Deciphering structural bases of intestinal and hepatic selectivity in targeting pregnane X receptor with indole-based microbial mimics.

Microbial metabolite mimicry is a new concept that promises to deliver compounds that have minimal liabilities and enhanced therapeutic effects in a host. In a previous publication, we have shown that microbial metabolites of L-tryptophan, indoles, when chemically altered, yielded potent anti-inflammatory pregnane X Receptor (PXR)-targeting lead compounds, FKK5 and FKK6, targeting intestinal inflammation. Our aim in this study was to further define structure-activity relationships between indole analogs and PXR, we removed the phenyl-sulfonyl group or replaced the pyridyl residue with imidazolopyridyl of FKK6. Our results showed that while removal of the phenyl-sulfonyl group from FKK6 (now called CVK003) shifts agonist activity away from PXR towards the aryl hydrocarbon receptor (AhR), the imidazolopyridyl addition preserves PXR activity in vitro. However, when these compounds are administered to mice, that unlike the parent molecule, FKK6, they exhibit poor induction of PXR target genes in the intestines and the liver. These data suggest that modifications of FKK6 specifically in the pyridyl moiety can result in compounds with weak PXR activity in vivo. These observations are a significant step forward for understanding the structure-activity relationships (SAR) between indole mimics and receptors, PXR and AhR.

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.

Methylindoles and Methoxyindoles are Agonists and Antagonists of Human Aryl Hydrocarbon Receptor.

Novel methylindoles were identified as endobiotic and xenobiotic ligands of the human aryl hydrocarbon receptor (AhR). We examined the effects of 22 methylated and methoxylated indoles on the transcriptional activity of AhRs. Employing reporter gene assays in AZ-AHR transgenic cells, we determined full agonist, partial agonist, or antagonist activities of tested compounds, having substantially variable EC50, IC50, and relative efficacies. The most effective agonists (EMAX relative to 5 nM dioxin) of the AhR were 4-Me-indole (134%), 6-Me-indole (91%), and 7-MeO-indole (80%), respectively. The most effective antagonists of the AhR included 3-Me-indole (IC50; 19 µM), 2,3-diMe-indole (IC50; 11 µM), and 2,3,7-triMe-indole (IC50; 12 µM). Reverse transcription polymerase chain reaction analyses of CYP1A1 mRNA in LS180 cells confirmed the data from gene reporter assays. The compound leads, 4-Me-indole and 7-MeO-indole, induced substantial nuclear translocation of the AhR and enriched binding of the AhR to the CYP1A1 promoter, as observed using fluorescent immunohistochemistry and chromatin immunoprecipitation assays, respectively. Molecular modeling and docking studies suggest the agonists and antagonists likely share the same binding pocket but have unique binding modes that code for their affinity. Binding pocket analysis further revealed that 4-methylindole and 7-methoxyindole can simultaneously bind to the pocket and produce synergistic interactions. Together, these data show a dependence on subtle and specific chemical indole structures as AhR modulators and furthermore underscore the importance of complete evaluation of indole compounds as nuclear receptor ligands.

Differential activation of human pregnane X receptor PXR by isomeric mono-methylated indoles in intestinal and hepatic in vitro models.

Dietary and microbial indoles can act as ligands and activators of pregnane X receptor (PXR), with implications in human intestinal health. In the current study, we examined the effects of simple mono-methylated indoles (MMIs) on the activity and function of PXR, using a series of human hepatic and intestinal cell models. Indoles 1-MMI and 2-MMI strongly induced CYP3A4 and MDR1 mRNAs in human intestinal adenocarcinoma cells LS180, but not in primary human hepatocytes. The levels of CYP3A4 mRNA were increased by 1-MMI and 2-MMI in wild type, but not in PXR-knock-out human hepatic progenitor HepaRG cells, implying the involvement of PXR in CYP3A4 induction by MMIs. Utilizing reporter gene assay, we observed dose-dependent activation of PXR by all MMIs, and their efficacies and potencies were comparable. Tested MMIs also displayed moderate antagonist effects on PXR, revealing about partial agonist effects of these compounds. As demonstrated using the Chromatin immunoprecipitation assay (ChIP),1-MMI increased PXR occupancy of the CYP3A4 promoter. Time-Resolved Fluorescence Resonance Energy Transfer revealed that MMIs are weak ligands of human PXR. Collectively, we show that MMIs are ligands and partial agonists of human PXR, which induce PXR-regulated genes in human intestinal cells.

