In-vitro-Charakterisierung telmisartan-basierter Substanzen für die selektive Modulation des Peroxisomen-Proliferator-aktivierten Rezeptors gamma (PPARγ)

Table of contents ............................................................................................................. iii Abstract .......................................................................................................................... vii Zusammenfassung .......................................................................................................... ix Abbreviations ................................................................................................................... xi List of figures ................................................................................................................. xiii List of tables ................................................................................................................... xv 1 Introduction .......................................................................................................... 16 1.1 Energy metabolism and glucose homeostasis ....................................................... 16 1.2 Adipose tissue ........................................................................................................ 17 1.3 Obesity and related health conditions .................................................................... 19 1.3.1 Prevalence .................................................................................................. 19 1.3.2 Metabolic Syndrome .................................................................................... 20 1.3.3 Treatment .................................................................................................... 24 1.4 Peroxisome Proliferator-Activated Receptors (PPARs) ......................................... 25 1.4.1 PPAR family and structure .......................................................................... 25 1.4.2 Mechanism .................................................................................................. 27 1.4.3 Function ....................................................................................................... 29 1.5 PPARγ activation ................................................................................................... 31 1.5.1 Endogenous ligands .................................................................................... 31 1.5.2 Synthetic ligands ......................................................................................... 32 Table of contents iv 1.5.3 Selective PPARγ Modulators (SPPARγMs) ................................................ 33 1.6 Aim of this study ..................................................................................................... 36 2 Material and Methods .......................................................................................... 37 2.1 Material .................................................................................................................. 37 2.1.1 Compounds ................................................................................................. 37 2.1.2 Chemicals and substances ......................................................................... 39 2.1.3 Kits ............................................................................................................... 41 2.1.4 Biological material ....................................................................................... 41 2.1.5 Plasmids ...................................................................................................... 42 2.1.6 Cofactor peptides ........................................................................................ 42 2.1.7 qPCR primers .............................................................................................. 43 2.1.8 Equipment and consumables ...................................................................... 44 2.1.9 Computer software and internet programs .................................................. 47 2.2 Methods ................................................................................................................. 48 2.2.1 Cell-lines ...................................................................................................... 48 2.2.1.1 Culture and differentiation of 3T3-L1 cells ............................................... 48 2.2.1.2 Culture of COS-7 cells ............................................................................. 48 2.2.1.3 Freezing and thawing of adherent cell-lines ............................................ 49 2.2.2 Bacterial cells .............................................................................................. 49 2.2.2.1 Generation of chemically competent bacterial cells ................................. 49 2.2.2.2 Transformation and plasmid extraction .................................................... 50 2.2.3 Luciferase assay ......................................................................................... 50 2.2.3.1 Transient transfection and stimulation of COS-7 cells ............................. 50 2.2.3.2 Measurement of luciferase activity .......................................................... 