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«Titel der Dissertation „Combined in silico/in vitro screening tools for identification of new insulin receptor ligands“ Verfasserin Dipl.-Ing. ...»

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Titel der Dissertation

„Combined in silico/in vitro screening tools for identification

of new insulin receptor ligands“


Dipl.-Ing. (FH) Daniela Digles

angestrebter akademischer Grad

Doktorin der Naturwissenschaften (Dr.rer.nat.)

Wien, 2011

Studienkennzahl lt. Studienblatt: A 091 490

Dissertationsgebiet lt. Studienblatt: Dr.-Studium der Naturwissenschaften Molekulare Biologie Betreuerin / Betreuer: Univ.-Prof. Dr. Gerhard F. Ecker Acknowledgements This work would not have been possible without the help and support by a lot of people. First, I would like to thank my supervisor Prof. Ecker, who caught my fascination for pharmacoinformatics. He was always the source of good advise and new ideas, and encouraged me to present my work on conferences. I also want to thank my second supervisor Prof. Dirsch, for suggesting this interesting topic and providing me the opportunity to perform the experimental part of this work myself in her lab. Although both are very busy with their work, they always found time for discussions and meetings.

I had the luck to work not only in one, but in two fantastic groups, the Pharmacoinformatics Research Group at the Department of Medicinal Chem- istry and the Molecular Targets Group at the Department of Pharmacognosy.

I am very thankful to Dominik Kaiser and Michael Demel for their patience and help with all the questions I had at the beginning of my thesis and to Christoph Waglechner for supporting me while I was learning programming.

As already during my diploma thesis, Elke Heiß was always available for ques- tions and discussions regarding the cell culture work. Also, she and Marta Pinto gave me valuable feed-back on the manuscript of this thesis. Renate Baumgartner supported me at the beginning of the experimental work, espe- cially on the topic of PTP1B. Matthias Kramer shared his knowledge about the handling and differentiation of adipocytes with me.

But the members of both groups did not only support me in scientific questions, but also provided the basis of a friendly environment where I found myself welcome. Here a special thank goes to Ishrat Jabeen and Yogesh Aher for a lot of discussions, cooking and insights into different cultures. Also to Andrea Schiesaro, with whom I shared the office during most of the time and all other neighbours I had in the last years.

Ein ganz besonderer Dank gilt meiner Familie für ihre Liebe und ihre persönliche, als auch finanzielle Unterstützung. Meiner Mutter Brigitte, für ihre Motivation und Zuversicht. Meinem Vater Günther, der mein Interesse i an Computern geweckt und gefördert hat. Meinem Bruder Dominik, für unsere spätabendlichen Diskussionen über Programmiersprachen und andere Themen. Und ganz besonders Martin, der mir die ganze Zeit über zur Seite gestanden ist, mich nach Rückschlägen immer wieder aufgemuntert hat und mir ein Ruhepol in all der Hektik meiner Arbeit war und ist.

I am very thankful to the Austrian Academy of Sciences for supporting my work with a DOC-fFORTE-fellowship.

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1.1 Diabetes mellitus Insulin is a hormone involved in the maintenance of normal blood glucose levels. When blood glucose levels are elevated, for example due to the intake of a meal, insulin is secreted by the b-cells of the pancreas. Insulin then leads to the uptake of glucose into insulin sensitive tissue (liver, muscle and fat), thus reducing the blood glucose levels.

Diabetes mellitus is a chronic disease which is characterized by the lack of, or resistance to, insulin action and consequently elevated blood glucose levels.

Health surveys carried out by Statistik Austria in 2006/2007 showed that 390 000 people in Austria are suffering from it, with 91% of them receiving treatment or medication.1 The number of cases increases with age. From people older than 75 years, 23% of females and 19% of males had encountered diabetes, while the average is 6%. Worldwide, the number of cases in the year 2000 was estimated to be approximately 171 millions, and due to the increasing aging and urbanization of the population this number is expected to double until 2030.2 Diabetes mellitus is categorized in different types, with type 1 and 2 being the most prominent ones. Type 1 diabetes mellitus is characterized by an absolute deficiency of insulin. Here, the b-cells of the pancreas are destroyed by the immune system. This stops the production of insulin, which then needs


to be provided from external sources. In contrast, type 2 diabetes mellitus (also non-insulin-dependent diabetes mellitus or mature onset diabetes) is characterized by a relative insulin deficiency. This relative deficiency can be caused by a decrease of insulin production, but more importantly by a resistance of the target cells to insulin. The resulting hyperglycemia increases the risk of microvascular damage such as retinopathy, nephropathy and neuropathy, as well as of macrovascular complications like ischaemic heart disease and stroke.

