雙氧水響應的硼酸結構用於牽制降鈣素類澱粉纖維的形成

Abstract

人類降鈣素(human calcitonin , hCT)是一種由32個胺基酸所組成的肽類激素，由甲狀腺的濾泡旁細胞所分泌，主要的作用是透過抑制破骨細胞的活性及腎臟、腸道對鈣離子的再吸收來降體內低血鈣濃度，因此可被用於治療骨質相關疾病，如骨質疏鬆症和佩吉特氏病，然而hCT容易於水溶液中形成類澱粉蛋白纖維(amyloid fibril)，這些纖維狀的聚集體會造成hCT生理功能下降，使hCT在作為藥物的使用上受到限制，目前臨床上則以低聚性的鮭魚降鈣素(salmon calcitonin, sCT)來取代hCT，但由於sCT與hCT序列的同源性低，造成部分患者服藥後產生嚴重的副作用。本研究工作試圖以分子嫁接方式來穩定hCT的構型，以便有效抑制hCT的聚集。我們使用硼酸化合物(NPC-BA)與hCT序列中兩個胺基酸側鏈(胺基)反應來”鎖住”hCT構型，並在需要該胜肽時，利用雙氧水”解開”，使恢復其原始狀態。在研究的第一部分，我們根據NPC-BA與胺基酸側鏈的反應性嘗試將其嫁接在hCT序列上N端(N-terminus)及Lys-18上，然而，由於兩連接位點過遠及N端反應性較Lysine低的原因，導致NPC-BA未能成功嫁接於hCT上。因此，我們將序列中Gln-14及Phe-22分別置換成Lysine，再將N端官能基乙醯化，合成兩條hCT的變異體，Ac-hCT-Q14K及Ac-hCT-F22K來做為與NPC-BA嫁接的胜肽。這種方式不僅成功將小分子NPC-BA分別嫁接於兩變異體上外，我們也證明雙氧水的響應也能將其”解開”，而硫磺素-T螢光動力學實驗也證實Ac-hCT-Q14K 和 Ac-hCT-F22K 相較於 hCT 具有更優異的抗聚集能力，同時其生物活性與hCT相比大致維持不變，然而溶解度差的問題是未來我們仍需要進行優化的部分。Human calcitonin (hCT) is a peptide hormone composed of 32 amino acids, secreted by the parafollicular cells of the thyroid gland. Its primary function is to lower blood calcium levels by inhibiting osteoclast activity and reducing calcium reabsorption in the kidneys and intestines. Due to these physiological roles, hCT has been employed in the treatment of bone-related diseases such as osteoporosis and Paget’s disease. However, hCT has a high tendency to form amyloid fibrils in an aqueous solution, leading to a loss of biological activity and limiting its therapeutic application. Currently, salmon calcitonin (sCT), which has a low aggregation propensity, is used as a clinical alternative. Nevertheless, the low sequence homology between sCT and hCT often results in serious side effects in patients. This study aims to stabilize the conformation of hCT through molecular conjugation in order to effectively suppress its aggregation. A boronic acid–containing compound, NPC-BA, was employed to"lock" the conformation of hCT by reacting with amine groups on the side chains of two amino acids. Upon demand, the conformation can be “unlocked” using hydrogen peroxide, thereby restoring the peptide to its original state. In the first part of the study, based on the reactivity of NPC-BA toward amino acid side chains, we attempted to conjugate it to the N-terminus and Lys-18 of hCT. However, due to the long spatial distance between the two conjugation sites and the lower reactivity of the N-terminal amine compared to lysine, the conjugation was unsuccessful. To address this issue, Gln-14 and Phe-22 in the hCT sequence were substituted with lysine, and N-terminal acetylation was introduced to construct two hCT variants, Ac-hCT-Q14K and Ac-hCT-F22K, providing closer conjugation points and more reactive lysine residues. This strategy successfully enabled NPC-BA to be conjugated to both variants. Moreover, the boronic acid–based linkage could be cleaved by hydrogen peroxide, effectively restoring the native peptide structure. Thioflavin-T fluorescence kinetic assays demonstrated that Ac-hCT-Q14K and Ac-hCT-F22K exhibited superior anti-aggregation properties compared to native hCT, while retaining comparable levels of biological activity. However, poor solubility remains a limitation, and further optimization will be necessary in future studies.