51 Table of contents v 2.2.4 Oil-Red O staining ....................................................................................... 52 2.2.5 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) ..... 53 2.2.6 Reverse transcription and qPCR ................................................................. 54 2.2.7 Microarray .................................................................................................... 55 2.2.7.1 Sample preparation and labeling ............................................................. 55 2.2.7.2 Hybridization ............................................................................................ 57 2.2.7.3 Data analysis ........................................................................................... 59 2.2.8 Glucose uptake ........................................................................................... 60 2.2.9 Statistical analysis ....................................................................................... 60 2.2.10 Lipophilicity .................................................................................................. 61 3 Results .................................................................................................................. 62 3.1 Morphological changes during 3T3-L1 differentiation ................................. 62 3.2 Triglyceride accumulation in 3T3-L1 adipocytes ......................................... 63 3.3 Expression of PPARγ2 mRNA in 3T3-L1 preadipocytes and adipocytes .... 64 3.4 PPARγ activation by partial and full agonists .............................................. 65 3.4.1 PPARγ transactivation by partial and full agonists ...................................... 65 3.4.2 Differentiation of 3T3-L1 cells by partial and full agonists ........................... 68 3.4.3 Lipophilicity of specific partial and full agonists ........................................... 69 3.4.4 Cofactor interaction of partial and full agonists ........................................... 70 3.4.5 Gene expression during adipogenesis induced by partial and full agonists .................................................................................................................. 75 3.4.5.1 Expression of PPARγ target genes at day 6 during 3T3-L1 differentiation .......................................................................................................... 75 3.4.5.2 Transcriptome analysis at day 6 during 3T3-L1 differentiation ................ 78 Table of contents vi 3.4.6 Glucose uptake with partial and full agonists .............................................. 85 3.5 PPARγ activation – importance of position 5 and 6 of the telmisartan scaffold ................................................................................................................... 87 3.5.1 Relevance of position 5 and 6 of the telmisartan scaffold for PPARγ transactivation ........................................................................................................ 87 3.5.2 Relevance of position 5 and 6 of the telmisartan scaffold for the differentiation of 3T3-L1 cells ................................................................................. 92 3.5.3 Lipophilicity of agonists with different moieties at position 5 and 6 ............. 93 3.5.4 Relevance of position 5 and 6 for cofactor interaction ................................ 94 4 Discussion .......................................................................................................... 101 4.1 Telmisartan-derived structures are selective PPARγ modulators ............. 101 4.2 Side effects of PPARγ activation ............................................................... 104 4.3 Dual pharmacology of telmisartan-derived structures ............................... 106 4.4 Further application options ........................................................................ 107 4.5 Conclusion and outlook ............................................................................. 109 References ................................................................................................................... 110 Appendix A - List of publications and poster presentations Appendix B – AcknowledgmentThe peroxisome proliferator-activated receptor gamma (PPARγ) is the ‘master regulator’ of adipogenesis and adipocyte gene expression, and represents an effective pharmacological target for the treatment of insulin resistance and type 2 diabetes mellitus. Activation and modulation of its function plays an important role on overall treatment effectiveness and the side effect profile. Full PPARγ agonists, known as thiazolidinediones (TZDs) are currently used in the clinic. Unfortunately, they are associated with diverse side effects, like weight gain and an increased risk for cardiovascular events. To circumvent these side effects associated with the full agonism, intensive investigation was performed during the last years to create drugs which exhibit a combination of partial activation and selective receptor modulation. Besides its blood pressure lowering property, the AT1-receptor blocker telmisartan was shown to be a partial agonist of PPARγ with beneficial metabolic effects in vitro and in vivo. In previous work of our group, comprehensive structure- activity relationship studies were