化學蛋白質體學搭配質譜技術分析Sunitinib之標的候選蛋白質

Abstract

化學蛋白質體學是一門新興的研究領域，主要目的在於鑑定與驗證活性小分子之標的蛋白質，利用化合物與蛋白質間特異性的相互作用力成為一個強而有力的搜尋技術。本篇論文目的為在人類急性骨髓性白血病(AML)細胞株MV4-11中找到sunitinib之標的蛋白質。先前文獻中顯示出小分子藥物sunitinib有抑制AML細胞株中FLT3磷酸化的作用，進而有效地抑制掉MV4-11的增殖組成。利用化學蛋白質體學的策略，sunitinib合成的生物素標記藥物固定於鏈黴卵白素粒子上形成親和性管柱，建立活性為基礎的探針於MV4-11細胞株中抓取特定蛋白質。實驗設計中導入negative控制組，其結構與biotinylated sunitinib相似但不具有藥理活性，做為背景值來減少非特異性蛋白質之鍵結。經過親和性層析適當的洗滌與洗脫過程，被洗脫之蛋白質以一維膠體電泳(SDS-PAGE)蛋白質和液相層析串聯式質譜儀(LC-MS/MS)進行分析和蛋白質資料庫比對鑑定出候選標的蛋白質。MV4-11細胞裂解液中有248個標的蛋白質被鑑定到；他們大部分可能為sunitinib之新奇作用物和標的蛋白質。藉由GeneGo路徑分析，在muscle contraction relaxin和development VEGF signaling via VEGFR2 generic cascades這兩項路徑中，RAP-1A和MAP2K1被認為是藥物目標物而且他們與血管的生成有關係，造成影響MV4-11細胞增殖與存活的原因。

Chemical proteomics is an emerging field that the goal is to identify and validate target proteins for bioactive small molecules. It utilizes the specific interaction of compound and the target proteins to be a powerful screening technology. The main purpose of this study is to identify sunitinib target proteins in acute myeloid leukemia (MV4-11) cell lines. It has been reported that sunitinib inhibits phosphorylation of FLT3 in AML cell lines, which potently inhibits constitutive proliferation of MV4-11. Using strategy of chemical proteomics, the synthesized biotin-tagged sunitinib was immobilized on streptavidin beads to make affinity column as an activity-based probe that captures the target proteins in MV4-11 cells. A negative control, which structure was similar to biotinylated sunitinib with no pharmacological activity, was designed as a background for eliminating the impact resulting from non-specific binding proteins. After appropriate wash and elution steps in the affinity chromatography, the eluted proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Two hundred forty eight sunitinib-binding proteins were identified in MV4-11 cell lysate. They are potential interactors and targets of sunitinib. These sunitinib-binding proteins were further analyzed by GeneGo and found they were associated with muscle contraction relaxing and development VEGF signaling via VEGFR2 cascades signaling pathways. Among them, RAP-1A and MAP2K1 are considered drug targets and related to angiogenesis. They are also contributed to the effects on MV4-11 cell proliferation and survival.