The reasons for diabetes are diverse and can include genetic as well as environmental causes. Major risk factors for type 2 diabetes mellitus are obesity and the lack of physical exercise, but also genetic factors have been shown to play a role in several subtypes of the disease.3 A recent study showed that a high fat diet fed to male rats can lead to impaired insulin secretion and glucose tolerance in their female offspring, which could also show a role of epigenetics in type 2 diabetes.4, 5 But the exact mechanism in which insulin resistance evolves are not clear. Two main theories are currently available.6 The first is that excess lipids can not be stored in fat tissue anymore and thus accumulate in muscle and liver cells instead, causing toxic effects in these cells. The other theory states that adipocytes release inflammatory cytokines, which then cause insulin resistance in other tissues.

Although lifestyle modifications such as a low-fat diet and increased physical exercise can already lead to a improved insulin sensitivity, additional medication is necessary in most of the cases. The classical treatments of type 2 diabetes mellitus include 4 main classes. The sulfonylureas increase the patient’s insulin secretion from the pancreas by increasing the b-cell’s glucose sensitivity. Representatives of this class are glibenclamide, gliclazide, glipizide and glimepiride. The biguanides (for example metformin and phenformin) reduce the hepatic glucose production. Thiazolidinediones (glitazones) are thought to be agonists of peroxisome proliferator-activated receptor-g (PPARg), thus enhancing the action of insulin. Examples for this class of compounds are pioglitazone and rosiglitazone. Rosiglitazone was recommended to be taken off the market by the European Medicines Agency in 2010 due to an increased risk of cardiovascular complications.7, 8 Acarbose is 3

1.1. DIABETES MELLITUS an inhibitor of a-glucosidase, which diminishes the blood glucose levels after meals by inhibiting the uptake of glucose in the gut. Structures of selected anti-diabetic drugs are shown in figure 1.1.

–  –  –

Insulin and its analogues, which are generally used as treatment in type 1 diabetes, can also be used in some cases of type 2 diabetes.

Treatments with anti-diabetic compounds have sometimes severe sideeffects, including weight gain, hypoglycemia, gastrointestinal problems, lactic acidosis, edema and anemia. Since type 2 diabetes mellitus is often associated with obesity, new approaches with a loss of weight, or at least no additional weight gain would be beneficial. Several newer targets are currently under investigation, leading already to some new drugs approved for the market.

Among these are amylin analogs, peroxisome proliferator-activated receptora/g (PPAR-a/g) agonists, sodium-dependent glucose transporter inhibitors and fructose bisphosphatase inhibitors.9, 10 One target exemplified here in more detail are incretin mimetics and enhancers. Incretins are hormones increasing the insulin secretion, thus showing glucose lowering activity. They


are said to aid the regeneration of insulin-secreting cells in the pancreas and to show heart protecting properties. Examples for incretin mimetics are exenatide, liraglutide, taspoglutide and lixisenatide, which are analogues of Glucagon-Like Peptide-1 (GLP-1). Endogenous incretins are rapidly degraded by dipeptidyl peptidase 4 (DPP-4). The gliptins (e.g. vildagliptin, sitagliptin, saxagliptin) are inhibitors of DPP-4 and thereby enhance the activity of the incretins.

1.2 The insulin receptor The physiologic responses to the presence of insulin in the blood stream are mainly initiated by the binding of insulin to its receptor, which then leads to the activation of several signalling cascades. In the following sections, a brief overview on the structure and activation mechanism of the insulin receptor, the main downstream signalling pathways as well as possible reasons for insulin resistance is given.

