Cannabidiol promotes intestinal cholesterol uptake mediated by Pregnane X receptor.

Background: Stevioside is used as a sweetener in food additives and pharmaceutical products. There is a growing concern about the adverse effects posed by stevioside usage. Stevioside is hydrolyzed into the aglycone steviol that is absorbed into the body. Recent studies suggest that steviol, but not stevioside, activates Pregnane X receptor (PXR) in human hepatic cells. In addition to the role in xenobiotic metabolism, the atherogenic and dyslipidemic effects of PXR have been revealed. Controversially, steviol not only increases the mRNA expression of PXR direct downstream gene CYP3A4, but also inhibits the enzymatic activities of CYP3A4. Thus, it is urgent to elucidate the molecular mechanisms by which steviol activates PXR signaling and to assess the possible adverse effects of steviol on pro-atherosclerotic events, such as dyslipidemia. Objective: Our study aims to explore the molecular mechanisms by which exposure to steviol activates human PXR and increases the risk of dyslipidemia. Methods: Both human hepatic and intestinal cells were used to test if steviol was a PXR agonist via transfection assay. The key residues within PXR’s ligand-binding pocket that steviol interacted with were investigated using a computational docking study with site-directed mutagenesis assay. Human intestinal cells were treated with steviol and/or PXR antagonist resveratrol (RES) to estimate the function of steviol in cholesterol uptake. Individual pairwise comparisons and other comparisons were analyzed with Student’s t-test and two-way ANOVA, respectively. Results: Steviol was a more potent agonist of human PXR than mouse PXR. Steviol promoted PXR to dissociate from its nuclear co-repressors. The key amino acids that were essential for the agonistic effects of steviol within PXR’s ligand binding pocket were established. Mechanistically, steviol induced gene expression of key intestinal cholesterol transporters, which led to increased cholesterol uptake by intestinal cells. Future studies in mice are needed to explore if exposure to steviol alters in vivo lipid profiles via PXR signaling. Our study provides potential evidence for the future risk assessment of steviol on cardiovascular disease. Keywords: Steviol, PXR, cholesterol uptake, dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

Differential activation of human pregnane X receptor PXR by isomeric mono-methylated indoles in intestinal and hepatic in vitro models

Pregnane X Receptor Activators Inhibit Human Hepatic Stellate Cell Transdifferentiation In Vitro

Pharmacotherapy of central nervous system (CNS) disorders is impaired by the drug efflux transporter, P-glycoprotein, which limits drug penetration across the blood-brain barrier into the CNS. One strategy to increase brain drug levels is to modulate P-glycoprotein regulation. This approach requires understanding of the mechanisms that control transporter expression and function. One mechanism through which P-glycoprotein is regulated is the nuclear receptor, pregnane X receptor (PXR). Xenobiotics including drugs activate PXR and induce P-glycoprotein, which potentially affects pharmacokinetics/pharmacodynamics of coadministered drugs. Because rodent models are not suitable to predict xenobiotic interactions with human PXR, in a porcine model, we studied functional similarities between pig and human PXR. We used brain capillary endothelial cells from pig to study the effect of PXR activation on P-glycoprotein. To activate PXR, we used the PXR ligands, rifampicin, hyperforin, and pregnenolone-16alpha-carbonitrile (PCN), and measured abcb1 mRNA with quantitative polymerase chain reaction, P-glycoprotein expression with Western blotting, and P-glycoprotein transport activity with a calcein assay. We provide first proof of principle that the human PXR ligands, rifampicin and hyperforin, but not the rodent PXR ligand, PCN, activate pig PXR at the blood-brain barrier and induce mRNA, protein expression, and transport activity of P-glycoprotein. Our data indicate functional similarities between human and pig PXR that suggest the pig model could be useful for predicting xenobiotic-PXR interactions in humans. Because PXR is crucial in controlling drug efflux transporters, our findings will contribute to a better understanding of the regulation of blood-brain barrier function, which could potentially have important clinical implications for the treatment of CNS disorders.

