Pregnane X Receptor Ligand-binding Domain Research Articles

The F-box-only protein 44 regulates pregnane X receptor protein level by ubiquitination and degradation.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug-drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.

Development and Experimental Validation of Regularized Machine Learning Models Detecting New, Structurally Distinct Activators of PXR.

The pregnane X receptor (PXR) regulates the metabolism of many xenobiotic and endobiotic substances. In consequence, PXR decreases the efficacy of many small-molecule drugs and induces drug-drug interactions. The prediction of PXR activators with theoretical approaches such as machine learning (ML) proves challenging due to the ligand promiscuity of PXR, which is related to its large and flexible binding pocket. In this work we demonstrate, by the example of random forest models and support vector machines, that classifiers generated following classical training procedures often fail to predict PXR activity for compounds that are dissimilar from those in the training set. We present a novel regularization technique that penalizes the gap between a model’s training and validation performance. On a challenging test set, this technique led to improvements in Matthew correlation coefficients (MCCs) by up to 0.21. Using these regularized ML models, we selected 31 compounds that are structurally distinct from known PXR ligands for experimental validation. Twelve of them were confirmed as active in the cellular PXR ligand-binding domain assembly assay and more hits were identified during follow-up studies. Comprehensive analysis of key features of PXR biology conducted for three representative hits confirmed their ability to activate the PXR.

Discrepancy in interactions and conformational dynamics of pregnane X receptor (PXR) bound to an agonist and a novel competitive antagonist

Pregnane X receptor (PXR) is a nuclear receptor with an essential role in regulating drug metabolism genes. While the mechanism of action for ligand-mediated PXR agonism is well-examined, its ligand-mediated inhibition or antagonism is poorly understood. Here we employ microsecond timescale all-atom molecular dynamics (MD) simulations to investigate how our newly identified dual kinase and PXR inhibitor, compound 100, acts as a competitive PXR antagonist and not as a full agonist. We study the PXR ligand binding domain conformational changes associated with compound 100 and compare the results to the full agonist SR12813, in presence and absence of the coactivator. Furthermore, we complement our research by experimentally disclosing the effect of eight key-residue mutations on PXR activation. Finally, simulations of P2X4 inhibitor (BAY-1797) in complex with PXR, which shares an identical structural moiety with compound 100, provide further insights to ligand-induced PXR behaviour. Our MD data suggests ligand-specific influence on conformations of different PXR-LBD regions, including α6 region, αAF-2, α1-α2′, β1′-α3 and β1-β1′ loop. Our results provide important insights on conformational behaviour of PXR and offers guidance how to alleviate PXR agonism or to promote PXR antagonism.

PXR and Metabolism

Pregnane X receptor (PXR) is a nuclear receptor initially characterized as a xenobiotic-sensing receptor that transcriptionally induces drug-metabolizing enzymes and transporters to control the disposition of xenobiotics. Emerging data reveal PXR's broader roles in regulating glucose and lipid metabolism to control metabolic homeostasis. In addition, there has been evidence of crosstalk among the PXR-regulated metabolism of drugs, lipids, and glucose. Such crosstalk might play roles in the development of diseases such as diabetes and cancer, and the therapeutic and toxic effects of drugs. The activity of PXR can be modulated by many structurally diverse chemicals. However, potent and selective chemical modulators of PXR was lacking. By using chemical biology, structural biology, and pharmacology approaches, we have developed a series of potent, selective and structurally related PXR modulators (i.e., agonists, antagonists, and inverse agonists). We discovered that subtle structural modifications of the compounds, or a mutation within the PXR ligand-binding domain can convert an antagonist to an agonist. While ligand binding affinity does not predict PXR's cellular activity, there is a correlation between ligand-induced positioning of the activation function 2 (AF-2) helix and PXR's transcriptional activity. We are using these novel chemical probes to investigate the broad metabolic function of PXR, with a goal to use them to develop therapies for PXR-regulated diseases.

