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.

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

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The regulation of gonadotropin releasing hormone (GnRH) receptor numbers in the pituitary is a crucial control point in reproduction. Pituitary sensitivity to GnRH can be directly correlated with GnRH receptor levels, which can be regulated at transcriptional and post-transcriptional level. The proximal promoter of the mouse GnRH receptor gene contains two cis elements bearing the consensus sequence for a Steroidogenic Factor-l (SF -1) binding site. The distal site has previously been shown to be involved in basal and tissue-specific transcriptional regulation, whereas the function of the proximal site was not established. SF-I, a member of the nuclear receptor superfamily of transcription factors, is involved in the transcriptional regulation of a large number of genes involved in steroidogenesis and reproduction. The consensus SF-I binding site can serve as a binding site for several members of the nuclear receptor superfamily. The aim of this study was to investigate the binding of SF-I protein from the aT3-1 gonadotrope cell line to the two putative SF-I binding sites in the mouse GnRH receptor promoter in vitro, in order to provide supporting evidence for their functional roles in GnRH receptor gene regulation. It was shown by Western blotting that SF-I and Nur77, another nuclear receptor transcription factor, are both expressed in aT3-1 cells, in a manner that is influenced by cell culture conditions. Gel mobility shift assays using specific antibodies showed that both SF-I and Nur77 protein in aT3-1 nuclear extracts bind to both sites in a mutually exclusive fashion. As shown by competition assays using mutated versions of the two sites, Nur77 protein had different base pair requirements than that of SF-I protein for binding to the sites. Additionally, SF-I mRNA was shown by Northern blotting to be increased in aT3-1 cells in response to stimulation of the Protein Kinase A (PKA) pathway by forskolin. These results highlight unexpected degeneracy in so-called "consensus" nuclear receptor binding sites. Furthermore, since Nur77 protein is involved in the stress response of the hypothalamic-pituitary-adrenal (HPA) axis, the unexpected presence of Nur77 protein in a gonadotrope cell line has potentially important implications for cross-talk between the HPA and hypothalamic-pituitary-gonadal (HPG) axes. / AFRIKAANSE OPSOMMING: Daar bestaan 'n direkte verband tussen pituïtêre sensitiwiteit vir gonadotropien-vrystellingshormoon (GnRH) en GnRH-reseptorvlakke Die regulering van GnRH-reseptorvlakke op transkripsionele en post-transkripsionele vlak in die pituïtêre klier is belangrik by die beheer van voortplantingsfunksies. Die proksimale promotor van die GnRH-reseptorgeen in die muis bevat twee cis elemente met die konsensus volgorde vir 'n Steroidogenic Factor-l (SF-I) bindingsetel. Die distale element is betrokke by basale en weefsel-spesifieke transkripsionele regulering, maar die funksie van die proksimale element is nog nie vasgestel nie. SF-1 is 'n lid van die superfamilie van selkernreseptore en is betrokke by die transkripsionele regulering van gene verantwoordelik vir steroïedogenese en voortplanting. Die konsensus SF-I bindingsvolgorde kan dien as bindingsetel vir verskeie selkernreseptore. Ten einde 'n beter insig ten opsigte van die regulering van die GnRH reseptorgeen te verkry, is ondersoek ingestel na die binding van SF-I-proteïen, afkomstig van die aT3-1 pituïtêre gonadotroopsellyn, aan die twee moontlike SF-l bindingsetels in die GnRH-reseptor promotor, in vitro. Die Western-klad metode het getoon dat beide SF-l en Nur77, 'n ander selkernreseptor-transkripsiefaktor, in die aT3-1 sellyn uitgedruk word. Die uitdrukking is afhanklik van selkultuurtoestande. Elektroforetiese mobiliteitsessais met spesifieke antiliggame het getoon dat SF-l en Nur77 proteïene in aT3-1 selkernproteïenekstraksies eksklusief aan beide bindingsetels bind. Nur77 proteïen benodig ander basispare as SF-l proteïen om aan die bindingsetels te bind. Hierdie resultate dui op onverwagse degenerasie in sogenaamde "konsensus" selkernreseptor-bindingsvolgordes. Die Northern-kladmetode het ook getoon dat SF-l mRNA vlakke in aT3-1 selle styg wanneer die proteïenkinase A (PKA) pad gestimuleer word met forskolin. Aangesien Nur77 proteïen betrokke is by die stres-respons van die hipotalamus-pituïtêre klier-adrenale (HP A) aksis, hou die onverwagse teenwoordigheid van Nur77 proteïen in 'n gonadotroop-sellyn potensieel belangrike inplikasies in vir kommunikasie tussen die HPA-aksis en die hipotalamus-pituïtêre klier-gonadale (HPG) aksis.

