| Recently, the research showed that the decrease of the function and quantity of pancreaticβ-cells caused by high glucose concentration was the primary cause for type 2 diabetes. So the function and quantity of pancreaticβ-cells and the sensitivity of peripheral tissues to insulin is essential for improving type 2 diabetes. Exendin-4 as an agonist of GLP-1R, it can active some element for signal transduction coupled with GLP-1R and execute physiological regulation, including the proliferation, regeneration, promoting differentiation from progenitor cell. Because of its capability for improving pancreaticβ-cells radically, exendin-4 was regarded as one of the most promising drug for type 2 diabetes.Exendin-4 is a peptide of 39 amino acids residues that was originally isolated from the salivary secretions of lizard Heloderma Suspectum in American Southwest. Exendin-4 displays similar functional properties to native GLP-1. Exendin-4 is consisted of three regions: the N-terminal region comprising residues 1~9 is an irregular coil and responsible for activating GLP-1R; the central region comprising residues 10~30 is a regularα-helix and significant region for binding GLP-1 receptor; the C-terminal region comprising residues 31~39 formed a compact hydrophobic cluster centered with Trp25 ofα-helix, called Trp-cage.Because of its glucose-lowering efficiency, exendin-4 as the first incretin mimetics had been researched for Type 2 diabetes by Amylin Pharmaceuticals and Eli Lilly Inc. and named Byetta. Exenatide can promote insulin secretion, increaseβ-cells, and inhibit gastric emptying and glucagon secretion. Another hand, it is responsible for weight loss and reducing risk of hypoglycemia. Exenatide was approved by FDA on April 28, 2005. As a peptide drug, Exenatide wasn't administrated as an oral way. It made diabetes patients very inconvenient. The aim of this study was that provide some structural information for designing the oral chemical drug through acquiring a high-active short peptide. So we were going to acquire the high-active short peptide from two aspects:1. Designing the structural mimeticsBased on the structural information of exendin-4, we designed some mimetics under remaining the N-terminus for activating receptor as below:(1) Structural mimetics of Ex1 and Ex2: Ex1 was designed by deleting N-terminal helix (LSKQMEEEA) and consisted of 30 amino acids; Ex2 was designed by replacing the N-terminal helix of exendin-4 with 3 Alas and consisted of 33 amino acids.(2) Structural mimetics of Ex3: Ex3 was designed by deleting the 11 amino acids residues and replacing Gln13 with Tyr13, it was consisted of 28 amino acids.(3) Structural mimetics of Ex4 and Ex5: Ex4 was designed by replacing the C-terminal helix (EEEAVRL) of exendin-4 with 3 Alas and consisted of 35 amino acids. Ex5 was designed by replacing Gln13 with Tyr13 based on the sequence of Ex4.(4) Structural mimetics of T1 and T2: T1 consisted of 29 amino acids was designed through connecting N-terminus active region of exendin-4 with Tc5b. T2consisted of 32 amino acids was designed through connecting N-terminus active region of exendin-4 with Tc5b by three Alas. These structural mimetics was investigated from the proliferation assay ofβ-cell, stability against DPPⅣ, circular dichroism (CD) and fluorescence spectroscopy assay. We drawed some conclusion: (1) N-terminal helix (LSKQMEEEA) was more important than C-terminal helix (EEEAVRL) for activating GLP-1R.(2) Replacing some sequence with 3 Alas was an effective method for optimizing exendin-4. We got two high-activity mimetics, Ex4 and Ex5. (3) Replacing Gln13 with Tyr13 was an important site for optimizing exendin-4. It was helpful to increase the helical tendency, bioactivity and stability.2. Screening the phage 12-mer peptide libraryUsing the technique of phage display, a receptor-binding region on the surface of RINm-5F cell was used as the target to screen peptide ligands for the receptor in a phage 12-mer peptide library. During the screening course, we increased the Tween-20 concentration (0.3%) in the washing buffer and eluted specially with high concentration exendin-4 (0.5mmol/L) from the second round in order to acquire some peptides binding specially for GLP-1R. After four rounds screening, there was a good enrichment of special phage clone and DNA sequencing revealed a group of closely related peptides from the third and the fourth round of selection, including SVGMKPSPR,SV~VGMKPSPR,SV~MKPSPR and LT~PGPR.We screened the active peptide in the five peptides containing the motif of SV~MKPSPR and two peptide containing the t motif of LT~PGPR. Finally, we got a more active 12-mer peptide named Ex-12PA. The bioactivity of Ex-12PA was slightly higher than that of exendin-4 and higher than KS-12 that researcher of Nankai University acquired by screening the 12-mer peptide library. We thought that the existence of His at the sequence terminus of Ex-12PA caused its high-activity because His in the exendin-4 and GLP-1 was the important amino acid for activating receptor. However, Ex-12PA displayed a faint hypoglycemia activity in the mice. The possible reason was that there was a hydrolytic site (Gln-Pro) in the sequence of Ex-12PA. It made a less stability and caused transient efficacy.The sequence of Ex-12PA built a good foundation for designing the oral diabetes drug. Other regular motifs which were no bioactivity were significant for the researching the mechanisms of binding GLP-1R and designing the short mimetics of exendin-4.For detecting the bioactivity of memtics exactly and efficiently, we improved the MTT assay and we built a quantitative assay based on the characteristic of binding nGLP-1R expressed in procaryon system.Glucose toxicity caused by chronic hyperglycemia would lead to decrease the quantity of pancreaticβ-cells and inhibit insulin secretion, which was the serious problem for type 2 diabetes patients. To investigate whether exendin-4 can protect pancreaticβ-cells in the chronic hyperglycemia and whether Ex-12PA screened from phage library had same role on protecting pancreaticβ-cells, we detected the pathway of signal transduction bcl-2/ bax ---Caspase-3. Furthermore, we tested pdx-1 and insulin gene related with the fuction of pancreaticβ-cells.Preliminary results show: (1) Chronic hyperglycemia induced the decrease of bcl-2 and increase of bax,β-cells trended to apoptosis. The addition of exneind-4 and Ex-12PA can upregulate bcl-2 and downregulate Bax to maintain the proportional balance of bax/bcl-2. So exendin-4 and Ex-12PA inhibited the apoptosis through regulating the rate of bax/bcl-2, but also Ex-12PA had a little stronger than exendin-4. At the same time, exendin-4 and Ex-12PA also inhibit the bioactivity of Caspase-3, but had a little stronger than Ex-12PA. (2)Exendin-4 and Ex-12PA improved the function of RINm-5F cells by increasing the mRNA level of pdx-1 in chronic hyperglycemia. Ex-12PA had a stronger capability to regulate pdx-1 gene. However, the mRNA level of insulin didn't changed. So exendin-4 and Ex-12PA had a same protective mechanism for inhibiting pancreaticβ-cells apoptosis and improving the function of pancreaticβ-cells in hyperglycemia.In summary, we optimized the central helical region of exendin-4 successfully and got some mimetics that had same bioactivity with exendin-4. Another hand, we utilized the GLP-1 receptor on the surface of RINm-5F cell as the target to screen peptide ligands for the receptor to screen ligands from a phage 12-mer peptide library and acquired a 12-mer peptide Ex-12PA. A further research showed that Ex-12PA had a same protective mechanism to pancreaticβ-cells with exendin-4 in hyperglycemia. The successful structure optimization of exendin-4 and acquiring Ex-12PA built a solid foundation for further designing peptoid for oral drug of diabetes. At the same time, the establishment of two complementary activity assay system will provide a technical platform for detecting the bioactivity of memitics quantitatively.
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