Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry
ABSTRACT: The human pregnane X receptor (PXR), a master regulator of drug metabolism, has important 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 to exploit 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, 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 human intestinal organoids and Caco-2 expressing the human PXR gene. These confocal images show FKK6 reduces NfKB translocation to the nucelus of intestinal organoids and caco-2 incubated with pro-inflammatory cocktail.
Project description: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.
Project description:The human pregnane X receptor (PXR), 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, 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 mine underexploited regions of chemical space.
Project description: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-? 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.
Project description: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 ?-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.
Project description:Intestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.
Project description:BACKGROUND:The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. METHODS:We stimulate a total of 106 colonic biopsies from 19 Crohn's disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-?B activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) mRNA levels by qPCR and in cell culture models by NF-?B reporter-driven luciferase activity and Western blot for signal transduction elements. RESULTS:We observe a strict inverse correlation between colonic epithelial PXR levels and NF-?B target gene expression in colonic biopsies from Crohn's disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-?B activation in IBD. The correlation between colonic epithelial PXR levels and NF-?B target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-?B-dependent signal transduction whereas conversely NF-?B signaling reduces levels of PXR expression. CONCLUSIONS:Our data indicate that the PXR is a major and clinically relevant antagonist of NF-?B activity in the intestinal epithelial compartment during inflammatory bowel disease.
Project description:BACKGROUND AND PURPOSE: P-glycoprotein (P-gp) is an important efflux transporter that supports the barrier function of the gut against invading antigens and against administered drugs. Since glucocorticoids, such as budesonide, are frequently used during inflammatory bowel disease we investigated how budesonide influences P-gp expression in different intestinal cell lines. EXPERIMENTAL APPROACH: LS180 and Caco-2 cells were incubated with budesonide and changes in P-gp expression were determined on mRNA, protein and functional level. The mRNA expression levels of glucocorticoid receptor (GR) and pregnane X receptor (PXR) were determined in these cell lines. PXR receptor was transiently transfected into Caco-2 cells. KEY RESULTS: Budesonide showed an induction of P-gp in LS180 cells and a down-regulation in Caco-2 cells. Expression levels of nuclear receptors revealed high expression of PXR only in LS180 cells and exclusive expression of GR in Caco-2 cells. Mifepristone, an anti-glucocorticoid, could not reverse the down-regulation of P-gp by budesonide in Caco-2 cells. In PXR-transfected Caco-2 cells the budesonide-mediated down-regulation of P-gp was abolished. Furthermore the expression of cytochrome P450 3A4 (CYP3A4), another PXR target gene, was induced in PXR-transfected Caco-2 cells after budesonide treatment. CONCLUSIONS AND IMPLICATIONS: Budesonide has the potential to influence MDR1 expression in vitro. In LS180 cells, the induction of MDR1 by budesonide probably is mediated via PXR. The mechanism of the down-regulation in Caco-2 cells still remains unclear, but GR does not seem to be involved. Further studies are required to evaluate how budesonide alters P-gp expression in vivo.
Project description:The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the <i>in vivo</i> human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.
Project description:The regeneration of intestinal epithelial are maintained by continuous differentiation and proliferation of intestinal stem cells (ISCs) under physiological and pathological conditions. However, little is known about the regulatory effect of intestinal microbiota on its recovery ability to repair damaged mucosal barrier. In this study, we established intestinal organoids and lamina propria lymphocytes (LPLs) co-cultured system, plus mice experiments, to explore the protective effect of Lactobacillus reuteri D8 on integrity of intestinal mucosa. We found that only live L. reuteri D8 was effective in protecting the morphology of intestinal organoids and normal proliferation of epithelial stained with EdU under TNF-? treatment, which was also further verified in mice experiments. L. reuteri D8 colonized in the intestinal mucosa and ameliorated intestinal mucosa damage caused by DSS treatment, including improvement of body weight, colon length, pathological change, and proliferation level. The repair process stimulated by L. reuteri D8 was also accompanied with increased numbers of Lgr5+ and lysozyme+ cells both in intestinal organoids and mice intestine. Furthermore, we demonstrated that D8 metabolite indole-3-aldehyde stimulated LPLs to secret IL-22 through aryl hydrocarbon receptor (AhR) and then induced phosphorylation of STAT3 to accelerate proliferation of intestinal epithelial, thus recovering damaged intestinal mucosa. Our findings indicate L. reuteri protects intestinal barrier and activates intestinal epithelial proliferation, which sheds light on treatment approaches for intestinal inflammation based on ISCs with probiotics Lactobacillus and daily probiotic consumption in heath foods.