Following the discovery of 2-(3-methoxyphenyl)-3,4-dihydroquinazoline-4-one and 2-(3-methoxyphenyl)quinazoline-4-thione as potent, but non-specific activators of the human Constitutive Androstane Receptor (CAR, NR1I3), a series of quinazolinones substituted at the C2 phenyl ring was prepared to examine their ability to selectively modulate human CAR activity. Employing cellular and in vitro TR-FRET assays with wild-type CAR or its variant 3 (CAR3) ligand binding domains (LBD), several novel partial human CAR agonists and antagonists were identified. 2-(3-Methylphenyl) quinazolinone derivatives 7d and 8d acted as partial agonists with the recombinant CAR LBD, the former in nanomolar units (EC50 = 0.055 μM and 10.6 μM, respectively). Moreover, 7d did not activate PXR, and did not show any signs of cytotoxicity. On the other hand, 2-(4-bromophenyl)quinazoline-4-thione 7l possessed significant CAR antagonistic activity, although the compound displayed no agonistic or inverse agonistic activities. A compound possessing purely antagonistic effect was thus identified for the first time. These and related compounds may serve as a remedy in xenobiotic intoxication or, conversely, in suppression of undesirable hepatic CAR activation.

• Klíčová slova
• CAR, Gene activation, Metabolism, Nuclear receptors, Quinazolines, Xenobiotics,
• MeSH
• chinazoliny farmakologie   MeSH
• konstitutivní androstanový receptor * MeSH
• lidé MeSH
• ligandy MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidní receptory * agonisté   metabolismus   MeSH
• thioketony MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chinazoliny MeSH
• konstitutivní androstanový receptor * MeSH
• ligandy MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidní receptory * MeSH
• thioketony MeSH

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

• MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• konstitutivní androstanový receptor * agonisté   chemie   MeSH
• lidé MeSH
• myši MeSH
• pyridiny farmakologie   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• steroidní receptory * agonisté   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• konstitutivní androstanový receptor * MeSH
• pyridiny MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidní receptory * MeSH

AIM: To investigate the effect of resveratrol on biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats. METHODS: Resveratrol (RSV) or saline were administered to rats by daily oral gavage for 28 d after sham operation or reversible bile duct obstruction (BDO). Bile was collected 24 h after the last gavage during an intravenous bolus dose of the Mdr1/Mrp2 substrate azithromycin. Bile acids, glutathione and azithromycin were measured in bile to quantify their level of biliary secretion. Liver expression of enzymes and transporters relevant for bile production and biliary secretion of major bile constituents and drugs were analyzed at the mRNA and protein levels using qRT-PCR and Western blot analysis, respectively. The TR-FRET PXR Competitive Binding Assay kit was used to determine the agonism of RSV at the pregnane X receptor. RESULTS: RSV increased bile flow in sham-operated rats due to increased biliary secretion of bile acids (BA) and glutathione. This effect was accompanied by the induction of the hepatic rate-limiting transporters for bile acids and glutathione, Bsep and Mrp2, respectively. RSV also induced Cyp7a1, an enzyme that is crucial for bile acid synthesis; Mrp4, a transporter important for BA secretion from hepatocytes to blood; and Mdr1, the major apical transporter for xenobiotics. The findings were supported by increased biliary secretion of azithromycin. The TR-FRET PXR competitive binding assay confirmed RSV as a weak agonist of the human nuclear receptor PXR, which is a transcriptional regulator of Mdr1/Mrp2. RSV demonstrated significant hepatoprotective properties against BDO-induced cirrhosis. RSV also reduced bile flow in BDO rats without any corresponding change in the levels of the transporters and enzymes involved in RSV-mediated hepatoprotection. CONCLUSION: Resveratrol administration for 28 d has a distinct effect on bile flow and biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.

