Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4.

Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2(-/-)) mice showed a distinctly "leaky" gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2(-/-)Tlr4(-/-) mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.

Synergistically acting PLA2: peptide hemorrhagic complex from Daboia russelii venom.

Snake venoms are complex mixture of enzymatic and non-enzymatic proteins. Non-covalent protein-protein interaction leads to protein complexes, which bring about enhanced pharmacological injuries by their synergistic action. Here we report identification and characterization of a new Daboia russelii hemorrhagic complex I (DR-HC-I) containing phospholipase A2 (PLA2) and non-enzymatic peptide. DR-HC-I was isolated from the venom of D. russelii by CM-Shepadex-C25 and gel permeation chromatography. Individual components were purified and identified by RP-HPL chromatography, mass spectrometry and N-terminal amino acid sequencing. DR-HC-I complex was lethal to mice with the LD50 dose of 0.7 mg/kg body weight with hemorrhagic and neurotoxic properties. DR-HC-I complex consists of non-hemorrhagic PLA2 and neurotoxic non-enzymatic peptide. The non-enzymatic peptide quenched the intrinsic fluorescence of PLA2 in a dose dependent manner, signifying the synergistic interaction between two proteins. PLA2 and peptide toxin in a 5:2 M ratio induced skin hemorrhage in mice with MHD 20 µg. However, addition of ANS (1-Anilino-8-naphthalene sulfonate) to DR-HC-I complex inhibited skin hemorrhagic effect and also synergic interaction. But there was no impact on PLA2 due to this synergistic interaction, and indirect hemolytic or plasma re-calcification activity. However, the synergistic interaction of PLA2 and non-enzymatic peptide contributes to the enhanced venom-induced hemorrhage and toxicity of Daboia russellii venom.

Purification, characterization, and chemical modification of neurotoxic peptide from Daboia russelii snake venom of India.

Comprehensive knowledge of venom composition is very important for effective management of snake envenomation and antivenom preparation. Daboia russelii venom from the eastern region of India is the most neurotoxic among the four venom samples investigated. From the eastern D. russelii venom sample, neurotoxic peptide has been purified by combined method of ion exchange gel permeation chromatography and reversed phase high performance liquid chromatography. Molecular weight of Daboia neurotoxin III (DNTx-III) found to be 6,849 Da (as measured on matrix-assisted laser desorption/ionisation-time of flight mass spectrometer), and N-terminal amino acid sequences is I K C F I T P D U T S Q A. Approximate LD50 dosage was 0.24 mg/kg body weight. It produced concentration- and time-dependent inhibition of indirectly stimulated twitches of Rana hexadactyla sciatic nerve gastrocnemius muscle preparations. Chemical modification of DNTx-III tryptophan residue(s) reduced the twitch height inhibition property of toxin, signifying the importance of tryptophan residues for the neurotoxic function. This type of neurotoxic peptide is unique to east Indian regional D. russelii venom.

Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR.

BACKGROUND: Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation. RESULTS: Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. CONCLUSION: Ketoconazole genetically interacts with specific PXR surface residues. SIGNIFICANCE: A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.

Alleviation of gut inflammation by Cdx2/Pxr pathway in a mouse model of chemical colitis.

Pregnane X Receptor (PXR), a master regulator of drug metabolism and inflammation, is abundantly expressed in the gastrointestinal tract. Baicalein and its O-glucuronide baicalin are potent anti-inflammatory and anti-cancer herbal flavonoids that undergo a complex cycle of interconversion in the liver and gut. We sought to investigate the role these flavonoids play in inhibiting gut inflammation by an axis involving PXR and other potential factors. The consequences of PXR regulation and activation by the herbal flavonoids, baicalein and baicalin were evaluated in vitro in human colon carcinoma cells and in vivo using wild-type, Pxr-null, and humanized (hPXR) PXR mice. Baicalein, but not its glucuronidated metabolite baicalin, activates PXR in a Cdx2-dependent manner in vitro, in human colon carcinoma LS174T cells, and in the murine colon in vivo. While both flavonoids abrogate dextran sodium sulfate (DSS)-mediated colon inflammation in vivo, oral delivery of a potent bacterial ß-glucuronidase inhibitor eliminates baicalin's effect on gastrointestinal inflammation by preventing the microbial conversion of baicalin to baicalien. Finally, reduction of gastrointestinal inflammation requires the binding of Cdx2 to a specific proximal site on the PXR promoter. Pharmacological targeting of intestinal PXR using natural metabolically labile ligands could serve as effective and potent therapeutics for gut inflammation that avert systemic drug interactions.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice.

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.

In vivo and in vitro characterization of a first-in-class novel azole analog that targets pregnane X receptor activation.

The pregnane X receptor (PXR) is a master regulator of xenobiotic clearance and is implicated in deleterious drug interactions (e.g., acetaminophen hepatotoxicity) and cancer drug resistance. However, small-molecule targeting of this receptor has been difficult; to date, directed synthesis of a relatively specific PXR inhibitor has remained elusive. Here we report the development and characterization of a first-in-class novel azole analog [1-(4-(4-(((2R,4S)-2-(2,4-difluorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone (FLB-12)] that antagonizes the activated state of PXR with limited effects on other related nuclear receptors (i.e., liver X receptor, farnesoid X receptor, estrogen receptor α, peroxisome proliferator-activated receptor γ, and mouse constitutive androstane receptor). We investigated the toxicity and PXR antagonist effect of FLB-12 in vivo. Compared with ketoconazole, a prototypical PXR antagonist, FLB-12 is significantly less toxic to hepatocytes. FLB-12 significantly inhibits the PXR-activated loss of righting reflex to 2,2,2-tribromoethanol (Avertin) in vivo, abrogates PXR-mediated resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in colon cancer cells in vitro, and attenuates PXR-mediated acetaminophen hepatotoxicity in vivo. Thus, relatively selective targeting of PXR by antagonists is feasible and warrants further investigation. This class of agents is suitable for development as chemical probes of PXR function as well as potential PXR-directed therapeutics.

Alleviating cancer drug toxicity by inhibiting a bacterial enzyme.

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial ß-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial ß-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial ß-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.

Expanding the roles for pregnane X receptor in cancer: proliferation and drug resistance in ovarian cancer.

PURPOSE: We examined the presence of the pregnane X receptor (PXR) and its effects on ovarian cancer cells after activation by its cognate ligand. EXPERIMENTAL DESIGN: SKOV-3 and OVCAR-8 ovarian carcinoma cells were analyzed for expression of PXR by quantitative reverse transcription-PCR and Western blot. Human ovarian cancer tissue was also analyzed for PXR expression by immunochemistry. Ligand (agonist)-induced PXR target genes were analyzed in SKOV-3 cells by quantitative reverse transcription-PCR. SKOV-3 cell proliferation was assessed by MTT assay. In vivo confirmation of in vitro effects of PXR ligands were done in NOD.SCID mice carrying SKOV-3 xenografts. RESULTS: PXR is expressed in ovarian cancer cells. In SKOV-3 cells, PXR is functional and its activation by cognate ligands induces PXR target genes (CYP2B6, CYP3A4, and UGT1A1) but not MDR1 and MRP2. PXR activation in SKOV-3 cells induces cell proliferation and drug resistance. In mice harboring SKOV-3 xenografts, rifampicin (PXR agonist) induces cell proliferation and tumor growth. CONCLUSION: PXR activation, regardless of the type of ligand agonist present, promotes the "malignant" phenotype of cancer cells. These data serve as the basis for finding novel nontoxic inhibitors of PXR activation as a method to control cell growth and prevent induction of drug resistance.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

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.