Identification of novel pregnane X receptor (PXR) agonists by In silico and biological activity analyses and reversal of cigarette smoke-induced PXR downregulation

Cigarette smoke (CS) contains many toxins that collectively harm nearly every organ in the body, and smoking is a key risk factor for many chronic diseases. Aside from its toxic actions, CS may alter expression of the drug- and steroid-binding pregnane X receptor (PXR), which when activated upregulates expression of cytochrome P450 (CYP) enzymes, glutathione transferases (GSTs), and multidrug resistance protein 1 (MDR1), an adaptive metabolic array that mediates clearance of CS component toxins. We sought to identify new PXR agonists that may be useful for restoring PXR activity in conditions wherein it is suppressed, and their mechanisms of PXR binding and activation. PXR has a uniquely larger, hydrophobic, and highly flexible ligand-binding domain (LBD) vs. other nuclear receptors, enabling it to interact with structurally diverse molecules. We tested certain calcium channel blockers (CCBs) as a pharmacological subset of potential PXR ligands, analyzing by molecular docking methods, and identified a putative active site in the PXR LBD, along with the relevant bonds and bonding energies. We analyzed felodipine binding and agonist activity in detail, as it showed the lowest binding energy among CCBs tested. We found felodipine was a potent PXR agonist as measured by luciferase reporter assay, whereas CCBs with higher binding energies were less potent (amlodipine) or nearly inactive (manidipine), and it induced CYP3A4 expression in HepG2 cells, a known target of PXR agonism. Felodipine also both induced PXR mRNA in HepG2 hepatocytes and reduced CS extract-induced diminution of PXR levels, indicating it modulates PXR expression. The results illuminate mechanisms of ligand-induced PXR activation and identify felodipine as a novel PXR agonist.

Nelfinavir and Its Active Metabolite M8 Are Partial Agonists and Competitive Antagonists of the Human Pregnane X Receptor.

The HIV protease inhibitor nelfinavir is currently being analyzed for repurposing as an anticancer drug for many different cancers because it exerts manifold off-target protein interactions, finally resulting in cancer cell death. Xenosensing pregnane X receptor (PXR), which also participates in the control of cancer cell proliferation and apoptosis, was previously shown to be activated by nelfinavir; however, the exact molecular mechanism is still unknown. The present study addresses the effects of nelfinavir and its major and pharmacologically active metabolite nelfinavir hydroxy-tert-butylamide (M8) on PXR to elucidate the underlying molecular mechanism. Molecular docking suggested direct binding to the PXR ligand-binding domain, which was confirmed experimentally by limited proteolytic digestion and competitive ligand-binding assays. Concentration-response analyses using cellular transactivation assays identified nelfinavir and M8 as partial agonists with EC50 values of 0.9 and 7.3 µM and competitive antagonists of rifampin-dependent induction with IC50 values of 7.5 and 25.3 µM, respectively. Antagonism exclusively resulted from binding into the PXR ligand-binding pocket. Impaired coactivator recruitment by nelfinavir as compared with the full agonist rifampin proved to be the underlying mechanism of both effects on PXR. Physiologic relevance of nelfinavir-dependent modulation of PXR activity was investigated in respectively treated primary human hepatocytes, which showed differential induction of PXR target genes and antagonism of rifampin-induced ABCB1 and CYP3A4 gene expression. In conclusion, we elucidate here the molecular mechanism of nelfinavir interaction with PXR. It is hypothesized that modulation of PXR activity may impact the anticancer effects of nelfinavir. SIGNIFICANCE STATEMENT: Nelfinavir, which is being investigated for repurposing as an anticancer medication, is shown here to directly bind to human pregnane X receptor (PXR) and thereby act as a partial agonist and competitive antagonist. Its major metabolite nelfinavir hydroxy-tert-butylamide exerts the same effects, which are based on impaired coactivator recruitment. Nelfinavir anticancer activity may involve modulation of PXR, which itself is discussed as a therapeutic target in cancer therapy and for the reversal of chemoresistance.