Ο μεταγραφικός παράγοντας COUP-TF είναι ένας ορφανός πυρηνικός υποδοχέας, ο οποίος είναι πολύ σημαντικός κατά την εμβρυική ανάπτυξη. Στην παρούσα εργασία επιχειρήθηκε η μελέτη του φυσιολογικού ρόλου του COUP-TFII των θηλαστικών κατά την αναγέννηση του σκελετικού μυός, χρησιμοποιώντας ως μοντέλο διαφοροποίησης τη μυοβλαστική σειρά ποντικού C2C12, που προέρχεται από ενήλικα βλαστικά κύτταρα του ίδιου μυός. Ο mCOUP-TFII εμπλέκεται στη ρύθμιση της ανάπτυξης του καρδιαγγειακού συστήματος, της μυογένεσης και άλλων αναπτυξιακών διαδικασιών. Έχει δειχθεί ότι αναστέλλει τη διαφοροποίηση των μυοβλαστών C2C12 σε μυοσωλήνες, κυρίως μέσω καταστολής της έκφρασης των μεταγραφικών παραγόντων μυογένεσης MyoD και Myogenin. Επίσης, η έκφρασή του στους μυοβλάστες C2C12 καταστέλλεται με την έναρξη της διαφοροποίησης. Αδημοσίευτα αποτελέσματα του εργαστηρίου μας, έχουν δείξει ότι μέσω εναλλακτικού ματίσματος στο mRNA του PlCOUP-TF του αχινού προκύπτουν δύο ισομορφές του υποδοχέα που διαφέρουν ως προς ένα εξώνιο 21 αμινοξέων στην DBD. H μικρή ισομορφή είναι λειτουργικός μεταγραφικός παράγοντας και δρα ως ομοδιμερές, ενώ η μεγάλη ισομορφή και τα ετεροδιμερή των δυο ισομορφών εμφανίζουν αδυναμία πρόσδεσης στο DNA. Με αυτόν τον τρόπο, η μεγάλη ισομορφή ρυθμίζει την ποσότητα του λειτουργικού μεταγραφικού παράγοντα, δρώντας ως επικρατής κατασταλτική πρωτεΐνη. Αρχικά, μελετήθηκαν οι ιδιότητες πρόσδεσης του mCOUP-TFII και η δυνατότητα σχηματισμού ετεροδιμερών με τη μεγάλη ισομορφή του PlCOUP-TF (L.I.) μέσω δοκιμής in vitro πρόσδεσης (EMSA), δεδομένης της ικανότητας ετεροδιμερισμού του hCOUP-TFI με την ομόλογη πρωτεΐνη του αχινού. Βρέθηκε ότι ο mCOUP-TFII δύναται να προσδεθεί στο στοιχείο απόκρισης C1R με τη μορφή ομοδιμερών και έχει ικανότητα ετεροδιμερισμού με τη PlCOUP-TF L.I. Τα ομοδιμερή του mCOUP-TFII εμφάνιζαν μειούμενη πρόσδεση στο DNA, όσο αυξανόταν ο λόγος PlCOUP-TF L.I./mCOUP-TFII. Ο απώτερος στόχος της έρευνας ήταν η μελέτη του ρόλου του mCOUP-TFII στη μυϊκή διαφοροποίηση, μέσω της υπερέκφρασης της επικρατούς κατασταλτικής PlCOUP-TF L.I. σε καλλιέργειες κυττάρων C2C12. Η αρχική μας υπόθεση ήταν ότι η PlCOUP-TF L.I. δύναται να δράσει ως επικρατής κατασταλτική ισομορφή του mCOUP-TFII, μέσω σχηματισμού μη λειτουργικού ετεροδιμερούς μαζί του. Σε αυτή την περίπτωση, θα αναμενόταν η PlCOUP-TF L.I. να καταστείλει τη δράση του ενδογενούς mCOUP-TFII των κυττάρων C2C12, με αποτέλεσμα την επαγωγή της διαφοροποίησης των μυοβλαστών. Αντιθέτως, η υπερέκφραση του mCOUP-TFII, θα αναμενόταν να συντελέσει στη διατήρηση της αδιαφοροποίητης κατάστασης των μυοβλαστών. Δεν παρατηρήθηκε διαφορά στις δράσεις των δυο COUP-TFs, ωστόσο, από τα πειράματα αυτά δεν μπορεί να εξαχθεί κάποιο ασφαλές συμπέρασμα αναφορικά με το ρόλο του mCOUP-TFII στη διαφοροποίηση των μυοβλαστών και τη δυνατότητα in vivo αλληλεπίδρασης μεταξύ των δυο υποδοχέων. / The transcription factor COUP-TF is an orphan nuclear receptor, which is very important during embryonic development. In the present dissertation, we attempted to study the physiological role of mammalian COUP-TFII during skeletal muscle regeneration, by use of the myoblast cell line C2C12, which originates from adult stem cells of skeletal muscle, as a model system of cell differentiation. mCOUP-TFII is involved in the regulation of cardiovascular system development, myogenesis and other developmental processes. It has been shown to inhibit the differentiation of C2C12 myoblasts to myotubes, mainly through suppression of the myogenic regulatory factors MyoD and Myogenin gene expression. Moreover, its expression in C2C12 myoblasts is suppressed at the onset of differentiation. Alternative splicing of the sea urchin PlCOUP-TF mRNA results in two isoforms, which differ by a 21 aa insertion in the DBD (unpublished data). The small isoform is a functional transcription factor that acts as a homodimer, while