Activation of pregnane X receptor (PXR) by clinical compounds during multidrug chemotherapy results in upregulation of the expression of PXR target genes, including cytochrome p450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), leading to chemoresistance. It is possible to overcome the PXR‐mediated chemoresistance by downregulating the upregulated PXR target genes by inhibiting the activated PXR. However, a selective and less‐toxic PXR antagonist has yet to be developed. In this regard, a clinical anticancer drug, with selective PXR antagonistic activity at its less‐toxic concentrations, would be beneficial. We sought to determine whether belinostat, a clinically‐used histone deacetylase inhibitor, inhibits the PXR target gene expression at its clinically relevant plasma concentrations (< ~100 μM) in human hepatocytes (primary hepatocytes & hepatocells) and intestinal cells (LS174T colon cancer cells). Rifampicin, an agonist of human PXR, was used to activate PXR. Cell viability and CYQUANT cell proliferation assays were performed to determine cytotoxicity and cell proliferation, respectively. Quantitative RT‐PCR assays were conducted to study the gene expression. CYP3A4 p450‐Glo and Rhodamine‐123 intracellular accumulation assays were performed to determine the function of CYP3A4 and MDR1, respectively. Belinostat, at its unbound therapeutic plasma concentrations (< ~5 μM) did not affect the viability of LS174T cells and the hepatocytes. Belinostat (1 & 3 μM) not only inhibited rifampicin‐induced gene expression of CYP3A4 and MDR1, but also attenuated rifampicin‐induced activity of CYP3A4 and MDR1. However, belinostat alone did not affect CYP3A4 or MDR1 gene expression. These results suggest that belinostat does not affect the basal expression of PXR target genes but downregulates the upregulated PXR target genes by inhibiting the ligand‐activated PXR. Notably, belinostat, at its PXR inhibiting concentrations, decreased rifampicin‐induced proliferation of LS174T cells, suggesting that belinostat suppresses PXR‐mediated proliferation of the cancer cells. Interestingly, belinostat failed to inhibit rodent PXR agonist pregnenolone‐16 alpha‐carbonitrile (PCN)‐induced expression of CYP3A1 (the rat analog of human CYP3A4) in rat primary hepatocytes, suggesting that belinostat exhibits species‐specific inhibition of PXR at unbound plasma therapeutic concentrations. Taken together, these results are consistent with the conclusion that belinostat, at its less‐toxic and clinically relevant unbound plasma concentrations, inhibits the ligand‐activated human PXR target gene expression. Future studies will determine the mechanisms of belinostat inhibition of PXR, and belinostat sensitization of the cancer cells to chemotherapy drugs with PXR agonistic activity.Support or Funding InformationThe authors would like to thank Drs. Coleman, Schwartz, and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Species differences in the induction and activation of the pregnane X receptor (PXR)

The pregnane X receptor (PXR) is a nuclear receptor significantly involved in the transcriptional regulation of drug-metabolizing enzymes and transporters. Interestingly, certain PXR ligands such as rifampin have been shown to readily induce human and rabbit but not rodent members of the cytochrome P450 3A. Because drugs of divergent chemical structures seem to be similarly affected, we hypothesized that specific amino acid residue(s) or domains in rat PXR affect receptor activation by certain human PXR ligands. To identify such a domain(s), an array of human-rat and rat-human chimeric PXR cDNAs in a tandem head-to-tail configuration were created using a random chimeragenesis method. Pharmacological characterization of these chimeras revealed a discreet segment within the ligand-binding domain of rat and human PXR to be essential for the rifampin effect. Within this region, the corresponding residues Leu308 and Phe305 of human and rat PXR, respectively, were found to be important for rifampin activation. Homology modeling derived from the recently determined crystal structure of human PXR indicates that these amino acids are located within or neighboring the flexible loop that forms part of the pore to the ligand-binding cavity. Rifampin, paclitaxel, and hyperforin sensitivity was conferred to rat PXR when Phe305 was converted to leucine, whereas attenuation of sensitivity was observed when Leu308 of human PXR was replaced with phenylalanine. Accordingly, our data provide compelling new insight into the importance of the amino acids comprising the pore to the ligand-binding cavity as a critical modulator of PXR response.

BackgroundThe pregnane X receptor (PXR) shows the highest degree of cross-species sequence diversity of any of the vertebrate nuclear hormone receptors. In this study, we determined the pharmacophores for activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs, using a common set of sixteen ligands. In addition, we compared in detail the selectivity of human and zebrafish PXRs for steroidal compounds and xenobiotics. The ligand activation properties of the Western clawed frog (Xenopus tropicalis) PXR and that of a putative vitamin D receptor (VDR)/PXR cloned in this study from the chordate invertebrate sea squirt (Ciona intestinalis) were also investigated.ResultsUsing a common set of ligands, human, mouse, and rat PXRs share structurally similar pharmacophores consisting of hydrophobic features and widely spaced excluded volumes indicative of large binding pockets. Zebrafish PXR has the most sterically constrained pharmacophore of the PXRs analyzed, suggesting a smaller ligand-binding pocket than the other PXRs. Chicken PXR possesses a symmetrical pharmacophore with four hydrophobes, a hydrogen bond acceptor, as well as excluded volumes. Comparison of human and zebrafish PXRs for a wide range of possible activators revealed that zebrafish PXR is activated by a subset of human PXR agonists. The Ciona VDR/PXR showed low sequence identity to vertebrate VDRs and PXRs in the ligand-binding domain and was preferentially activated by planar xenobiotics including 6-formylindolo-[3,2-b]carbazole. Lastly, the Western clawed frog (Xenopus tropicalis) PXR was insensitive to vitamins and steroidal compounds and was activated only by benzoates.ConclusionIn contrast to other nuclear hormone receptors, PXRs show significant differences in ligand specificity across species. By pharmacophore analysis, certain PXRs share similar features such as human, mouse, and rat PXRs, suggesting overlap of function and perhaps common evolutionary forces. The Western clawed frog PXR, like that described for African clawed frog PXRs, has diverged considerably in ligand selectivity from fish, bird, and mammalian PXRs.

