Methods for the preparation of novel antifungal saponins have been investigated in order to further explore their medicinal utility and provide the opportunity to synthesize their derivatives. Through this work, several partially protected stereoisomers of Cholestane, Androstane and Spirostane have been prepared which could be used for the synthesis of various saponin derivatives in order to discover novel saponin based antifungal agent. Various mono and disaccharide derivatives of these steroids have been synthesized and evaluated for their antifungal activity against four pathogenic fungal strains. Among the various derivatives maltose derivatives were found to have the best antifungal activity. However there is a need for more extensive SAR studies to discover compounds with better potency. Additionally, the branched oligosaccharide synthesis was explored in two parts. First, these results demonstrated that the central 2,3-branched portion can be synthesized efficiently from a partially protected glucopyranosyl acceptor since the C-2 and C-3 alcohols differ in their reactivity in glycosylation reactions. Second, a tagged sugar based strategy for synthesis of branched oliogosaccharides was developed, and found to be effective for general synthesis of branched oligosaccharides. Microwave assisted synthesis of cyclic imides have been explored this was a key precursor for the synthesis of our tag molecules which were required for synthesis of branched oligosachharides. A comparison of microwave versus conventional methods for synthesis of cyclic imides has been studied. The synthesis of tagged sugars and their selective deprotection to remove tag molecules were successfully explored in order to have proof of concept for its applicability towards synthesis of branched oligosaccharides. Benzylic mono and dibromination was achieved in very high yields using microwave conditions using environmentally friendly solvent in order to avoid use of carcinogenic carbon tetrachloride as solvent for this type of reactions. In addition reaction time was reduced to 30 minutes to 3 hours compared to convention methods, which needed more than 15 hours for the benzylic bromination reaction.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Ioannis Mouratidis Supervisor: Doc. PharmDr. Petr Pávek Ph.D. Testing of newly synthesized compounds with constitutive androstane receptor (CAR) Interactions of a diverse array of nuclear receptors with numerous isolated compounds (ligands) have been extensively investigated during the last years. The reason of this intense research activity is, of course, the wide therapeutic potential of the nuclear receptors super- family. It is known that they interact with the metabolism and excretion of various compounds, endogenous or exogenous that interfere with the homeostasic mechanisms of the living organisms. In my study, I tested the activation of a specific orphan nuclear receptor known as constitutive androstane receptor (CAR). I tested 11 structurally different compounds as well as a known inhibitor of CAR (clotrimazole) and an activator (CITCO). To test these interactions between these compounds and CAR, I used the method of the mammalian two- hybrid system, a method where ligand-protein interactions are studied in an environment close to that in vivo. The method is based on the Dual-Luciferase Reporter kit, which made the quantification of the CAR responsive luciferase reporter...

There are many causes of cholestasis, which results when the flow of bile acids is slowed or stopped. Bile acids are hydrophobic molecules synthesized from cholesterol in the liver, and when present in excess, are cytotoxic to cell membranes. Treatment options for cholestasis are limited, and if left untreated or inadequately treated, many patients will require a liver transplant; thus, underscoring the importance of successfully managing this disease. Activation of nuclear receptors in animal models has been shown to be hepatoprotective during bile acid-induced cholestasis; however, the mechanisms underlying the hepatoprotective effects are poorly understood. Therefore, the over-arching goal of this project is to glean an improved comprehension of the mechanisms of hepatoprotection during bile acid-induced cholestasis. All of the studies involve administration of CAR activators phenobarbital (PB), oltipraz (OPZ), 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene [TCPOBOP (TC)] or corn oil (CO) to C57BL/6 wild type (WT), or WT and CAR knockout (CAR-/-) mice prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Efflux transport proteins such as Mrps 3 and 4 are known to be up-regulated during cholestasis, and this was the first topic of exploration. Unexpectedly, the expression of efflux transporters was not consistently up-regulated in protected mice. However, a decrease in total liver bile acid concentrations was observed. These changes in hepatic bile acids indicated that bile acid biosynthesis may be relevant to hepatoprotection. Indeed decreases in total and individual bile acids correlated with hepatoprotection, and Cyp8b1 expression was also increased which could be suggestive of a shift in the bile acid biosynthesis pathway towards the formation of less toxic bile acid species. CAR may also have a role in cell death via apoptosis by altering Bcl-2 protein expression. Although apoptosis was decreased in hepatoprotected mice, an increase in the expression of Mcl-1 and Bcl-xL was not observed, suggesting hepatoprotection is not a direct result of CAR-induced Mcl-1 expression. These findings add significantly to the body of knowledge surrounding cholestatic liver disease and suggest that studies aimed toward manipulation of nuclear receptors are worthy of further exploration.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Ivana Šachová Supervisors: PharmDr. Jan Martin, PhD., Dipl. Ing. Mgr. Ferdinand Molnár, PhD. Title of diploma thesis: Preparation of strategy for studying interaction of human constitutive androstane receptor with steroid receptor coactivator-1 The aim of this study was to express wild type and mutated synthetic human SRC1 central domains in E. coli BL21 DE3 cells. Expressed recombinant proteins were purified afterwards and used for investigation of SRC1-CAR protein interaction using pull down experiments. The constitutive androstane receptor (CAR), a nuclear receptor (NR), is a transcriptional regulator, which influences the expression of various proteins, such as enzymes of biotransformation and transporters important for metabolism of both endogenous and exogenous compounds. CAR can be fully active only in presence of coactivators, such as steroid receptor coactivator-1 (SRC1). The most important fragment of SRC1 for binding to NRs is the central domain, made of three α-helical Leu-X-X-Leu-Leu (LXXLL) L1, L2, L3 motifs, in where L is a leucine and X represents any amino acid. hSRC1, as well as hCAR, can be successfully produced by expression of cDNA of interest in E. coli strains. In case of SRC1,...