the large isoform and the heterodimers of the two isoforms fail to bind DNA. In this way, the large isoform regulates quantitatively the functional transcription factor, acting as dominant negative protein. Initially, by using in vitro binding assays (EMSA), we studied the binding properties of mCOUP-TFII and the possibility of forming heterodimers with the large isoform of PlCOUP-TF (L.I.), provided that hCOUP-TFI has been reported to be able to heterodimerize with the sea urchin homologue. We found that mCOUP-TFII is capable of binding the C1R response element as a homodimer and of forming heterodimers with PlCOUP-TF L.I. The binding of mCOUP-TFII homodimers has been shown to reduce as the ratio PlCOUP-TF L.I./mCOUP-TFII increases. The ultimate goal of this research, was the study of the role of mCOUP-TFII in muscle differentiation via overexpression of the dominant negative PlCOUP-TF L.I. in C2C12 cell cultures. Our initial assumption was that PlCOUP-TF L.I. is capable of acting as a dominant negative isoform of mCOUP-TFII, by forming non functional heterodimers with it. In this case, PlCOUP-TF L.I. would be expected to suppress the action of endogenous mCOUP-TFII in C2C12 cells. In contrast, overexpression of mCOUP-TFII would be expected to contribute to the maintenance of myoblasts in an undifferentiated state. We did not observe any difference in the actions of the two COUP-TFs, however, we cannot report any result regarding either the role of mCOUP-TFII in myoblast differentiation or the ability of in vivo interaction between these receptors.

Chez les mammiferes, la lipogenese ou synthese de novo des acides gras joue un rôle essentiel a l'homeostasie energetique. Elle est particulierement active dans le foie. Le Liver X Receptor (LXR) est un recepteur nucleaire de classe II qui est implique dans la regulation de l'expression de genes importants dans cette voie metabolique. Au niveau hepatique, LXR regule directement l'expression de certains genes de la lipogenese et aussi l'expression des facteurs de transcription SREBP-1c et ChREBP intervenant respectivement dans la reponse hepatique a l'insuline et au glucose. Les ligands naturels de LXR sont les oxysterols, des derives oxygenes du cholesterol. Aussi, LXR est avant tout considere et connu comme un senseur du cholesterol. Au cours de ces travaux nous nous sommes interesses in vivo au role de LXR dans la regulation transcriptionnelle de la lipogenese hepatique en fonction de differents stimuli: pharmacologiques, inflammatoires et nutritionnels. Par une approche pharmacologique, nous avons etudie la regulation croisee avec entre LXR et le recepteur active par les proliferateurs de peroxisome (PPAR . Nous avons aussi montre que l'inflammation intestinale est un puissant inhibiteur de la lipogenese hepatique. Enfin, nous avons mis en evidence le role de LXR dans la regulation de la lipogenese en reponse a une carence en acides gras essentiels et a un regime riche en fructose. / In mammals, lipogenesis or de novo fatty acid synthesis plays an essential part in energy homeostasis. It is particularly active in the liver. The Liver X Receptor (LXR) is a class II nuclear receptor that regulates the expression of important genes involved in this pathway. In the liver, LXR directly controls the expression of lipogenic genes and also the expression of transcription factors such as SREBP-1c and ChREBP required for the hepatic response to insulin and glucose respectively. Natural ligands for LXR are oxysterols, which are oxygenated derivatives of cholesterol. Therefore, LXR is primarily considered and known as a cholesterol sensor. In this work, we were interested in the role of LXR in the transcriptional control of hepatic lipogenesis in vivo in response to distinct stimuli: pharmacological agonists, gut inflammation and changes in diet composition. Through a pharmacological study, we highlighted the cross-talk between LXR signaling and the regulation of the Peroxisome Proliferator Activated Receptor (PPAR ). We have also evidenced that experimentally induced colitis induces a potent inhibition of hepatic lipogenesis. Finally, we have shown that LXR is involved in the regulation of lipogenesis in response to essential fatty acid deficiency and to high fructose.