Structure-based amelioration of PXR transactivation in a novel series of macrocyclic allosteric inhibitors of HIV-1 integrase

Previous studies have identified a series of imidazo[1,2-a]pyridine (IZP) derivatives as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) and virus infection in cell culture. However, IZPs were also found to be relatively potent activators of the pregnane-X receptor (PXR), raising the specter of induction of CYP-mediated drug disposition pathways. In an attempt to modify PXR activity without affecting anti-HIV-1 activity, rational structure-based design and modeling approaches were used. An X-ray cocrystal structure of (S,S)-1 in the PXR ligand binding domain (LBD) allowed an examination of the potential of rational structural modifications designed to abrogate PXR. The introduction of bulky basic amines at the C-8 position provided macrocyclic IZP derivatives that displayed potent HIV-1 inhibitory activity in cell culture with no detectable PXR transactivation at the highest concentration tested.

Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles.

Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.

Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

QSAR development and profiling of 72,524 REACH substances for PXR activation and CYP3A4 induction

The Pregnane X Receptor (PXR) is a key regulator of enzymes, for example the cytochrome P450 isoform 3A4 (CYP3A4), and transporters involved in the metabolism and excretion of xenobiotics and endogenous compounds. Activation of PXR by xenobiotics causes altered protein expression leading to enhanced or decreased turnover of both xenobiotics and endogenous compounds. This can potentially result in perturbations of normal physiology and adverse effects. Identification of PXR activating and CYP3A4 inducing compounds is included in drug-discovery programs but we still need similar information for the remaining tens-of-thousands of man-made compounds to which humans are potentially exposed. In the present study, we used high-throughput in vitro assay results for 2816 drugs to develop four quantitative structure-activity relationship (QSAR) models with binary outputs for binding to the human PXR ligand binding domain, full-length human and rat PXR activation and human CYP3A4 induction, respectively. Rigorous cross- and blinded external validations demonstrated four robust and highly predictive models with balanced accuracies ranging from 75.4% to 92.7%. The models were applied to screen 72,524 substances pre-registered under the EU chemicals regulation, REACH, and the models could predict 52.5% to 71.9% of the substances within their respective applicability domains. These predictions can, for example, be used for priority setting and in weight-of-evidence assessments of chemicals. Statistical analyses of the experimental drug dataset and the QSAR-predicted set of REACH substances were performed to identify similarities and differences in frequencies of overlapping positive results for PXR binding, PXR activation and CYP3A4 induction between the two datasets.

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket.

Ligand-regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug-drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely "expanded" and "contracted", in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single "ligand-adaptable" conformational state. Ensemble-based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named "α8 pocket", whose opening-closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure-based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a "tightly-contracted" conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism.

Developing Adnectins That Target SRC Co-Activator Binding to PXR: A Structural Approach toward Understanding Promiscuity of PXR

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated “Adnectins”, derived from 10th fibronectin type III domain (10Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.

Carboxymefloquine, the major metabolite of the antimalarial drug mefloquine, induces drug-metabolizing enzyme and transporter expression by activation of pregnane X receptor.