CAR (Constitutive Androstane Receptor, NR1I3) et PXR (Pregnane X Receptor, NR1I2) sont deux récepteurs nucléaires dédiés à la reconna issance et à l'élimination de molécules lipophiles potentiellement toxiques pour l'organisme. Ces facteurs de transcription peuvent être activés par des ligands d'origines et de structures diverses (médicaments, polluants environnementaux, produits de l'alimentation et de phytothérapies). L'activation de ces récepteurs entraîne l'expression des gènes majeurs de la fonction de détoxication entéro-hépatique (CYP450, transférases, transporteurs) permettant l'élimination de ces toxiques. Dans ce travail, nous avons dans un premier temps 1) montré que CAR contrôle l'expression de Spot14, une protéine pro-lipogénique, et 2) nous avons identifié une nouvelle isoforme de PXR (PXR-small) codant uniquement pour le domaine de liaison des ligands de PXR. Nous avons pu déterminer les origines de transcription par 5'-RACE PCR et montrer que PXR-small représente environ 10% de l'ensemble des transcrits de PXR dans le tissu hépatique sain par une approche de PCR qua ntitative. Nous avons pu détecter sa présence par western-blot sur des extraits de protéines nucléaires issus de tissus hépatiques et de lignées cellulaires hépatiques. Par des expériences de gel retard, nous avons observé que cette nouvelle isoforme tronquée, qui ne code que pour le LBD de PXR, ne peut pas se lier à l'ADN. Des expériences de gènes rapporteurs suggèrent que cette isoforme se comporte comme un dominant négatif de PXR. Enfin, la présence d'un ilot CpG situé juste en amont de PXR-small suggère que cette nouvelle isoforme pourrait être régulée épigénétiquement par méthylation, notamment dans les cellules tumorales. / CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are two nuclear receptors devoted to the recognition and elimination of lipohilic molecules potentially toxic to the body.These transcription factors can be activated by ligands of different origins and structures (drugs, environmental pollutants, food products and herbal medicine...). The activation of these receptors leads to the expression of major genes of the detoxification process (CYP450, transferases, transporters) leading to the elimination of these toxics. In this work, we 1) showed that Spot14, a pro-lipogenic protein, is a target gene of CAR, then 2) we identified a novel isoform of PXR (PXR-small), coding only the ligand binding domain of PXR. By using 5'-RACE PXR, we established the origins of transcription of PXR-small and by quantitative PCR we observed that PXR-small represents about 10% of all PXR transcripts in human liver. By using western blo t, we detect its presence on nuclear protein extracts from liver tissues and hepatic cell lines. In Electromobility shift essays experiments, we observed that PXR-small cannot bind to DNA, while reporter essay experiments suggest that this isoform acts as a dominant negative of PXR. Finally, the presence of a CpG island just upstream of PXR-small suggests that this novel isoform might be regulated epigenetically by methylation, more particularly in tumor cells.