• Klíčová slova
• Azithromycin, Bile acids, Bile production, Pregnane X receptor, Resveratrol,
• MeSH
• ABC transportéry metabolismus   MeSH
• antiflogistika nesteroidní farmakologie   terapeutické užití   MeSH
• antioxidancia farmakologie   terapeutické užití   MeSH
• aplikace orální MeSH
• azithromycin farmakokinetika   MeSH
• cholestáza farmakoterapie   etiologie   patofyziologie   MeSH
• glutathion metabolismus   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   patofyziologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• potkani Wistar MeSH
• pregnanový X receptor MeSH
• resveratrol MeSH
• steroidní receptory agonisté   MeSH
• stilbeny farmakologie   terapeutické užití   MeSH
• žlučové kyseliny a soli chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ABC transportéry MeSH
• antiflogistika nesteroidní MeSH
• antioxidancia MeSH
• azithromycin MeSH
• glutathion MeSH
• pregnanový X receptor MeSH
• resveratrol MeSH
• steroidní receptory MeSH
• stilbeny MeSH
• žlučové kyseliny a soli MeSH

The pregnane X receptor (PXR) is a transcription factor regulating P-glycoprotein (P-gp; ABCB1)-mediated transport and cytochrome P450 3A4 (CYP3A4)-mediated metabolism of xenobiotics thereby affecting the pharmacokinetics of many drugs and potentially modulating clinical efficacy. Thus, pharmacokinetic drug-drug interactions can arise from PXR activation. Here, we examined whether the selective α1-adrenoreceptor blocker tamsulosin or the antagonist of muscarinic receptors tolterodine affect PXR-mediated regulation of CYP3A4 and of P-gp at the messenger RNA (mRNA) and protein level in an enantiomer-specific way. In addition, the effect of tamsulosin and tolterodine on P-gp activity was evaluated. We used quantitative real-time PCR, gene reporter assay, western blotting, rhodamine efflux assay, and calcein assay for determination of expression, activity, and inhibition of P-glycoprotein. The studied compounds significantly and concentration-dependently increased PXR activity in the ABCB1-driven luciferase-based reporter gene assay. We observed much stronger induction of ABCB1 mRNA by S-tamsulosin as compared to the R or racemic form. R or racemic form of tolterodine and R-tamsulosin concentration-dependently increased P-gp protein expression; the latter also enhanced P-gp efflux function in a rhodamine-based efflux assay. R-tamsulosin and all forms of tolderodine slightly inhibited P-gp. The effect on CYP3A4 expression followed the same pattern but was much weaker. Taken together, tamsulosin and tolterodine are demonstrated to interfere with P-gp and CYP3A4 regulation in an enantiomer-specific way.

• Klíčová slova
• Drug metabolism, Drug transporters, Enantiospecificity, PXR,
• MeSH
• ABC transportér, podrodina B, člen 4 MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• isomerie MeSH
• lidé MeSH
• LLC-PK1 buňky MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• P-glykoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• prasata MeSH
• pregnanový X receptor MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• steroidní receptory agonisté   metabolismus   MeSH
• sulfonamidy chemie   farmakologie   MeSH
• tamsulosin MeSH
• tolterodin tartarát chemie   farmakologie   MeSH
• transfekce MeSH
• upregulace MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• ABCB1 protein, human MeSH Prohlížeč
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP3A MeSH
• cytochrome P450 3A4, mouse MeSH Prohlížeč
• messenger RNA MeSH
• multidrug resistance protein 3 MeSH Prohlížeč
• P-glykoproteiny MeSH
• pregnanový X receptor MeSH
• steroidní receptory MeSH
• sulfonamidy MeSH
• tamsulosin MeSH
• tolterodin tartarát MeSH

Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/β-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/β-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3β (GSK3β) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the β-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3β inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a β-catenin-independent manner. In conclusion, GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

