Peptides for treatment of diabetes
A technology of amino acids and analogues, applied to peptides containing affinity tags, preparations for in vivo tests, peptides containing His tags, etc., can solve problems such as increased risk of hypoglycemia
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Embodiment 1
[0465] Example 1: Design of Peptides
[0466] Design of novel peptides following rational structure-activity studies. For FOL-005 (SEQ ID NO: 1), the peptide was designed around the RGD site, but mutated to create different structures that may interact with different integrins. A sequence similar to FOL-005 was identified in the domain of the third fibronectin type III repeat (TNfn3) in tenascin-C and found to be quite similar to the mutant RGD site of FOL-005. A peptide was designed from this sequence, denoted FOL-014. The X-ray crystal structure of the tenascin-3 TNfn3 domain was analyzed (PDB code 1TEN, Leahy et al. (1992) Science 258(5084):987-91). The FOL-014 (SEQ ID NO: 136) sequence spans the first β-turn structure and the entire 3rd β-sheet layer. FOL-014 variants were designed to allow structural modification and stabilization of three-dimensional molecular structures. Specifically, peptide variants cover the β-turn structural region with exposed side chains and s...
Embodiment 2
[0468] Example 2: FOL-005 and FOL-014 induce proliferation of INS-1 cells
[0469] To investigate whether FOL-005 and FOL-014 can induce the proliferation of β cells, we used INS-1 cells. Rat INS-1 cells were seeded in 96-well plates in RPMI medium with supplements, and after 2 hours, the medium was changed to RPMI without supplements. During proliferation experiments, cells were incubated under different test conditions (FOL-005, FOL-014, coated or in solution, 48 h incubation), and during the last 20 h of the culture period, cells were treated with 1 μ of Ci / Wells of [methyl-3H]thymidine-pulsed cells. Cells were then harvested onto glass fiber filters using a FilterMate harvester. Filters were air dried and bound radioactivity was measured using a liquid scintillation counter. To investigate whether FOL-005 affects β-cell proliferation, INS-1 cells were treated with increasing amounts of soluble FOL-005 (0.06-6 μM) over 48 hours and measured with radiolabeled thymidine i...
Embodiment 3
[0471] Example 3: FOL-005 protects β cells from glucotoxicity
[0472] Since glucotoxicity of pancreatic β-cells is a well-recognized process in type 2 diabetes, we next investigated the protective effect of FOL-005 on β-cells under glucotoxic conditions. First, we confirmed that 20 mM glucose after 48 h of exposure induced apoptosis in INS cells. High glucose (20 mM) containing RPMI medium induced more Annexin V positive cells and more Caspase-3 activity in INS cells compared to cells incubated with medium containing 5 mM glucose ( figure 2 A-B). INS-1 cells simultaneously exposed to 20 mM glucose and FOL-005 reduced apoptosis as detected by Annexin V staining and Caspase-3 activity ( figure 2 A-B). Apoptotic rate in INS-1 cells was measured by staining with Caspase-3 assay kit, or Annexin V apoptosis detection kit with 7-AAD. Caspase-3 activity was measured using fluorescence at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Caspase-3 activit...
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