Design And Evaluation Of Inhibitor With Cleaving Activity Against Human Islet Amyloid Polypeptide Aggregation

Type 2 diabetes mellitus is a great threat to human health and quality of life. Islet amyloid deposit is one of the hallmarks of type II diabetes mellitus. The human islet amyloid polypeptide (hIAPP), containing 37 amino acid residues, is the key component of the amyloid deposit. In the meanwhile hIAPP is crucial for amyloid formation. hIAPP is accumulated because of the imbalance of production and clearance caused by insulin resistance and ageing, which further leads to aggregation into amyloid deposit. The toxic aggregates and reactive oxygen species are generated during the aggregation which disturbs the cell membrane stability and causes oxidative stress accumulation. Cell damage then occurs. In this work we tried to find inhibitor targeting islet amyloid polypeptide toxicity. The inhibitor is supposed to interfere with aggregation process as well as reactive oxygen species generation and further hydrolyze islet amyloid polypeptide specifically.Following"recognizing-cleaving"strategy, inhibitor with cleavage activity was synthesized targeting hIAPP. Inhibitor was composed of two parts. Cyclen forming complex with copper ion was the cleavage motif and peptide derived from the core sequence of hIAPP was adopted as the recognition domain. These two parts were covalently linked. Inhibitor can bind to hIAPP via the recognition group. This inhibited self-assembly of hIAPP. Also cleavage on amide was anticipated.It was concluded from the results that the inhibitor increased cell viability and inhibited apoptosis of INS-1 cells by disturbing hIAPP aggregation process including dimmer formation, inhibiting and reversing effect. Hydrolysis on amide bond was detected as well. Cleavage occurred through multibinding of inhibitor to hIAPP. When uncomplexed with copper ion, the inhibitor can seize the copper ion already binding with hIAPP and inhibit H₂O₂ generation. These effects can also increase INS-1 cell viability. When exploring the inhibitory mechanism, it was revealed that both the recognition domain and the cleavage domain were of great importance for inhibitory effect. The recognition domain increased the inhibitory efficiency as well as specificity. It was also found that successful formation of cyclen-Cu(II) was crucial for cleavage effect. The inhibitory effect was confirmed to have two steps including inhibition followed by cleavage.The inhibitor designed according to"recognizing-cleaving"strategy holds the promise to eliminate hIAPP cytotoxicity through multifunctional approach, which shows the potential for the clinic use for treating type 2 diabetes mellitus.