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Browsing by Subject "homology modeling"

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  • Vuorinen, Anna (2010)
    11β-hydroxysteroid dehydrogenase/reductase (11β-HSD) enzymes 1 and 2 regulate the amount of cortisone and cortisol in human tissues. Since overexpression of 11β-HSD1 especially in the adipose tissue causes symptoms of metabolic syndrome, selective inhibition of 11β-HSD1 provides a way to treat this syndrome and type II diabetes. Inhibition of 11β-HSD2 causes cortisol-dependent mineralocorticoid activation, which leads to hypertensive side effects. There are several reported 11β-HSD1 inhibitors, for selective 11β-HSD2 inhibitition, only a few compounds have been developed. The difference between 11β-HSD1 and 2 ligand binding sites is unknown, which complicates the search of selective inhibitors to both of the enzymes. This study was done with two aims: (1) to identify the difference between the two isozymes, (2) to create pharmacophore models for selective 11β-HSD2 inhibitiors. These tasks were approached with computational methods: homology modeling, docking, ligand-based pharmacophore modeling and virtual screening. The homology model of 11β-HSD2 was constructed using SwissModeler and it showed satisfying superimposition both with is template 17β-HSD1 and 11β-HSD1. The difference between the enzymes could not be identified by visual inspections Therefore, seven compounds, of which six are 11β-HSD2 -selective, were docked both to 11β-HSD1 and 11β-HSD2 ligand binding sites using the program GOLD. The docking results revealed that the compounds orientate differently in the enzymes. To 11β-HSD1, the compounds were anchored similar than unselective compound carbenoxolone, whereas in 11β-HDS2, they adopted a flipped binding mode. The flipped binding mode in 11β-HDS2 enables hydrogen bonds to Ser310 and to Asn171, both residues that are only present in 11β-HSD2. Pharmacophore modeling and virtual screening were done using the program LigandScout3.0. The ligand-based pharmacophores were based on the six 11β-HSD2 selective compounds, which were also used for the docking studies. Both of the models consisted of six features (hydrogen bond acceptors, hydrogen bond donor and hydrophobic feature) besides the exclusion volumes. The most important features considering the 11β-HSD2 selectivity seem to be the hydrogen bond acceptor feature that could interact with the Ser310 and the hydrogen bond donor feature next to it. The interaction pair for this hydrogen bond donor feature was not observed in the homology model. However, a possibility of water molecule as an interaction pair was evaluated and it seems to be a possible solution to the problem. Since both of the models were able to find the selective 11β-HSD2 inhibitors and exclude the unselective ones from the test set database, they were employed for the screening of the database that consists of 2700 compounds stored at the University of Innsbruck. From the hits of these screenings ten compounds were selected and sent to biological testing. The results of the biological tests will decide how well the models represent the theory of the 11β-HSD2 selectivity.
  • Karhu, Lasse (2012)
    The orexinergic system is a central regulator for sleep-wake rhythm and energy homeostasis. Dysfunction of the system is at least one of the reasons behind narcolepsy, in addition to which insomnia, obesity and certain cancers could be treated by targeting orexin receptors. The orexin system in human comprises two receptor subtypes, orexin receptor 1 and 2 (OX₁R and OX₂R respectively) as well as two cognate ligands, peptides orexin-A and -B. In this study the focus is on OX₁R and orexin-A. The aims of the study are (1) to propose a binding mode for orexin-A to OX₁R and (2) to understand the molecular interactions of OX₁R leading to receptor activation. I order to create 3D molecular models of OX₁R, a sequence alignment of the eight G proteincoupled receptors (GPCRs) that have been crystallized up to date was first generated by ClustalX and adjusted based on the superimposition by SYBYL-X. Structurally conserved regions were deduced from the alignment and used to add the orexin receptors. Five different models built with MODELLER were selected for their large binding cavity among a large pool of models. These models were constructed based on the chemokine receptor 4 (PDB Id:3ODU), as such and a modified version where TM3 was moved by 1 Å further from the center of the binding cavity, from the β₂-adrenoceptor (PDB Id: 2RH1) and from the adenosine receptor A2A (PDB Id: 2YD0), as such and with rotamer changes to few binding site residues. Orexin-A with straight conformation found by NMR (PDB Id:1WSO) was docked to these models using ZDOCK and RDOCK. In addition, an in-house docking protocol was implemented, but could not be validated. Docking poses were scored by purpose built knowledge based scoring function and clustered. High scoring clusters were then used to converge to three different binding modes. As a result, we suggest that the binding site of OX₁R consists of two hydrophobic walls, one from TM3 and TM5, the other from TM6 and TM7. Binding modes include a hydrogen bond network between the ligand and especially binding site residues Gln1263.32, Thr2235.46, Asn3186.55, Lys3216.58 and Tyr3117.43. Based on the binding modes, it is suggested that the OX₁R is activated by similar binding site contraction as β-adrenoceptors and adenosine A2A. The contraction in could result from the hydrogen bonds between ligand, Gln1263.32, Thr2235.46 and Asn3186.55. The hydrogen bonding of Thr2235.46 can also disrupt interactions between TM5 and TM3, an interaction which is identified as an important factor in keeping the receptor in the inactive state. The role of other ligand residues would be to direct ligand binding and keep the ligand in the helical conformation.
  • Urvas, Lauri; Rinne, Maiju; Xhaard, Henri (2020)
    The orexin system is an important regulator of the sleep/wake cycle, and small molecule agonists of the orexin receptors could be beneficial for patients suffering from type 1 narcolepsy. To develop new therapeutics, understanding the interactions between the orexin peptides and their cognate receptors is crucial. The three-dimensional arrangement of the orexin-A peptide complexed to its cognate orexin-2 receptor has so far remained elusive. Here, we identify structurally conserved regions at the predicted binding site and conserved residues of the orexin-A peptide by comparing orexin 2 receptor sequences from nine diverse species as well as with insect allatotropin receptor, a distant relative, and orexin-A sequences from 10 different species. We also visualized the conservation of interaction networks in the orexin 2 receptor binding site by building homology models of the putative orexin receptor of Ciona intestinalis and the allatotropin receptor of Manduca sexta. Structural conservation in the binding site is concentrated on transmembrane segments 2-3-7, in particular the salt bridge between D2.65 and H7.39, and a hydrogen bond network between Q3.32-T2.61-Y7.43. Conservation in orexin-A is concentrated on the C-terminus, while the most conserved individual residues of the peptide are L20, G29, I30, and L31. Applying our conservation results into the analysis of two previously suggested binding modes for orexin-A shows that one of the two demonstrates better mirroring of the conserved residues between the peptide and the binding site. Finally, our data shows that the hydrophobic side of helix 1 of the amphipathic orexin-A should be seriously considered in the analysis of binding modes.