Le cancer colorectal est marqué par une importante mortalité dans les stades avancés du fait du fort taux de récidives tumorales après chimiothérapie. La prédiction de l'efficacité et de la toxicité des cytotoxiques s'impose comme un des enjeux majeurs de ces prochaines années. Parce que la majorité des anticancéreux sont pris en charge par les enzymes et transporteurs dont l'expression est contrôlée par le niveau d'expression et d'activation des xénosenseurs CAR et PXR, il est fort probable que ces xénosenseurs puissent représenter des facteurs prédictifs à prendre en compte dans la prise en charge des cancers. Notre équipe a récemment montré que les récepteurs des xénobiotiques PXR (NR1I2) (Raynal et al, 2010) et CAR (NR1I3) sont exprimés dans des lignées cellulaires et des tissus coliques humains. Leur surexpression dans les lignées coliques LS174T et T84 entraine leur résistance à l'irinotécan et à son métabolite actif le SN38 alors que leur inhibition antagonise cette résistance. Des dosages intra- et extra-cellulaires du SN38 et du SN38-G, ainsi que la quantification des ARNm des l'UGT1As et du transporteur MDR1, montrent que CAR et PXR augmentent le métabolisme détoxifiant et l'efflux du SN38. L'impact de la surexpression de ces xénosenseurs sur la viabilité des cellules LS174T à différentes classes de cytotoxiques (anti-métabolites, intercalants, inhibiteurs de topoisomérases, poisons du fuseau) a ensuite été évaluée. Nous avons observé que l'expression de CAR ou PXR conduit à une forte chimiorésistance au paclitaxel, au docétaxel et au 4-hydroxy-cyclophosphamide alors que PXR entraîne une sensibilisation marquée au cisplatine et au carboplatine en augmentant la quantité d'adduits de platine sur l'ADN. Les études du transcriptome de nos modèles cellulaires nous ont permis d'identifier les gènes cibles impliqués dans ces variations de cytotoxicité. Des études de confirmation par modulation pharmacologique ou ARNs interférents de ces gènes cibles sont en cours et nous permettront de préciser les mécanismes mis en jeu dans les variations de chimiosensibilité. Ces travaux devraient permettent de mieux appréhender le rôle des xénosenseurs CAR et PXR sur le métabolisme intra-tumoral des cytotoxiques et potentiellement sur la réponse à des chimiothérapies variées. / Colorectal cancer is characterized by high mortality in advanced stages due to the high rate of tumor recurrence after chemotherapy. The prediction of the efficacy and toxicity of cytotoxic drugs represents a major challenge in the coming years. Because the majority of cancer drugs are supported by the enzymes and transporters whose expression is controlled by the level of expression and activation of the xenosensors CAR (NR1I3) and PXR (NR1I2), it is likely that they may represent predictive factors in the management of cancer. Our team has recently shown that xenobiotic receptors PXR (Raynal et al, 2010) and CAR are expressed in cell lines and human colon tissues. Their overexpression in colon cancer cell lines LS174T and T84 leads to resistance to irinotecan and to its active metabolite SN38, while their inhibition reverse this resistance. Irinotecan metabolites detection assays of SN38 and SN38G, and the quantification of UGT1As and MDR1 mRNA, show that CAR and PXR increase the detoxifying metabolism and the efflux of SN38. The impact of overexpression of these xenosensors on LS174T cell viability to different classes of cytotoxic agents (anti-metabolites, DNA intercalators, topoisomerase inhibitors, antimitotic agents) was then evaluated. We observed that the expression of CAR or PXR results in a significant drug resistance to paclitaxel, docetaxel and 4-hydroxy-cyclophosphamide whereas PXR leads to a marked sensitization to cisplatin and carboplatin by increasing the amount of platinum adducts the DNA. Microarray studies of our cell models allowed us to identify the target genes potentially involved in these changes in cytotoxicity. Further studies by pharmacological modulation or interfering RNAs of these target genes are in progress and will allow us to clarify the mechanisms involved in the changes in chemosensitivity. This work should help us to understand the impact of CAR and PXR xenosensors on the intratumoral metabolism of cytotoxic drugs and potentially on the response to various chemotherapies.

