for miR-34-mediated suppression. We next demonstrated that overexpression of exogenous miR-34 or activation of the p53 pathway, which regulates endogenous miR-34 expression, decreased NR4A2 expression. Consistent with previous reports, overexpression of NR4A2 blocked the induction of p53 target genes, including mir-34a. This was a phenotypic effect, as NR4A2 overexpression could rescue cells from p53-induced inhibition of proliferation. In summary, our results are the first characterization of a cancer-related miRNA capable of regulating NR4A2 and suggest a network and possible feedback mechanism involving p53, miR-34, and NR4A2.

Small-molecule modulators of PXR and CAR.

Two nuclear receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), participate in the xenobiotic detoxification system by regulating the expression of drug-metabolizing enzymes and transporters in order to degrade and excrete foreign chemicals or endogenous metabolites. This review aims to expand the perceived relevance of PXR and CAR beyond their established role as master xenosensors to disease-oriented areas, emphasizing their modulation by small molecules. Structural studies of these receptors have provided much-needed insight into the nature of their binding promiscuity and the important elements that lead to ligand binding. Reports of species- and isoform-selective activation highlight the need for further scrutiny when extrapolating from animal data to humans, as animal models are at the forefront of early drug discovery. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Small-molecule modulators of the constitutive androstane receptor.

INTRODUCTION: The constitutive androstane receptor (CAR) induces drug-metabolizing enzymes for xenobiotic metabolism. AREAS COVERED: This review covers recent advances in elucidating the biological functions of CAR and its modulation by a growing number of agonists and inhibitors. EXPERT OPINION: Extrapolation of animal CAR function to that of humans should be carefully scrutinized, particularly when rodents are used in evaluating the metabolic profile and carcinogenic properties of clinical drugs and environmental chemicals. Continuous efforts are needed to discover novel CAR inhibitors, with extensive understanding of their inhibitory mechanism, species selectivity, and discriminating power against other xenobiotic sensors.

CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function.

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes.

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.

Small molecule inhibitors as probes for estrogen and androgen receptor action.

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor {alpha}.

The mechanisms responsible for 17ß-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

A new small molecule inhibitor of estrogen receptor alpha binding to estrogen response elements blocks estrogen-dependent growth of cancer cells.

Estrogen receptor alpha (ERalpha) plays an important role in several human cancers. Most current ERalpha antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERalpha binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERalpha binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency, and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI) (TPBM), inhibited ERalpha binding to the flcERE (IC(50) approximately 3 microm) and inhibited ERalpha-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER-specific, because progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ERalpha, TPBM inhibited 17beta-estradiol (E(2))-ERalpha (IC(50) 9 microm) and 4-hydroxytamoxifen-ERalpha-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E(2).ERalpha recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E(2)-dependent growth of ERalpha-positive cancer cells (IC(50) of 5 microm). TPBM is not toxic to cells and does not affect estrogen-independent cell growth. TPBM acts outside of the ER ligand binding pocket, does not act by chelating the zinc in ER zinc fingers, and differs from known ERalpha inhibitors. Using a simple high throughput screen for inhibitors of ERalpha binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERalpha-mediated gene expression and estrogen-dependent growth of cancer cells.