• Klíčová slova
• AhR, CTNNB1, CYP, GSK3β, PXR, crosstalk,
• MeSH
• beta-katenin agonisté   antagonisté a inhibitory   genetika   metabolismus   MeSH
• enzymová indukce účinky léků   MeSH
• GSK3B MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• indoly farmakologie   MeSH
• induktory cytochromu P450 chemie   metabolismus   farmakologie   MeSH
• inhibitory proteinkinas chemie   metabolismus   farmakologie   MeSH
• kinasa 3 glykogensynthasy antagonisté a inhibitory   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• organokovové sloučeniny farmakologie   MeSH
• oximy farmakologie   MeSH
• pregnanový X receptor MeSH
• pyridiny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• receptory aromatických uhlovodíků agonisté   chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• reportérové geny účinky léků   MeSH
• RNA interference MeSH
• signální dráha Wnt účinky léků   MeSH
• simulace molekulového dockingu MeSH
• steroidní receptory agonisté   genetika   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 chemie   genetika   metabolismus   MeSH
• transkripční faktory bHLH agonisté   chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• 6-bromoindirubin-3'-oxime MeSH Prohlížeč
• AHR protein, human MeSH Prohlížeč
• beta-katenin MeSH
• Chir 99021 MeSH Prohlížeč
• CTNNB1 protein, human MeSH Prohlížeč
• GSK3B protein, human MeSH Prohlížeč
• GSK3B MeSH
• GSK3iXV MeSH Prohlížeč
• indoly MeSH
• induktory cytochromu P450 MeSH
• inhibitory proteinkinas MeSH
• kinasa 3 glykogensynthasy MeSH
• organokovové sloučeniny MeSH
• oximy MeSH
• pregnanový X receptor MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• receptory aromatických uhlovodíků MeSH
• rekombinantní fúzní proteiny MeSH
• steroidní receptory MeSH
• systém (enzymů) cytochromů P-450 MeSH
• transkripční faktory bHLH MeSH

In the past decade, a large number of enantiopure drugs were introduced to clinical practice, since improved therapeutic effects were demonstrated for one of the enantiomers from originally racemic drug. While the therapeutic effects and safety of enantiopure drugs were tested prior to their approval, various biological enantiospecific activities of these, often "old" drugs, remain to be elucidated. In the current paper, we examined enantiospecific effects of clinically used enantiopure drugs containing one chiral center in the structure (i.e. zopiclone, tamsulosin, tolterodine, modafinil, citalopram) towards aryl hydrocarbon (AhR), glucocorticoid (GR) and pregnane X (PXR) receptors in human reporter cell lines. The cytotoxicity (IC50), agonist (EC50) and antagonist effects (IC50) of R-form, S-form and racemic mixture for each tested drugs were determined and compared in AhR-, GR- and PXR-gene reporter cell lines. Since AhR, GR and PXR are key regulators of drug metabolism, energy metabolism, immunity and play many other physiological functions, the data presented here might be of toxicological significance.

• Klíčová slova
• 2,3,7,8-tetrachlorodibenzo-p-dioxin, AhR, Chiral drugs, Cytotoxicity, DEX, Enantiospecificity, GR, Gene reporter assay, PXR, RIF, RU486, TCDD, Xenoreceptors, aryl hydrocarbon receptor, dexamethasone, glucocorticoid receptor, mifepristone, pregnane X receptor, rifampicin,
• MeSH
• azabicyklické sloučeniny chemie   farmakologie   MeSH
• benzhydrylové sloučeniny chemie   farmakologie   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• citalopram chemie   farmakologie   MeSH
• fenylpropanolamin chemie   farmakologie   MeSH
• kresoly chemie   farmakologie   MeSH
• lidé MeSH
• modafinil MeSH
• nádorové buněčné linie MeSH
• piperaziny chemie   farmakologie   MeSH
• pregnanový X receptor MeSH
• receptory aromatických uhlovodíků agonisté   antagonisté a inhibitory   metabolismus   MeSH
• receptory glukokortikoidů agonisté   antagonisté a inhibitory   metabolismus   MeSH
• reportérové geny genetika   MeSH
• stereoizomerie MeSH
• steroidní receptory agonisté   antagonisté a inhibitory   metabolismus   MeSH
• sulfonamidy chemie   farmakologie   MeSH
• tamsulosin MeSH
• tolterodin tartarát MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azabicyklické sloučeniny MeSH
• benzhydrylové sloučeniny MeSH
• citalopram MeSH
• fenylpropanolamin MeSH
• kresoly MeSH
• modafinil MeSH
• piperaziny MeSH
• pregnanový X receptor MeSH
• receptory aromatických uhlovodíků MeSH
• receptory glukokortikoidů MeSH
• steroidní receptory MeSH
• sulfonamidy MeSH
• tamsulosin MeSH
• tolterodin tartarát MeSH
• zopiclone MeSH Prohlížeč

Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.

