Targeting the Aryl Hydrocarbon Receptor with Microbial Metabolite Mimics Alleviates Experimental Colitis in Mice.

Targeting the aryl hydrocarbon receptor (AhR) is an emerging therapeutic strategy for multiple diseases (e.g., inflammatory bowel disease). Thermosporothrix hazakensis microbial metabolite 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a putative AhR endogenous ligand. To improve the chemical stability, we synthesized a series of ITE chemical mimics. Using a series of in vitro assays, we identified 2-(1H-indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH3) as a highly potent (EC50 = 1.6 nM) AhR agonist with high affinity (Ki = 88 nM). ITE-CONHCH3 triggered AhR nuclear translocation and dimerization of AhR-ARNT, enhanced AhR binding in the CYP1A1 promoter, and induced AhR-regulated genes in an AhR-dependent manner. The metabolic stability of ITE-CONHCH3 in a cell culture was 10 times higher than that of ITE. Finally, we observed protective effects of ITE-CONHCH3 in mice with DSS-induced colitis. Overall, we demonstrate and validate a concept of microbial metabolite mimicry in the therapeutic targeting of AhR.

Indole scaffolds as a promising class of the aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AhR), deemed initially as a xenobiotic sensor, plays multiple physiological roles and is involved in various pathophysiological processes and many diseases' etiology. Therefore, the therapeutic and chemopreventive targeting of AhR is a fundamental issue. To date, thousands of structurally diverse ligands of AhR have been identified. The bottleneck in targeting the AhR is that it is a Janus-faced player with beneficial vs. harmful effects in the ligand-specific context. A distinct structural class of the AhR ligands is those with indole-based scaffolds. The present review summarizes the knowledge on the existing indole-derived AhR ligands, comprising natural and dietary compounds, synthetic compounds including clinically used drugs, endogenous intermediary metabolites, and catabolites produced by human microbiota. The examples of novel, indole ring containing, rational design based AhR ligands are presented. The molecular, in vitro, and in vivo effects are described.

Indole microbial intestinal metabolites expand the repertoire of ligands and agonists of the human pregnane X receptor.

The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 µM) and IAD (IC50 10 µM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

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.

Targeting the pregnane X receptor using microbial metabolite mimicry.

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.

Mono-methylindoles induce CYP1A genes and inhibit CYP1A1 enzyme activity in human hepatocytes and HepaRG cells.

Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 µM to 23.8 µM and from 3.4 µM to 11.4 µM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.

Belinostat, at Its Clinically Relevant Concentrations, Inhibits Rifampicin-Induced CYP3A4 and MDR1 Gene Expression.

Activation of human pregnane X receptor (hPXR) has been associated with induction of chemoresistance. It has been proposed that such chemoresistance via cytochrome P450/drug transporters can be reversed with the use of antagonists that specifically abrogate agonist-mediated hPXR activation. Unfortunately, proposed antagonists lack the specificity and appropriate pharmacological characteristics that allow these features to be active in the clinic. We propose that, ideally, an hPXR antagonist would be a cancer drug itself that is part of a "cancer drug cocktail" and effective as an hPXR antagonist at therapeutic concentrations. Belinostat (BEL), a histone deacetylase inhibitor approved for the treatment of relapsed/refractory peripheral T-cell lymphoma, and often used in combination with chemotherapy, is an attractive candidate based on its hPXR ligand-like features. We sought to determine whether these features of BEL might allow it to behave as an antagonist in combination chemotherapy regimens that include hPXR activators. BEL represses agonist-activated hPXR target gene expression at its therapeutic concentrations in human primary hepatocytes and LS174T human colon cancer cells. BEL repressed rifampicin-induced gene expression of CYP3A4 and multidrug resistance protein 1, as well as their respective protein activities. BEL decreased rifampicin-induced resistance to SN-38, the active metabolite of irinotecan, in LS174T cells. This finding indicates that BEL could suppress hPXR agonist-induced chemoresistance. BEL attenuated the agonist-induced steroid receptor coactivator-1 interaction with hPXR, and, together with molecular docking studies, the study suggests that BEL directly interacts with multiple sites on hPXR. Taken together, our results suggest that BEL, at its clinically relevant therapeutic concentration, can antagonize hPXR agonist-induced gene expression and chemoresistance.