以液晶感測器篩選抗胰島類澱粉蛋白聚集的小分子

Abstract

澱粉樣蛋白纖維(amyloid fibrils)的形成及其在細胞外累積所造成的細胞毒性是許多類澱粉沉積症的共同特徵，胰島類澱粉蛋白(islet amyloid polypeptide, IAPP)就是其中具有高傾向聚集形成澱粉樣纖維的胜肽之一。IAPP在人體中與胰島素由胰島β細胞共同分泌，在特定的環境下會發生錯誤的聚集摺疊形成具有細胞毒性的寡聚物與不可溶的澱粉樣纖維，影響β細胞正常功能，是導致第二型糖尿病(Type 2 diabetes, T2D)的重要成因之一，在過去研究中，許多文獻都曾提及此症患者的胰島周邊可發現該蛋白沉積。因此，對聚集有抑制效果的分子或材料陸續被開發出來，然而他們所使用的篩選工具都過於耗時且花費成本過高，在本研究中，我們期望利用液晶感測器可以用於快速篩選對IAPP有抑制效果的小分子，並利用一個只會形成寡聚體但不會形成澱粉樣纖維的IAPP變異體 (I26P-IAPP)，來進一步討論液晶感測器的響應機制。在研究的第一部分，我們嘗試利用會帶負電兩親性離子的十二烷基硫酸鈉(sodium dodecyl sulfate, SDS)作為界面活性劑誘導液晶呈現整齊排列，藉由IAPP與SDS間的電荷相互作用，單體會在界面吸附形成高濃度環境，使得IAPP開始聚集，並透過蛋白質體積的變化影響液晶排列，產生光學訊號暗到亮的轉變。然而，在結果中發現SDS與液晶間誘導作用的穩定性不足以無法克服小分子本身結構中具有的氫鍵或苯環結構對液晶排列的干擾。因此，在第二部分的研究中我們嘗試以對液晶分子會有更強誘導的4-正辛氧基聯苯-4-甲酸(4’-n-octyloxybiphenyl-4-carboxylic acid, 8OCBA)作為液晶摻雜物，除了長碳鏈端的疏水性作用，其與液晶分子間會有π-π堆積作用。以此分子摻雜的液晶系統在500 nM IAPP的濃度下就能表現出蛋白質聚集的現象，透過利用這項獨特的功能，我們可以評估分子阻礙 IAPP 聚集的能力並比較其抑制效果，達到快速簡單篩選的目的。The formation of amyloid fibrils which cause cellular toxicity are commonly seen in amyloidosis. Islet amyloid polypeptide(IAPP)is one of the peptides with a high tendency to form amyloid which may contribute to the development of type 2 diabetes(T2D).IAPP is co-secreted with insulin by pancreatic beta cells in the human body. Under specific conditions, it undergoes erroneous aggregation and folding, forming cytotoxic oligomers and insoluble amyloid fibers. This behavior impairs the normal function of beta cells, ultimately contributing to the development of T2D. Previous studies have noted the presence of protein deposits around the pancreatic islets in patients with this disease. In the past, many amyloid inhibitors including small molecules and materials were developed, but the process is time-consuming and high cost. In this study, we aim to develop a liquid crystal(LC)-based sensor system for quick screening of molecules that are capable of inhibiting the aggregation of IAPP. In parallel, we utilize a non-amyloidogenic IAPP variant, I26P-IAPP to investigate the detection mechanism of LC-based sensors. In the initial stage of our research, we explored the use of sodium dodecyl sulfate(SDS), an amphiphilic surfactant that carries the negatively charge , to induce ordered alignment of liquid crystals. Through the interaction between IAPP and SDS, IAPP monomers were adsorbed at the interface, creating a high-concentration environment conducive to IAPP aggregation. Changes in protein volume then influenced the arrangement of LCs, resulting in a transition from dark to bright optical signals. However, during the experiment, we encountered challenges in mitigating the interference of small molecules on the LC arrangement, possibly due to hydrogen bonds or aromatic interactions. We surmise that the induction effect between SDS and liquid crystals may not be sufficiently robust. Subsequently, in the latter part of our study, we turned to utilize 4'-n-octyloxybiphenyl-4-carboxylic acid(8OCBA)as a liquid crystal dopant with stronger inducing properties. In addition to its hydrophobic effect at the end of the long carbon chain, 8OCBA exhibits π-π stacking interactions with liquid crystal molecules. LC systems doped with this molecule demonstrated protein aggregation phenomena observed at a very low concentration of 500 nM IAPP. By utilizing this unique feature, we can evaluate molecules for their capability to hinder IAPP aggregation and compare the effectiveness of their inhibitory effects. The LC sensor streamlines a rapid and simple screening process.