• MeSH
• aktivace transkripce * účinky léků   MeSH
• genetické inženýrství metody   MeSH
• knihovny malých molekul analýza   MeSH
• lidé MeSH
• luciferasy genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• objevování léků metody   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• reportérové geny MeSH
• rychlé screeningové testy metody   MeSH
• sekvenční delece MeSH
• steroidní receptory * agonisté   antagonisté a inhibitory   genetika   metabolismus   MeSH
• steroidy farmakologie   MeSH
• transfekce MeSH
• transkripční faktory genetika   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• knihovny malých molekul MeSH
• luciferasy MeSH
• rekombinantní proteiny MeSH
• steroidní receptory * MeSH
• steroidy MeSH
• transkripční faktory MeSH

Drugs for weight loss have been in use for nearly hundred years. Orlistat (Xenical) is a non-centrally acting anti-obesity drug that inactivates gastric and intestinal lipases, thus, preventing absorption of dietary triglycerides. There are reports indicating that Orlistat reduces bioavailability of Cyclosporin to a clinically relevant degree. Since Cyclosporin is metabolized by cytochrome P450 CYP3A4, we examined whether interaction between Orlistat and Cyclosporin involves induction of CYP3A4. Human Caucasian colon adenocarcinoma cells LS174T and primary cultures of human hepatocytes were used, as in vitro models of intestinal and hepatic cells, respectively. Treatment of LS174T cells for 24h with Orlistat (1-100mg/L) did not cause induction of CYP3A4 mRNA levels as compared to control cells while Orlistat (100mg/L) slightly induced CYP3A4 mRNA in human hepatocytes. Rifampicin, a model CYP3A4 inducer, significantly induced CYP3A4 mRNA in both types of cells. The level of CYP3A4 protein in human hepatocytes was increased by Orlistat after 48h, while rifampicin strongly induced CYP3A4 protein level. In addition, Orlistat moderately dose-independently activated pregnane X receptor (PXR) in LS174T cells transiently transfected with p3A4-luc reporter construct containing the basal promoter (-362/+53) with proximal PXR response element and the distal xenobiotic responsive enhancer module (-7836/-7208) of the CYP3A4 gene 5'-flanking region. In conclusion, we report here that Orlistat is weak PXR activator and CYP3A4 inducer in human hepatocytes, but it has no effect on CYP3A4 in intestinal cells, implying no role of CYP3A4 induction in the interaction between Orlistat and Cyclosporin in absorption process.

• MeSH
• biologická dostupnost MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• hepatocyty cytologie   účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• laktony farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádorové buněčné linie MeSH
• orlistat MeSH
• pregnanový X receptor MeSH
• promotorové oblasti (genetika) MeSH
• steroidní receptory agonisté   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP3A MeSH
• inhibitory enzymů MeSH
• laktony MeSH
• messenger RNA MeSH
• orlistat MeSH
• pregnanový X receptor MeSH
• steroidní receptory MeSH

Structural modification of 20-hydroxyecdysone (20E) based on photochemical transformation yielded dimeric ecdysteroid 7alphaH,7'alphaH-bis-[(20R,22R)-2beta,3beta,20,22,25-pentahydroxy-5beta-cholest-8(14)-en-6-one-7-yl] as a main product. Its structure was determined by detailed NMR analysis. Furthermore, two new monomeric analogues: 14-epi-20-hydroxyecdysone and 14-deoxy-14,18-cyclo-20-hydroxyecdysone were identified in addition to the earlier described 14-deoxy and 14-hydroperoxy derivatives of 20E. Formation of the specific and so far unique ecdysteroid dimer has not been observed in earlier photo-transformation studies. The transformed dimeric analogue of 20-hydroxyecdysone retained the high agonistic activity on the ecdysone receptor in the B(II)-bioassay compared with the original 20E.

• MeSH
• dimerizace MeSH
• Drosophila melanogaster MeSH
• ekdysteroidy analogy a deriváty   MeSH
• ekdysteron chemická syntéza   chemie   MeSH
• fotochemie metody   MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• spektrofotometrie infračervená MeSH
• steroidní receptory agonisté   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ecdysone receptor MeSH Prohlížeč
• ekdysteroidy MeSH
• ekdysteron MeSH
• steroidní receptory MeSH