1.2.1 Structure of the insulin receptor The insulin receptor (IR, INSR) is a receptor protein-tyrosine kinase (EC These enzymes pass on signals from their extracellularly bound ligands to the inside of the cell by transferring phosphate groups from donor molecules such as ATP to tyrosine residues of their substrates. Receptors belonging to the same subfamily as the insulin receptor are the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related protein (INSRR).11 The human insulin receptor precursor (UniProt-ID: P06213) consists of a short signal peptide and two subunits of the insulin receptor, the a- and the b-chain. Numbering of the amino acids is varying, depending on whether the signal peptide is included or excluded. In the present work, the numbering without the signal peptide is used. To get the UniProt numbering, 27 has to be added to the amino acid number. Two different isoforms are produced by alternative splicing: the isoform Long (HIR-B) and the isoform Short (HIR-A), which misses 12 amino acids in the a-chain.


1.2. THE INSULIN RECEPTOR During the maturation process, the insulin receptor precursor is N-glycosylated and intra- and intermolecular disulfide bridges are formed in the endoplasmic reticulum. The a- and b-chains are subsequently cleaved at the trans-Golgi network and further glycosylation occurs before the mature receptor is finally transported to the plasma membrane. The functional form of the insulin receptor consists of two disulfide-linked a,b-dimers. The asubunits are extracellular and contain the insulin binding site. The two b-monomers each have a single transmembrane helix. The C-terminal region is intracellular and contains the kinase domain which is responsible for the activity of the insulin receptor (figure 1.2) Figure 1.2: Schematic representation of the insulin receptor.

X-ray structures of the extracellular domain,12 as well as the intracellular kinase domain13–15 have been resolved. A structure of the whole insulin receptor was determined using electron microscopy.16 1.2.2 Activation of the insulin receptor The first step of the activation of a receptor tyrosine kinase is in general the binding of its ligand to the extracellular domain of the receptor. In many cases this is thought to stabilize the dimerized state of two receptor monomers. Dimerization is necessary to bring the two kinase domains


close to each other, so that trans-phosphorylation of the subunits can occur.

But dimerization alone is not the only prerequisite for the activation of the receptor.17 Binding of the ligand leads to a conformational change, which subsequently leads to the activation of the intracellular kinase domain. For the epidermal growth factor receptor (EGFR) it was shown, that it can exist as an inactive dimer on the cell surface without its ligand.18 Binding of the ligand might lead to a movement in the transmembrane and juxtamembrane region, bringing the two kinase domains in the right distance for autophosphorylation. Another important structural feature in many kinases is the so called activation-loop. Structural rearrangement of this loop which is often associated with the phosphorylation of an amino acid residue can be necessary for activation of the intrinsic kinase.

In the case of the insulin receptor, the dimerization is not necessary as the subunits are already linked by disulfide bonds. Binding of insulin to the extracellular part of its receptor induces a conformational change followed by trans-phosphorylation of Tyr1158, Tyr1162 and Tyr1163 within the activation-loop. In the inactive state, the activation-loop sterically blocks the access for the protein substrate and the ATP binding pocket. Phosphorylation of the tyrosine residues leads to a conformational change of the activation-loop (see figure 1.3), which exposes the binding site and activates the tyrosine kinase domain.13, 14 Recent crystallographic studies identified a possible binding pocket for an insulin receptor activator binding to the intracellular domain.19 They showed the possible role of an additional tyrosine residue (Tyr984) for the activation of the insulin receptor. This tyrosine, which is conserved in all insulin receptor proteins, seems to be important for the autoinhibition of the kinase domain. Tyr984 is positioned in the juxtamembrane region next to the kinase domain. Figure 1.4 shows the N-terminal lobe of the insulin receptor kinase domain in the active and inactive conformation. In a crystal structure of the inactive state, Tyr984 is located in a hydrophobic pocket of the tyrosine kinase domain, whereas in the active state it is not. The active and inactive structures of the insulin receptor also show a movement of the aC helix in the N-terminal lobe of the kinase domain. Stabilization 7

1.2. THE INSULIN RECEPTOR Figure 1.3: Comparison of the active (1IR3, green) and inactive (1IRK, red) conformation of the insulin receptor kinase domain. An ATP analogue and a substrate peptide bound to the activated state are depicted as space filling molecule and a black ribbon, respectively. Phosphorylated tyrosine residues on the activation loop are depicted in stick representation.


Figure 1.4: Comparison of the active (1IR3, green) and inactive (1P14, red) conformation of the N-terminal lobe of the insulin receptor kinase domain.

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