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.

Extra- and intracellular distribution of cytokinins in the leaves of monocots and dicots.

The plant hormones cytokinins are a convenient target of genetic manipulations that bring benefits in biotechnological applications. The present work demonstrates the importance of the subcellular compartmentalization of cytokinins on the model dicot plant Arabidopsis thaliana and monocot crop Hordeum vulgare. The method of protoplast and vacuole isolation combined with precise cytokinin analysis and recovery assay of a vacuolar marker protein were used to quantify the contents of individual cytokinin forms in the leaf extracellular space, cell interior and vacuole. The data obtained for wild type plants and in each case a specific mutant line allow comparing the effect of genetic manipulations on the hormone distribution and homeostatic balance of cytokinins in the modified plants.

Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress.

Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific ß-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance.

Whole transcriptome analysis of transgenic barley with altered cytokinin homeostasis and increased tolerance to drought stress.

Cytokinin plant hormones have been shown to play an important role in plant response to abiotic stresses. Herein, we expand upon the findings of Pospísilová et al. [30] regarding preparation of novel transgenic barley lines overexpressing cytokinin dehydrogenase 1 gene from Arabidopsis under the control of mild root-specific promotor of maize ß-glycosidase. These lines showed drought-tolerant phenotype mainly due to alteration of root architecture and stronger lignification of root tissue. A detailed transcriptomic analysis of roots of transgenic plants subjected to revitalization after drought stress revealed attenuated response through the HvHK3 cytokinin receptor and up-regulation of two transcription factors implicated in stress responses and abscisic acid sensitivity. Increased expression of several genes involved in the phenylpropanoid pathway as well as of genes encoding arogenate dehydratase/lyase participating in phenylalanine synthesis was found in roots during revitalization. Although more precursors of lignin synthesis were present in roots after drought stress, final lignin accumulation did not change compared to that in plants grown under optimal conditions. Changes in transcriptome indicated a higher auxin turnover in transgenic roots. The same analysis in leaves revealed that genes encoding putative enzymes responsible for production of jasmonates and other volatile compounds were up-regulated. Although transgenic barley leaves showed lower chlorophyll content and down-regulation of genes encoding proteins involved in photosynthesis than did wild-type plants when cultivated under optimal conditions, they did show a tendency to return to initial photochemical activities faster than did wild-type leaves when re-watered after severe drought stress. In contrast to optimal conditions, comparative transcriptomic analysis of revitalized leaves displayed up-regulation of genes encoding enzymes and proteins involved in photosynthesis, and especially those encoded by the chloroplast genome. Taken together, our results indicate that the partial cytokinin insensitivity induced in barley overexpressing cytokinin dehydrogenase contributes to tolerance to drought stress.

Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants.

Transgenic barley: a prospective tool for biotechnology and agriculture.

Barley (Hordeum vulgare L.) is one of the founder crops of agriculture, and today it is the fourth most important cereal grain worldwide. Barley is used as malt in brewing and distilling industry, as an additive for animal feed, and as a component of various food and bread for human consumption. Progress in stable genetic transformation of barley ensures a potential for improvement of its agronomic performance or use of barley in various biotechnological and industrial applications. Recently, barley grain has been successfully used in molecular farming as a promising bioreactor adapted for production of human therapeutic proteins or animal vaccines. In addition to development of reliable transformation technologies, an extensive amount of various barley genetic resources and tools such as sequence data, microarrays, genetic maps, and databases has been generated. Current status on barley transformation technologies including gene transfer techniques, targets, and progeny stabilization, recent trials for improvement of agricultural traits and performance of barley, especially in relation to increased biotic and abiotic stress tolerance, and potential use of barley grain as a protein production platform have been reviewed in this study. Overall, barley represents a promising tool for both agricultural and biotechnological transgenic approaches, and is considered an ancient but rediscovered crop as a model industrial platform for molecular farming.

Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility.

Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.