Steroid hormone receptors (SHRs) are well validated therapeutic targets in a number of diseases. Current therapies competitively antagonise the ligand binding domain (LBD), blocking activation of the receptor and downstream signalling pathways. However cross-reactivity can be seen amongst the antagonists of different SHRs eliciting unwanted side effects. Additionally the acquisition of resistance to current therapies in diseases such as prostate cancer limits their use. The amino-terminal domain (NTD) of SHRs provides an alternative target for antagonism by allowing potential therapies to block receptor transactivation and inhibit interactions with co-activator proteins. Reduced homology between different SHR NTDs also increases the specificity of drug interactions. However development of targeted therapies using rational drug design has been hindered by its intrinsically disordered structure. Establishing cell lines which stably express a SHR responsive reporter gene alongside variants of SHRs lacking the LBD provides a method by which small molecules specifically targeting the NTD of each receptor can be identified. This assay has been designed to overcome the barriers to drug discovery that are presented by an intrinsically disordered protein. The project follows the design, development, optimisation and implementation of a high throughput screening assay with the potential to identify novel small molecule inhibitors of SHRs. The applications of these inhibitors are highlighted throughout, with specific reference to their potential to inhibit the androgen receptor in prostate cancer.

7 Abstract With the first appearance of life on Earth, organisms had to adapt to an ever-changing surrounding environment in order to survive. Since the emergence of metazoan multi- cellularity, subsets of cells could adapt to perform specific biological tasks beneficial to the whole organism, necessitating not only spatiotemporal regulation of gene expression during development, but also integration of tissue specific needs with overall organis- mal status. Within the set of evolutionary conserved regulatory systems, the family of nuclear receptor (NR) transcription factors stands out due to its high degree of evolu- tionary conservation, plasticity and uniqueness to the metazoan kingdom, regulating gene expression in response to, or in the absence of a ligand by genomic and non- genomic actions. With an increasing number of different compounds being recognized as ligands to NRs, it is now thought that ancient NRs were probably characterized by low ligand binding specificity, eventually serving as environmental sensors, integrating nutrient availability and gene expression at the base of metazoan evolution. Characteri- zation of the NR network in one of the simplest metazoan organisms, Trichoplax ad- haerens, revealed not only a functional network and sub-specialization of NR dependent gene regulation, but...

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La signalisation stéroïdienne médiée par des récepteurs nucléaires est impliquée dans de nombreux processus ayant trait au développement des animaux. La compréhension de ces phénomènes est importante pour répondre à des questions de santé publique, d’agronomie ou de biologie de la conservation. Ceci nécessite de connaître et de mettre en relation l’évolution des récepteurs qui fixent ces stéroïdes et des voies de synthèse qui produisent les stéroïdes. Mon travail s’est articulé autour de trois grands axes. 1. La mise à jour des relations de parenté entre les récepteurs nucléaires impliqués dans la fixation des stéroïdes, mais aussi de ceux qui sont impliqués dans la régulation de la stéroïdogenèse, pour comprendre quand et dans quel contexte cette machinerie est apparue. 2. La démonstration que les enzymes impliquées dans la stéroïdogenèse étaient apparues indépendamment par recrutement d’enzymes à spécificité de substrat plus large impliquées dans la détoxification des xénobiotiques. 3. L'élucidation des relations de parenté entre des voies métaboliques, montrant que les voies de la stéroïdogenèse avaient évolué comme des voies de dégradation du cholestérol. Ces résultats aboutissent à un modèle dans lequel la signalisation hormonale des animaux à symétrie bilatérale serait l’héritière de voies de détoxification de molécules stéroïdiennes contenues dans leur alimentation. Ce modèle expliquerait le couplage entre l’accumulation de nutriments et la maturation sexuelle, ainsi que les nombreux dérèglements touchant à la fois le métabolisme et la reproduction dus aux perturbateurs endocriniens ou à certaines molécules thérapeutiques. / Nuclear receptor mediated steroid signaling is involved in many processes in metazoandevelopment, such as puberty in vertebrates, molting in insects and entry into infective stage in some parasitic nematodes. Understanding those phenomena is important regarding public health, agronomical and conservation biology issues. This necessitates to know and to explore the interactions between the evolution of steroid-binding receptors and steroid-synthesizing pathways. My work was articulated around three major parts. First, using the historical expertise of the laboratory, I updated the relationships between nuclear receptors that are involved in steroid binding, but also from all those that are involved in steroidogenesis regulation, in order to elucidate when and in which context this machinery has arisen. Second, using a classical comparative genomic approach, I showed that the steroidogenetic enzymes have appeared independently by duplication from xenobiotic-metabolizing enzyme with a wider range of substrate specificity.Third, I explored the relationships between metabolic pathways using tools from comparative anatomy. This has confirmed and completed the previous results, showing that steroidogenetic pathways have evolved with the pattern of cholesterol degradation pathways.The synthesis of all these results has led to an evolutionary model where hormonal signaling in bilaterian animals has been inherited from the detoxification of dietary sterols. This model may explain the coupling between nutrient accumulation and sexual maturation, and also the link between metabolic disorders and endocrine disruption due to environmental chemicals or drugs.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Draský Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacological influence of nuclear receptors in diabetes mellitus therapy Nuclear receptors belong to the superfamily of transcription factors, their main functions include regulating the expression of target genes. In my work I focused mainly on the group of orphan receptors, namely the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). A common feature of these receptors is their activation by a specific ligand. Both CAR and PXR have an essential function as biological sensors of hydrophobic xenobiotics when they induce enzymes I and II. phase of metabolism. They are also essential in the regulation of gluconeogenesis, insulin response, adipogenesis, cholesterol homeostasis, fatty acids, triglycerides and glycogen. The aim of this experimental work was to introduce a luciferase reporter assay method for two DNA constructs containing the promoter region of the PEPCK and CYP7A1 genes. We used the known agonist rifampicin and the antagonist SPA70 to activate/deactivate PXR. We used CITCO as a CAR receptor agonist. We first verified the functionality of the luciferase reporter gene assay...