analyzed to reveal the importance of different parts of the telmisartan structure and various moieties. These studies were the foundation for the here presented thesis, investigating the biochemical and biological effects of the most promising structures. Telmisartan-derived PPARγ agonists (agonists 1, 2 and 3) with partial and full agonism were selected and analyzed for differential cofactor interaction, differential gene expression and glucose uptake. The degree of PPARγ activity was characterized by a differentiation assay performed with 3T3-L1 cells and by a PPARγ transactivation assay using Cos-7 cells transiently transfected with pGal4-hPPARγDEF, pGal5-TK-pGL3 and pRL-CMV. Interaction with the coactivators SRC1, PGC-1α and TRAP220, and the corepressor NCoR1 were determined using TR-FRET assays. Furthermore, microarray and qPCR analyses were performed to elucidate the gene expression profile of 3T3-L1 cells after stimulation with telmisartan-derived compounds. Moreover, glucose uptake was measured in mature adipocytes differentiated from 3T3- L1 cells after incubation with the indicated compounds and 3H-labeled deoxy-glucose. Telmisartan-derived PPARγ agonists demonstrated a distinct cofactor interaction and mRNA expression profile from their corresponding control substances pioglitazone and telmisartan. In addition, compounds with new modifications on the telmisartan structure were characterized for their PPARγ activation potential and cofactor interaction. These compounds contain a benzimidazole (agonists 4-5 and 4-6), benzothiophene (agonists 5-5 and 5-6) or benzofuran (agonists 6-5 and 6-6) moiety either at position 5 or 6 of the benzimidazole core structure. The shift of the benzofuran or benzothiophene moiety from position 5 to position 6 increased both, potency and efficacy, and also increased cofactor recruitment/ release. Conclusively, all new agonists demonstrated in vitro characteristics of SPPARγMs, and therefore, present promising results for future in vivo experiments.Der Peroxisomen-Proliferator-aktivierte Rezeptor gamma (PPARγ) ist der „master regulator“ der Fettzelldifferenzierung und Adipozytengenexpression, und repräsentiert ein effektives pharmakologisches Ziel für die Behandlung von Insulinresistenz und Diabetes Typ 2. Die Aktivierung und Modellierung der Rezeptorfunktion spielt eine bedeutende Rolle in der Gesamteffizienz der Behandlung und dem Nebenwirkungsprofil. Die volle Aktivierung von PPARγ durch Thiazolidindione (TZDs), welche in der Klinik eingesetzt werden, ist mit verschiedenen Nebenwirkungen, wie Gewichtszunahme und einem erhöhtem Risiko für Herz-Kreislauf Erkrankungen, verbunden. In den letzten Jahren wurde intensive Forschung durchgeführt, um Medikamente mit verbesserter Wirkung und Verträglichkeit herzustellen, welche PPARγ partiell und selektiv aktivieren und modulieren. Neben seiner blutdrucksenkenden Wirkung ist der AT1-Rezeptorblocker Telmisartan auch ein partieller Agonist für PPARγ mit vielversprechenden metabolischen Eigenschaften in vitro und in vivo. In vorhergehenden Arbeiten unserer Gruppe wurden umfangreiche Studien zur Struktur-Wirkbeziehung durchgeführt, um die Bedeutung der Struktureinheiten von Telmisartan und unterschiedlicher Seitengruppen im Hinblick auf ihre PPARγ aktivierenden Effekte aufzudecken. Diese Studien lieferten die Grundlage für diese Arbeit, bei der biochemische und biologische Effekte der interessantesten Substanzen untersucht wurde. Zunächst wurden unterschiedliche telmisartanbasierte PPARγ Agonisten (Agonist 1, 2 und 3) ausgewählt und bezüglich selektiver Kofaktorwechselwirkungen, differentieller Genexpression und Glukoseaufnahme untersucht. Der Grad der PPARγ Aktivität wurde mit Hilfe eines Differenzierungsassays unter Verwendung von 3T3-L1 Zellen, und eines PPARγ Transaktivierungsassays mit Cos-7 Zellen, welche transient mit pGal4-hPPARgDEF, pGal5-TK-pGL3 und pRL-CMV transfiziert wurden, ermittelt. Wechselwirkungen mit den Koaktivatoren SRC1, PGC-1α und TRAP220, und dem Korepressor NCoR1 wurden mit TR-FRET untersucht. Microarray und qPCR Analysen wurden durchgeführt, um das Genexpressionsprofil von 3T3-L1 Zellen nach Stimulation mit telmisartanbasierten Substanzen zu ermitteln. Außerdem wurde die Glukoseaufnahme von ausgereiften Adipozyten der 3T3-L1 Zelllinie nach Inkubation mit den Substanzen und 3H-markierter Deoxyglukose gemessen. Die telmisartanbasierten Substanzen zeigten, im Vergleich zu den Kontrollen Pioglitazon und Telmisartan, abweichende Kofaktorwechselwirkungen und ein differenzielles mRNA-Expressionsprofil. Desweiteren wurden Substanzen mit neuen Modifizierungen der Telmisartanstruktur bezüglich ihrer PPARγ- aktivierenden Eigenschaft und Kofaktorwechselwirkungen charakterisiert. Diese Substanzen haben eine Benzimidazol (Agonist 4-5 und 4-6), Benzothiophen (Agonist 5-5 und 5-6), oder Benzofuran (Agonist 6-5 und 6-6) Seitengruppe entweder an Position 5 oder Position 6 des Benzimidazolgrundkörpers. Die Verlagerung der Benzothiophen oder Benzofuran Seitengruppe von Position 5 nach Position 6 verstärkte sowohl Potenz und Effizienz, als auch Kofaktorrekrutierung. Alle neuen telmisartanbasierten Substanzen zeigten die in vitro Eigenschaften selektiver PPARγ Modulatoren, und sind daher vielversprechende Substanzen für zukünftige in vivo-Experimente