硫磺素T的衍生物作為替代螢光探針用於偵測人類胰島類澱粉蛋白的聚集

Abstract

許多胜肽或蛋白質的不可逆錯誤折疊被指出與一些疾病有高度相關。例如，人類胰島類澱粉蛋白 (human islet amyloid polypeptide, hIAPP) 是一個由37個胺基酸所組成的賀爾蒙胜肽，容易發生聚集形成高度具有規則性結構的聚集體，稱為類澱粉蛋白纖維，並沉積於胰島β細胞的外部，造成β細胞的凋亡，此聚集體在大多數第二型糖尿病的病患中被找到。硫磺素T (Thioflavion T, ThT) 是一種普遍用於檢測類澱粉蛋白纖維生成的螢光探針。然而，ThT的放光波段與生物組織中自發螢光物質訊號重疊。合成的小分子或是奈米材料使用在一般抑制效果的測試實驗時，也有可能因本身的吸光範圍而影響到ThT的偵測結果，造成實驗結果的誤判。這激勵了我們想以ThT為結構骨架並進一步合成出另一系列的螢光探針，透過不同官能基的修飾使得放光波長能夠靠近近紅外光的範圍，以克服以上所述的難題。AmySP-4-Nap-Ene為該系列其中一個可放光在近紅外光區的螢光探針，它可辨識hIAPP形成的類澱粉蛋白纖維而大幅增長螢光強度，對於hIAPP纖維的結合親和力還更優於ThT。爾後，我們還利用全內反射式螢光顯微鏡觀察到螢光探針與hIAPP纖維結合時的放光。將其與大鼠胰島瘤細胞共培育48小時後也沒有發現明顯的細胞毒性。初步研究結果顯示，此探針具有檢測類澱粉蛋白纖維存在的潛力。

Many irreversible misfolding of peptides or proteins may be related to some severe diseases. Human islet amyloid polypeptide (hIAPP), a 37-amino acid residue peptide hormone, is prone to aggregate and form highly ordered amyloid fibrils. The deposit of hIAPP amyloid fibrils found outside the pancreatic β-cells of patients with type 2 diabetes is considered to be closely related to β-cell apoptosis. Thioflavin T (ThT) is a general fluorescent probe used to detect amyloid fibril formation. However, the fluorescence emission range of ThT overlaps the autofluorescence from biological matter in tissue. When the synthetic compounds or nanomaterials are used in the general inhibition effect tests, their own absorbance band may interfere with ThT and result in misjudge. That encourages us to synthesize a series of ThT derivatives to overcome these problems. We aim to modify these probes and force these probes to emit in the near-infrared (NIR) range. AmySP-4-Nap-Ene (maximum λem at 685 nm) is one of these synthetic probes and shows a significant fluorescence enhancement toward hIAPP amyloid fibrils. We found that the binding affinity of AmySP-4-Nap-Ene to hIAPP fibrils is better than that of ThT. Afterward, we observed the fluorescence of the probe upon binding to hIAPP fibrils by using the total internal reflection fluorescence microscope. This probe did not induce significant cytotoxicity when incubated with the rat insulinoma cell (INS-1) within 48 h. Preliminary test results indicate that this probe would be used in the detection of amyloid fibrils.