Crystal structure of the PXR–T1317 complex provides a scaffold to examine the potential for receptor antagonism

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Pregnane X receptor (PXR) is an orphan nuclear receptor that regulates the expression of genes encoding drug-metabolizing enzymes and transporters. In addition to affecting drug metabolism, potent and selective PXR agonists may also have therapeutic potential by removing endogenous and exogenous toxins. In this article, we report the synthesis and identification of novel PXR agonists from a library of peptide isosteres. Compound S20, a C-cyclopropylalkylamide, was found to be a PXR agonist with both enantiomer- and species-specific selectivity. S20 has three chiral carbons and was resolved into its two enantiomers. The individual S20 enantiomers exhibited striking mouse/human-specific PXR activation, whereby enantiomer (+)-S20 preferentially activated hPXR, and enantiomer (-)-S20 was a better activator for mPXR. As a human PXR (hPXR) agonist, (+)-S20 was more potent and efficacious than rifampicin. Mutagenesis studies revealed that the ligand binding domain residue Phe305 is critical for the preference for the (-)-S20 enantiomer by the rodent PXR. Treatment of S20 induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays, in primary human hepatocytes, and in "humanized" hPXR transgenic mice. To our knowledge, S20 represents the first compound whose enantiomers have opposite species preference in activating a xenobiotic receptor. The stereoselectivity may be used to guide the development of safer drugs to avoid drug-drug interactions or to achieve human-specific therapeutic effects when a xenobiotic receptor is being used as a drug target.

The most common clinical implication for the activation of the human pregnane X receptor (PXR) is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in Pxr-null mice using a BAC clone containing the complete human PXR gene and 5'- and 3'-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an approximately 60% decrease was observed for both the maximal midazolam serum concentration (C(max)) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1'-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off‐target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first‐in‐class non‐cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR‐specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro‐inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

BackgroundThe pregnane X receptor (PXR) is a xenobiotic sensing nuclear receptor that is activated by a diverse array of substances, including environmental toxins, pharmaceutical compounds and metabolites released from the intestinal microbiota. While the PXR's prototypical role is to regulate the expression of the drug metabolizing/detoxifying genes in the liver and intestinal epithelium, we and others have reported that it plays a role in regulating inflammatory signaling. More specifically, the PXR can inhibit NFκB‐dependent inflammatory signaling and regulates the function/expression of innate immune receptors, including TLR4 and NLRP3. In the current study, we sought to characterize the function of the PXR in macrophages. Given the interactions between the PXR and innate immune receptors described in other non‐immune cell types, we hypothesized that its activation would modulate NLRP3 inflammasome activation and the resulting processing/release of IL‐1β.AimsTo evaluate the effect of PXR activation of NLRP3 inflammasome activation and to determine the mechanism(s) whereby the PXR modulates inflammasome activity.MethodsMouse peritoneal macrophages and PMA‐differentiated THP‐1 cells were used to assess NLRP3 inflammasome activation. Due to species‐specific ligand interactions with the PXR, in mouse studies we used pregnenolone 16α‐carbonitrile (PCN), whereas in THP‐1 experiments, we used the human PXR agonists, rifaximin and SR12813. To test if PXR activation modulated NLRP3 inflammasome activation, LPS‐primed macrophages or PMA‐differentiated THP‐1 were pretreated for one hour with PXR agonists and then challenged with ATP (5mM), a known NLRP3 inflammasome activator. Caspase‐1 cleavage and IL‐1β secretion were measured to assess inflammasome activation. In some experiments, extracellular ATP was measured following PXR agonist treatment.ResultsWhile PXR activation had no effect on ATP‐induced NLRP3 inflammasome activation, each PXR agonist alone stimulated the cleavage of caspase‐1 and the secretion of IL‐1ββ, reminiscent of inflammasome activation. In PXR−/− macrophages, these responses were absent, supporting a direct role of the PXR in these responses. Deletion of NLRP3 in mouse macrophages and THP‐1 cells attenuated PXR agonist‐induced caspase‐1 cleavage and IL‐1β secretion, suggesting that PXR activation triggers NLRP3 inflammasome activation. Furthermore, PXR driven responses were attenuated following caspase‐1 inhibition. Mechanistically, NLRP3 activation by the PXR did not involve ROS production, nor was it sensitive to chelation of intracellular calcium. However, treating cells with apyrase, to catabolize extracellular ATP, or selective inhibition of the P2X7 receptor attenuated PXR agonist‐induced caspase‐1 activation and IL‐1β secretion. Interestingly, we subsequently found that PXR activation led to a rapid (within 15 seconds) increase in extracellular ATP, reaching levels previous described in NLRP3 inflammasome activation models. This response was absent in PXR−/− cells, supporting a direct role of the PXR in ATP release.ConclusionsTaken together, our data suggest that activation of the xenobiotic sensing PXR triggers the release of ATP, which in turn causes NLRP3 inflammasome activation in macrophages. These findings suggest a novel mechanism whereby non‐microbial/non‐viral agents of environmental origin can stimulate innate immune responses and contribute to inflammatory disease.Support or Funding InformationCrohn's and Colitis CanadaNSERCLloyd Sutherland Investigatorship in IBD/GI researchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.