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Klára Dohnalová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Inhibitory effect of SPA70 on hPXR activation This work focuses on pregnane X receptor (PXR) and its antagonists. PXR is a ligand-activated nuclear receptor that plays a major role in detoxification of xenobiotics and protecting the organism from their toxic effects. Recent evidence also shows endogenous action of PXR in the metabolism of lipids, glucose and bile acids. However, PXR activation could be harmful, since induction of biotransformation enzymes by PXR agonists may result in reduced treatment efficacy, increased toxicity of drug metabolites and resistance to chemotherapeutic agents. Recent research has been intensively focused on PXR antagonists capable of abolishing these unfavourable effects. Recently discovered human PXR antagonist SPA70 has a promising potential for future usage. In this study, we investigated the inhibitory effect of SPA70 on activated PXR. To activate PXR we used agonists binding directly to PXR (rifampicin, hyperforin, SR12813) and also agonists activating PXR indirectly via cell signalling pathways (U0126, PD184352, PD0325901). Experiments were performed using luciferase...

Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos. / Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

Foi definido que elementos regulatórios da expressão atrial-específica do promotor da SMyHC3 estão contidos em um elemento complexo de resposta a receptores nucleares (ECRRN). Ensaios de transativação celular indicam que alguns receptores nucleares se ligam nesta região. A partir destes ensaios verificamos a ativação do promotor por um receptor nuclear, o COUP-TFII. Ele regula muitos processos biológicos, como angiogênese e o próprio desenvolvimento atrial. Através da deleção do ECRRN observamos que o promotor não era ativado por COUP-TFII, indicando a sua ligação nessa região. Verificamos ainda que somente o domínio de ligação ao ligante do COUP-TFII é capaz de ativar o promotor, sugerindo a necessidade de uma interação com outros RNs para ativar o promotor. Uma análise proteômica indica que a maioria dos interactores de COUP-TFII está relacionada com complexos reguladores da transcrição e com a via de sinalização do receptor de andrógenos (AR). Ensaios de transativação celular mostram que juntos, COUP-TFII e AR, são capazes de aumentar a ativação do promotor. / It was determined that regulatory elements of the atrial-specific expression of the promoter SMyHC3 are contained in a complex nuclear receptor response element (CNRRE). Cellular transactivation assays indicated certain nuclear receptors (NR) can bind in this region. From these trials, was observed the promoter activation by a nuclear receptor, COUP-TFII. It regulates many biological processes such as angiogenesis and atrial development. Deletion of CNRRE resulted in no activation of the promoter by COUP-TFII, indicating their connection in this region. We also verified that only the ligand binding domain of COUP-TFII is able to activate the promoter, suggesting interaction with other NRs to activate it. A proteomic analysis revealed that most of COUP-TFII partners relates to complexes of transcription regulators and the androgen receptor (AR) signaling pathway. Cell transactivation assays showed that together, COUP - TFII and AR, are able to increase promoter activation.

