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We examined the effects of gut microbial catabolites of tryptophan on the aryl hydrocarbon receptor (AhR). Using a reporter gene assay, we show that all studied catabolites are low-potency agonists of human AhR. The efficacy of catabolites differed substantially, comprising agonists with no or low (i3-propionate, i3-acetate, i3-lactate, i3-aldehyde), medium (i3-ethanol, i3-acrylate, skatole, tryptamine), and high (indole, i3-acetamide, i3-pyruvate) efficacies. We displayed ligand-selective antagonist activities by i3-pyruvate, i3-aldehyde, indole, skatole, and tryptamine. Ligand binding assay identified low affinity (skatole, i3-pyruvate, and i3-acetamide) and very low affinity (i3-acrylate, i3-ethanol, indole) ligands of the murine AhR. Indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, and i3-acetamide induced CYP1A1 mRNA in intestinal LS180 and HT-29 cells, but not in the AhR-knockout HT-29 variant. We observed a similar CYP1A1 induction pattern in primary human hepatocytes. The most AhR-active catabolites (indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, i3-acetamide) elicited nuclear translocation of the AhR, followed by a formation of AhR-ARNT heterodimer and enhanced binding of the AhR to the CYP1A1 gene promoter. Collectively, we comprehensively characterized the interactions of gut microbial tryptophan catabolites with the AhR, which may expand the current understanding of their potential roles in intestinal health and disease.

The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1 . As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR. The aryl hydrocarbon receptor regulates the expression of genes involved in many cell processes and its dysregulation has been implicated in different diseases. Here, the authors identify dietary monoterpenoid carvone as an atypical non-competitive antagonist of human aryl hydrocarbon receptor and demonstrate that it can protect against ultraviolet skin damage in female mice.

The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

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. Synopsis This study demonstrates that microbial metabolite mimicry can expand the chemical space in drug discovery. Chemical mimics of microbial indoles interacting with a host nuclear receptor provides a novel and non‐toxic therapeutic approach for treating inflammatory conditions of the intestine. The hybrid structure based (HSB) method utilized the interactions of both IPA and indole in the ligand‐binding domain (LBD) of PXR. The resulting pharmacophore was screened, and ranking by individual docking score resulted in core indole structures that were simplified using intermediates of their synthetic pathway. The two identified lead molecules FKK5 and FKK6 directly bound the ligand‐binding pocket of human PXR and induced a PXR‐dependent gene expression profile in cells and tissues. Mice expressing the human PXR gene (hPXR) significantly expressed PXR target genes upon dosing with FKK6. FKK6 abrogated inflammation in a chemical model of murine colitis in a PXR‐ dependent manner. Graphical Abstract This study demonstrates that microbial metabolite mimicry can expand the chemical space in drug discovery. Chemical mimics of microbial indoles interacting with a host nuclear receptor provides a novel and non‐toxic therapeutic approach for treating inflammatory conditions of the intestine.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

Chronic intestinal inflammation significantly contributes to the development of colorectal cancer (CRC) and remains a pertinent clinical challenge, necessitating novel therapeutic approaches. Indole-based microbial metabolite mimics FKK6, which is a ligand and agonist of the pregnane X receptor (PXR), was recently demonstrated to have PXR-dependent anti-inflammatory and protective effects in a mouse model of dextran sodium sulfate (DSS)-induced acute colitis. Here, we examined the therapeutic potential of FKK6 in a mouse model (C57BL/6 FVB humanized PXR mice) of colitis-associated colon cancer (CAC) induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). FKK6 (2 mg/kg) displayed substantial anti-tumor activity, as revealed by reduced size and number of colon tumors, improved colon histopathology, and decreased expression of tumor markers (c-MYC, β-catenin, Ki-67, cyclin D) in the colon. In addition, we carried out the chronic toxicity (30 days) assessment of FKK6 (1 mg/kg and 2 mg/kg) in C57BL/6 mice. Histological examination of tissues, biochemical blood analyses, and immunohistochemical staining for Ki-67 and γ-H2AX showed no difference between FKK6-treated and control mice. Comparative metabolomic analyses in mice exposed for 5 days to DSS and administered with FKK6 (0.4 mg/kg) revealed no significant effects on several classes of metabolites in the mouse fecal metabolome. Ames and micronucleus tests showed no genotoxic and mutagenic potential of FKK6 . In conclusion, anticancer effects of FKK6 in AOM/DSS-induced CAC, together with FKK6 safety data from tests and chronic toxicity study, and comparative metabolomic study, are supportive of the potential therapeutic use of FKK6 in the treatment of CAC.

Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we performed the initial in vitro pharmacological profiling of FKK6. A complex series of cell-free and cell-based assays were employed. The organic synthesis, and advanced analytical chemistry methods were used. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. FKK6 was rapidly metabolized by cytochromes P450, notably by CYP3A4 in human liver microsomes. Two oxidized FKK6 derivatives, including N6-oxide and C19-phenol, were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. The PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development. What is already known: Microbial metabolite mimic FKK6 is a hPXR agonist with anti-inflammatory properties in mice and human.The in vitro PXR binding, absorption, and metabolism have not been completely characterized. What this study adds: PXR selectivity with unique binding mode, high intestinal cell permeability, rapid and complex microsomal metabolism.Initial testing for predicted metabolites shows reduced potency as PXR agonists. Clinical significance: PXR effects of FKK6 are predicted to be predominantly in the intestines.FKK6 has a suitable pharmacological profile supporting its potential preclinical development. Microbial metabolite mimic FKK6 is a hPXR agonist with anti-inflammatory properties in mice and human. The in vitro PXR binding, absorption, and metabolism have not been completely characterized. PXR selectivity with unique binding mode, high intestinal cell permeability, rapid and complex microsomal metabolism. Initial testing for predicted metabolites shows reduced potency as PXR agonists. PXR effects of FKK6 are predicted to be predominantly in the intestines. FKK6 has a suitable pharmacological profile supporting its potential preclinical development.

ABSTRACT Carvones, the constituents of essential oils of dill, caraway, and spearmint, were reported to antagonize the human aryl hydrocarbon receptor (AhR); however, the exact molecular mechanism remains elusive. We show that carvones are non-competitive allosteric antagonists of the AhR that inhibit the induction of AhR target genes in a ligand-selective and cell type-specific manner. Carvones do not displace radiolabeled ligand from binding at the AhR, but they bind allosterically within the bHLH/PAS-A region of the AhR. Carvones did not influence a translocation of ligand-activated AhR into the nucleus. Carvones inhibited the heterodimerization of the AhR with its canonical partner ARNT and subsequent binding of the AhR to the promotor of CYP1A1. Interaction of carvones with potential off-targets, including ARNT and protein kinases, was refuted. This is the first report of a small dietary monoterpenoids as a new class of AhR non-competitive allosteric antagonists with the potential preventive and therapeutic application. Competing Interest Statement The authors have declared no competing interest.

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 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. Footnotes * Data included at the request of reviewers