Malaria patients are frequently coinfected with HIV and mycobacteria causing tuberculosis, which increases the use of coadministered drugs and thereby enhances the risk of pharmacokinetic drug-drug interactions. Activation of the pregnane X receptor (PXR) by xenobiotics, which include many drugs, induces drug metabolism and transport, thereby resulting in possible attenuation or loss of the therapeutic responses to the drugs being coadministered. While several artemisinin-type antimalarial drugs have been shown to activate PXR, data on nonartemisinin-type antimalarials are still missing. Therefore, this study aimed to elucidate the potential of nonartemisinin antimalarial drugs and drug metabolites to activate PXR. We screened 16 clinically used antimalarial drugs and six major drug metabolites for binding to PXR using the two-hybrid PXR ligand binding domain assembly assay; this identified carboxymefloquine, the major and pharmacologically inactive metabolite of the antimalarial drug mefloquine, as a potential PXR ligand. Two-hybrid PXR-coactivator and -corepressor interaction assays and PXR-dependent promoter reporter gene assays confirmed carboxymefloquine to be a novel PXR agonist which specifically activated the human receptor. In the PXR-expressing intestinal LS174T cells and in primary human hepatocytes, carboxymefloquine induced the expression of drug-metabolizing enzymes and transporters on the mRNA and protein levels. The crucial role of PXR for the carboxymefloquine-dependent induction of gene expression was confirmed by small interfering RNA (siRNA)-mediated knockdown of the receptor. Thus, the clinical use of mefloquine may result in pharmacokinetic drug-drug interactions by means of its metabolite carboxymefloquine. Whether these in vitro findings are of in vivo relevance has to be addressed in future clinical drug-drug interaction studies.

The role of residues T248, Y249 and T422 in the function of human pregnane X receptor

The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates the expression of numerous drug-metabolizing enzymes. Some posttranslational mechanisms modulate its transcriptional activity. Although several kinases have been shown to directly phosphorylate this receptor, little is known about phosphorylation sites of PXR. In the present work, we examined T248, Y249 and T422 putative phosphorylation sites determined based on in silico consensus kinase site prediction analysis. T248 and T422 residues are critical for the interaction of the PXR ligand-binding domain and the activation function-2 (AF2) domain. Site-directed mutagenesis analysis was performed to generate phospho-deficient and phospho-mimetic mutants. We examined transactivation activity of the PXR mutants in gene reporter assays, formation of PXRmutant/RXRα heterodimer, binding of PXR mutants to the CYP3A4 gene response element DR3 and CYP3A4 expression in HepG2 cells after expression of the mutants. We found that T248D mutant activated CYP3A4 transactivation constitutively regardless of the presence or absence of a ligand. Contrary, T248V mutant exhibited low basal and ligand-inducible transactivation capacity as compared to wild-type PXR. Dose-response analysis revealed reduced ligand-dependent transactivation potency of PXR Y249D mutant. Transactivation of the CYP3A4 promoter was abolished with T422A/D mutants. All PXR mutants formed heterodimer with RXRα at a similar level to that observed with wild-type PXR. The ability to bind to DNA in vitro was substantially decreased in case of T248D, T422D and T248V mutants. Our data thus indicate that phosphorylation of T248, Y249 and T422 residues may be critical for the both basal and ligand-activated function of PXR.

Rifampicin - Independent Interactions Between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Co-Activator and Co-Repressor Proteins

The pregnane X receptor (PXR), a member of the nuclear receptor family, regulates the transcription of target genes whose products are involved in the metabolism of endogenous and exogenous compounds, including many pharmaceutical compounds. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for co-activator proteins, while decreasing its affinity for co-repressors. Here, we report the use of steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain (PXR-LBD) and a fluorescently labeled, peptide fragment of the steroid receptor co-activator 1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of about 5 μM and 2 s−1, respectively, were obtained both in the presence and absence of rifampicin. Additionally, TIRFM was used to examine the interaction between PXR-LBD and peptide fragments of the co-repressor proteins, nuclear receptor co-repressor (NCoR) and silencing mediator for retinoid or thyroid receptors (SMRT), in the presence and absence of rifampicin. Equilibrium dissociation constants of about 170 μM and 70 μM were obtained for NCoR and SMRT, respectively, in the presence and absence of rifampicin. These results indicate that rifampicin does not alter the affinity of PXR-LBD for co-activator and co-repressor fragments, thereby suggesting that the mechanism of PXR action differs markedly from that of other nuclear receptors.

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s(-1), respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.

Activation of the human nuclear xenobiotic receptor PXR by the reverse transcriptase-targeted anti-HIV drug PNU-142721.