Os receptores nucleares constituem uma superfamília de fatores de transcrição regulados pela interação com hormônios. Esta superfamília inclui, por exemplo, os receptores de hormônio tireoidiano, estrogênio, androgênio, glicocorticóide e mineralocorticóide. Neste trabalho, empregamos técnicas de biologia estrutura e bioinformática para estudar as interações entre alguns dos membros da família de receptores nucleares e seus respectivos ligantes. Para o receptor de hormônio tireoidiano, foi demonstrado, através da análise das estruturas cristalográficas das duas isoformas do receptor ligados aos tiromimético Triac, que os componentes entálpicos visíveis nas estruturas não explicam a seletividade do ligante. Dados de dinâmica molecular confirmaram que a seletividade do hormônio tem um importante componente entrópico. Empregando a técnica de dinâmica molecular, estudamos a ligação do receptor de mineralocorticóide humano à aldosterona, ao cortisol, à espironolactona e à cortisona e simulamos ainda o efeito da mutação S810L, conhecida por converter a atividade antagonista da cortisona e da espironolactona em agonista. A análise das simulações revelou um perfil de ligações de hidrogênio similar na ligação do receptor selvagem ao cortisol e à aldosterona. A cortisona perde, por conta da inserção de uma hidroxila na posição 11, uma ligação de hidrogênio importante com a Asn770 e, por isso, tem menor energia potencial de ligação. A espironolactona perde a mesma ligação de hidrogênio ao mesmo tempo em que aumenta o número de contatos de van der Waals pela inserção do grupo tioacetil na posição 7. A mutação S810L simulada no complexo com cortisona, cortisol e espironolactona não interfere no padrão de ligações de hidrogênio estabelecidas entre o receptor e os ligantes, mas altera a mobilidade de uma das regiões propostas como rota de dissociação. Propomos, portanto, que a mutação interfere na cinética de dissociação dos ligantes e não no padrão de interações estabelecidas no equilíbrio. Simulações de dissociação induzida do ligante confirmam esta proposição. Na última etapa, utilizamos os modelos experimentalmente determinados para o receptor ativado por proliferadores peroxissomais gama para a busca de novos ligantes através da técnica de docking molecular. Neste trabalho, utilizamos uma base de dados com aproximadamente um milhão de compostos. Destes, quatro foram selecionados após o docking molecular e testados experimentalmente. Um dos compostos testados se mostrou ativo neste receptor, apresentando uma atividade de 60-70% da atividade da rosiglitazona, conhecido agonista total do PPARg. / Nuclear receptors are a superfamily of hormone-regulated transcriptional factors. This superfamily includes, for example, the receptor for thyroid hormone, estrogen, androgen, gluco and mineralocorticoid. In this work, we used structural biology and bioinformatic tools to study the interactions between some members of the nuclear receptor superfamily and its respective ligands. We showed by the analysis of the crystal structures of both thyroid hormone receptor isoforms bound to the thyromimetic Triac that the enthalpic components visible in the structures do not explain the ligand selectivity. Molecular dynamics simulation data confirmed later that the hormone selectivity has an important entropic component. Using the molecular dynamics simulation, we studied, in a second stage, the interaction between the human mineralocorticoid receptor bound to aldosterone, cortisol, spironolactone and cortisone and also simulated the effects of the mutations S810L, known to convert the antagonist properties of spironolactone and cortisone in an agonist activity. The analysis of the simulations showed a similar profile in hydrogen bonds established between the wild type receptor bound to cortisol and aldosterone. Cortisone looses an important hydrogen bond with Asn770 because of the insertion of a carbonyl group in the 11 position and shows a decreased binding potential energy. Spironolactone loses the same interaction but has an increased number of van der Waals contacts because of the insertion of a tioacetyl group in the 7 position. The mutant S810L simulated in complex with cortisol, cortisone and spironolactone showed that the mutation do not interfere with the hydrogen bond profile established between the receptor and the ligands but changes the mobility of a region in the receptor previously proposed as a ligand dissociation route. Ligand unbinding simulations through steered molecular dynamics (SMD) confirm that aldosterone and cortisol unbind differentially and the mutation S810L alters the unbinding profile. We then propose that the mutation changes the kinetics of ligand association/dissociation without changing the profile of the interactions established in the equilibrium. In the last stage, we used the experimentally determined structural model of the peroxissome proliferator-activated receptor gamma to search for novel ligands using the molecular docking technique. For this work, we used a database containing about 1 million compounds. Among those, four compounds were selected after the docking computation and experimentally tested. One of these compounds was found to be active in the receptor, showing about 60-70% of the agonistic activity of rosiglitzone, a known PPARg total agonist.

Ng, Sin Man. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.