Thesis (Ph. D.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

<p>U prvom delu ove doktorske disertacije je izvr&scaron;ena<br />sinteza novih derivata androstana sa D-laktonskom<br />funkcijom ili D-seko derivata, sa modifikacijama u Ai/<br />ili B-prstenovima. U drugom delu je ispitana<br />antiproliferativna aktivnost odabranih<br />novosintetizovanih jedinjenja prema humanim<br />tumorskim ćelijskim linijama (MCF-7, MDA-MB-231,<br />PC-3, HeLa, HT-29, K562) kao i prema zdravim<br />ćelijama fetalnih fibroblasta pluća (MRC-5).</p> / <p>In first part of this work was achieved synthesis of&nbsp;some new androstane derivatives with D-lactone&nbsp;function and new D-seco derivatives with modification&nbsp;in A and/or B rings. In the second part the&nbsp;antiproliferative activity of selected newly synthesized&nbsp;compounds by human carcinoma cell lines (MCF-7,&nbsp;MDA-MB-231, PC-3, HeLa, HT-29, K562) and the&nbsp;healthy cells of fetal lung fibroblasts (MRC-5 ) was&nbsp;examined.</p>

<p>U prvom delu ove doktorske disertacije je izvr&scaron;ena sinteza&nbsp;novih 17&alpha;-pikolil i 17(E)-pikoliniliden androstanskih&nbsp;derivata, sa modifikacijama u A- i/ili B-prstenovima. U&nbsp;drugom delu rada je ispitana antiproliferativna aktivnost&nbsp;odabranih novosintetizovanih jedinjenja prema humanim&nbsp;tumorskim&nbsp; ćelijskim linijama (MCF-7, MDA-MB-231,&nbsp;PC-3, HeLa, HT-29 i A549) kao i prema zdravim ćelijama&nbsp;fetalnih fibroblasta pluća (MRC-5).</p> / <p>In the first part of this dissertation, the synthesis of new&nbsp;17&alpha;-picolyl and 17(E)-picolinylidene androstane&nbsp;derivatives with modifications in A and/or B rings, were&nbsp;presented. The second part of the work consisted on&nbsp;investigation of antiproliferative activity of selected newly&nbsp;synthesized compounds toward human carcinoma cell lines&nbsp;(MCF-7, MDA-MB-231, PC-3, HeLa, HT-29 and A549),&nbsp;as well as the healthy fetal lung fibroblasts (MRC-5).</p>

Transcription factors activated by exogenous or endogenous stimuli alter gene expression with major effects on chromatin accessibility and the epigenome. This thesis investigates that impact of environmental chemical and hormonal exposure on liver chromatin accessibility in a mouse liver model. Exposure to the constitutive androstane receptor (CAR)-specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induces localized changes in chromatin accessibility at several thousand DNase hypersensitive sites (DHS). Activating histone marks, associated with enhancers and promoters, were induced by TCPOBOP and were highly enriched at opening DHS. Opening DHS were highly enriched for CAR binding sites and nuclear receptor direct repeat-4 motifs. These DHS were also enriched for the CAR heterodimeric partner RXRA, binding by CEBPA and CEBPB, and motifs for other liver-specific factors. Thus, TCPOBOP alters the enhancer landscape through changes in histone marks and by mechanisms linked to induced CAR binding. In other studies, the impact of pituitary growth hormone (GH) secretion patterns on chromatin accessibility changes associated with sex-biased liver gene expression was examined. In adult male liver, the transcription factor STAT5 is directly activated by each successive plasma GH pulse. In female liver, STAT5 is persistently activated by the near-continuous stimulation by plasma GH. A majority of the ~4,000 GH-regulated, sex-biased DHS have chromatin marks characteristic of enhancers and were enriched for proximity to sex-biased gene promoters. Chromatin accessibility is thus a key feature of sex-differential gene expression. Two major classes of male-biased DHS were identified: dynamic male-biased DHS, almost all bound by STAT5, which undergo repeated cycles of chromatin opening and closing induced by each GH pulse; and static male-biased DHS, whose accessibility is unaffected GH/STAT5 pulses and whose sex bias results from these chromatin sites being more closed in female liver. Sites with STAT5 binding showed greater chromatin opening, many of which also contain the STAT5 motif. Finally, the effect of a single GH pulse on hypophysectomized male mouse liver was investigated to identify DHS responsive to the male, pulsatile-GH, secretion pattern. These studies demonstrate that widespread epigenetic changes associated with target gene expression are induced by xenobiotics and hormones regulating liver gene expression. / 2022-01-31T00:00:00Z