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Synthesis and biochemical characterization of hybrid analogues of human insulin and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Koberová, Monika (referee)
The ever-increasing occurrence of diabetes mellitus brings about the need for development of new therapeutic agents to provide adequate treatment for patients. An important element in this research area is elucidation how insulin works, mainly in connection with insulin-like growth factors (IGF-1 and IGF-2), which show significant structural homology to each other. In addition, their respective receptors - insulin receptor (IR) and receptor for IGF-1 and IGF-2 (IGF-1R) - exhibit very high similarity. As a result, IGF-1 and IGF-2 can bind to IR and insulin can bind to IGF-1R. Of a particular importance is the high affinity binding of IGF-2 to the isoform A of IR. Unlike insulin, which predominantly mediates glucose entry into cells, IGFs induce growth or mitogenic effects. The finding which structural determinants in insulin and IGFs are responsible for the differences in the activation of their cognate receptors could provide an explanation for different functional responses upon binding of these hormones to different target cells. Understanding of this mechanism could also help in the development of functionally selective analogues of these hormones. The aim of this study was the synthesis and characterization of analogues of human insulin extended at the C terminus of the B chain with the amino...
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Phenotyping of proteolytic activities enabled by fluorogenic libraries
Pospíšil, Šimon ; Míšek, Jiří (advisor) ; Hocek, Michal (referee)
This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.
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Preparation of a new insulin analog in order to study the interaction of the hormone with insulin receptor isoforms,
Halamová, Tereza ; Jiráček, Jiří (advisor) ; Obšilová, Veronika (referee)
Insulin acts as a key hormone in the blood glucose levels maintaining mechanisms. Outside this metabolic function it also has a growth hormone functionality. The interaction of insulin with the two existing insulin receptor isoforms - IR-A and IR-B, which are variously represented in the human body is determining insulin. IR-A, supposed to be mainly responsible for the mitogenic function of insulin, is located in the brain or lymphatic cancer and fetal tissue, whereas IR-B, performing metabolic function is located in adipose and muscle tissue. Present aim is to design such insulin analogs that would preferentially bind to IR-B, and could thus more efficiently carry out physiological metabolic function of insulin necessary for patients with diabetes. Based on the recently solved 3D structure of insulin bound to IR, it was found that the C-terminus of the B-chain of insulin must undergo conformational change bending it in about 90ř, for efficient binding to IR. The aim of this thesis was the preparation and characterization of two insulin analogs with bridging C-terminus of the B-chain in positions B26-B29 and B27-B29 using disulfide bridge. This could fix a bended structure of the B chain end and could help to increase the affinity of IR and specificity for IR-B. The preparation was carried out...
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Interaction studies of insulin, IGF-1/2 and IGF-1 analogue with insulin and IGF-1 receptors
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Insulin-like growth factors 1 and 2 (IGF-1/2) are single-chain peptides exerting homology (in both amino-acid sequence and tertiary structure) to insulin. The main function of these peptides is promoting celular growth, proliferation and differentiation. Both insulin and insulin-like growth factors mediate their function through membrane receptors - insulin receptor (isoforms A and B) and IGF-1 receptor. All these receptors are members of the tyrosinkinase family of receptors and they exert the same subunit and domain composition. The activation of insulin and IGF-1 receptors is tightly associated with activation of two intracellular signaling pathways. The PI3-K/Akt pathway is involved in the glucose transport to the cell, induction of proliferation or inhibition of apoptosis, while the Ras/MAPK pathway is involved mainly in the induction of cell growth and differentiation. Due to the structure similarity in both the ligands and receptors, every ligand can activate different receptors (with different potency) and the signaling pathways associated with these receptors. Thus, the functions of IGFs and insulin, the same as their receptors, are overlapping. The distinct function of the concrete ligand can be distinguished by the different tissue distribution of both isoforms of insulin receptor and...
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Synthesis and biochemical characterization of hybrid analogues of human insulin and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Koberová, Monika (referee)
The ever-increasing occurrence of diabetes mellitus brings about the need for development of new therapeutic agents to provide adequate treatment for patients. An important element in this research area is elucidation how insulin works, mainly in connection with insulin-like growth factors (IGF-1 and IGF-2), which show significant structural homology to each other. In addition, their respective receptors - insulin receptor (IR) and receptor for IGF-1 and IGF-2 (IGF-1R) - exhibit very high similarity. As a result, IGF-1 and IGF-2 can bind to IR and insulin can bind to IGF-1R. Of a particular importance is the high affinity binding of IGF-2 to the isoform A of IR. Unlike insulin, which predominantly mediates glucose entry into cells, IGFs induce growth or mitogenic effects. The finding which structural determinants in insulin and IGFs are responsible for the differences in the activation of their cognate receptors could provide an explanation for different functional responses upon binding of these hormones to different target cells. Understanding of this mechanism could also help in the development of functionally selective analogues of these hormones. The aim of this study was the synthesis and characterization of analogues of human insulin extended at the C terminus of the B chain with the amino...
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