Bone is a dynamic tissue, where bone forming osteoblasts and bone resorbing osteoclasts maintain homeostasis. Research into bone toxicology has largely focused on pharmaceutical side effects adversely affecting bone development. However, many environmental toxicants can regulate bone homeostasis. Recently, the nuclear receptor peroxisome proliferator activated receptor gamma (PPARγ) has emerged as an important target of environmental toxicants. PPARγ dimerizes with the retinoid-X receptor alpha (RXRα), is a central transcription factor in adipogenesis, and in bone can transdifferentiate osteoblasts into adipocytes by suppressing osteogenic pathways. The central hypothesis of this dissertation is that environmental chemicals can adversely affect bone homeostasis by activating nuclear receptors in bone cells – particularly osteoblasts and osteoclasts – to perturb cellular differentiation and function. Three study aims were developed to test and refine this hypothesis. First, a set of structurally diverse environmental PPARγ agonists were individually applied to mouse primary bone marrow mesenchymal stromal cell cultures undergoing osteogenic differentiation. In vitro PPARγ ligand treatment suppressed osteogenesis and stimulated adipogenesis. Organotin compounds (tributyltin, triphenyltin) in particular more efficaciously suppressed osteogenesis. The second aim characterized the effects of in vivo tributyltin exposure on bone microarchitecture in female C57Bl/6 mice. Tributyltin exposure resulted in a thinner cortical bone, but significantly increased trabecular mineralization. Further analyses suggested that tributyltin did not suppress osteoclast numbers but rather changed osteoclast function, minimally attenuating the resorptive function and enhancing their ability to generate osteogenesis-stimulating factors. Furthermore, tributyltin activated not only PPARγ, but also RXR and liver X receptors. The third aim established the utility of Generalized Concentration Addition in modeling PPARγ activation by mixtures of full and partial PPARγ agonists. A complex mixture of multiple phthalate compounds activated an in vitro PPARγ reporter assay, and the individual dose-responses of each compound were used to construct modeled responses. The comparisons of empirical data and model predictions supported the use of Generalized Concentration Addition in modeling a complex mixture of environmental PPARγ agonists. Together, these studies support and establish important toxicological mechanisms related to PPARγ and RXRα activation in different aspects of bone biology and provide a basis for studying mixture effects of PPARγ agonists.

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Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Previously, two orthogonal ligand receptor pairs (OLRP) were developed as potential molecular switch systems by modifying nuclear receptors, ligand-activated transcription factors, to bind and activate gene expression with the synthetic ligand LG335 and not with the natural ligand 9-cis retinoic acid (9cRA). The two OLRP previously discovered were RXR variant 130 (I268A, I310A, F313A, and L436F) (also known as GR130) and the RXR variant QCIMFI (Q275C, I310M, and F313I) and (also known as GRQCIMFI). The OLRP were further developed into molecular switches to provide controlled gene expression and potentially benefit gene therapy applications by replacing the DNA binding domain (DBD) with a Gal4 DBD, a yeast transcription factor. Both molecular switches are able to bind Gal4 RE in response to LG335 and activate expression of a luciferase or GFP reporter gene in either a two- or one-component system. When characterizing the GR130 variant in the two-component system, no activation was observed with the natural ligand 9cRA, and the variant displayed a 19±5-fold activation and a 50 nM EC50 value in the presence of LG335. When the GRQCIMFI variant was evaluated in the two-component system, activation was observed in the presence of LG335 with a 10 nM EC50 value and a 6±2-fold induction, and 9cRA induced activation only at the highest concentration. The GRQCIMFI variant was also characterized with the one-component system containing the reporter gene GFP in a transient transfection as well as through retroviral transduction, displaying green fluorescence in 30% of the cells in the presence of 10 µM LG335. Several attempts were made to improve the molecular switch system. The VP16 activation domain was fused to GRQCIMFI in an effort to increase the fold induction; however, the addition of the VP16 created a constitutively active protein. Another approach to improve the molecular switch incorporated error-prone PCR to discover a new variant, Q275C, I310M, F313I, L455M (QCIMFILM), which displayed a 10-fold increase in sensitivity towards LG335 with a 5 nM EC50 value. Examination of the L455 position in the crystal structure of RXR revealed this residue is located outside of the ligand binding pocket on helix 12 (H12), but is able to significantly enhance receptor function. In fact, the single variant, L455M, was able to enhance receptor activation, compensate for a nonfunctional variant, as well as influence coactivator association. The long-term goal of this research is to develop a gene regulation system that would be used in human gene therapy trials. In the process of creating this system a deeper assessment of the nuclear receptor structure and function is made, which can be used for the enhancement and development of transcriptional regulation mechanisms.

Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.