The human pregnane X receptor (PXR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. PXR responds to a structurally diverse variety of endogenous and xenobiotic compounds, and coordinates the expression of genes central to the metabolism and excretion of potentially harmful chemicals, including human therapeutics. The reverse transcriptase inhibitor PNU-142721 has been designed to treat human immunodeficiency virus (HIV) infection. Although this compound has anti-HIV activity, it was established using cell-based assays that PNU-142721 is an efficacious PXR agonist. We present here the 2.8 Å resolution crystal structure of the human PXR ligand-binding domain in complex with PNU-142721. PXR employs one hydrogen bond and fourteen van der Waals contacts to interact with the ligand, but allows two loops adjacent to the ligand-binding pocket to remain disordered in the structure. These observations highlight the role structural flexibility plays in PXR's promiscuous responses to xenobiotics. The crystal structure also explains why PNU-173575, a thiomethyl metabolite of PNU-142721, exhibits enhanced PXR activation relative to the unmodified compound and why PNU-142721 can also activate rat PXR. Taken together, the results presented here elucidate the structural basis for PXR activation by PNU-142721 and related chemicals.

Nuclear Receptor (PXR), Ligand and Co-Activator Interactions Measured by Total Internal Reflection Fluorescence Microscopy

The pregnane X receptor (PXR) is a member of the nuclear receptor family. PXR acts in a ligand-dependent manner, in concert with its dimerization partner, the retinoid X receptor, and co-regulators, to modulate the expression of proteins that metabolize endogenous and exogenous compounds. Here, we report the use of total internal reflection fluorescence microscopy (TIRFM) for examining interactions between the ligand-binding domain of PXR (PXR-LBD), its ligands, and a peptide derived from a co-activator. TIRFM is a surface-specific technique that enables us to study the behavior of fluorescent species close to or at interfaces. Our experimental system consists of biotinylated PXR-LBD immobilized on a fused silica substrate coated with NeutrAvidin and ovalbumin, a ligand (e.g., the antibiotic rifampicin) and a fluorescently labeled, 25 amino acid fragment of the steroid receptor co-activator-1 (F-SRC-1). Using TIRFM, we measured the surface-associated fluorescence as a function of the F-SRC-1 concentration at fixed ligand concentrations. These curves were fit to a model of single-site binding to obtain apparent equilibrium constants. The apparent equilibrium constants as a function of ligand concentration were fit to an appropriate model to obtain binding constants for the ligand/PXR-LBD interaction, F-SRC-1 binding to apo and ligand-bound PXR-LBD and ligand binding to F-SRC-1-bound PXR-LBD. The approach yielded four, previously unmeasured, binding constants. These values indicate that the increase in PXR's affinity for its co-activator peptide upon binding an activating ligand is modest. This observation implies that for gene expression to be significantly upregulated, the signal arising from the activation of PXR must be amplified downstream - possibly when co-activators like SRC-1 start recruiting the cell's transcription machinery. Kinetic data obtained by combining TIR illumination with fluorescence recovery after photobleaching and/or fluorescence correlation spectroscopy may also be discussed.

Microcantilever-based Nanomechanical Studies of the Orphan Nuclear Receptor Pregnane X Receptor-Ligand Interactions

Human pregnane X receptor (PXR) is of vital importance in pharmaceutical and exogenous compound metabolism within the body. This provides strong motivation for investigating this orphan receptor’s activation by various pharmaceuticals, xenobiotics, and endocrine disrupting chemicals (EDCs). A nanomechanical transducer is developed to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the sensitive, label-free study of ligand interaction with the receptor. PXR shows real-time, reversible responses when exposed to a specific pharmaceutical, EDCs, and xenobiotic ligands. Three EDCs binding interactions are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR. PXR LBD was exposed to rifampicin, a potent PXR activator, with various injection and recovery times to study their interaction. A two protein array of PXR and estrogen receptor ? (ER-?) directly compares the nanomechanical responses of these receptors with rifampicin on a single platform.