Suramin藥物阻絕 hFGF1 和 FGFR2 D2 domain 之間的結合並降低下游訊號生物活性

Suramin Blocks Interaction between Human FGF1 and FGFR2 D2 Domain and Reduces Downstream Signaling Activity

吳宗憲(Wu, Zong Sian)

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2016年)

摘要

人類纖維母細胞生長因子蛋白(hFGF)家族，是由22種以上結構具有高度相似性的人類纖維母細胞生長因子蛋白所組成，而人類纖維母細胞生長因子蛋白(hFGF1)為hFGF家族蛋白中的一份子，其結構主要由β-sheet組成，是一個具有廣泛病理及生理活性的蛋白質。FGFR2是人類纖維母細胞生長因子受體蛋白(FGFR)家族成員之一，一直以來主要被認為是誘發hFGF1的重要受體，一旦hFGF1與FGFR2結合，將會誘發多個生物訊號傳遞，進而促進細胞增生、細胞分化、促進血管生成、以及促進傷口癒合等。
Suramin在之前研究，已被證實可抑制生長因子，是一種生長因子拮抗劑，因此在本篇研究，我們選用suramin當作hFGF1與FGFR2 D2 domain的阻絕藥物。本實驗希望能夠進一步了解hFGF1-FGFR2 D2 domain與hFGF1-Suramin之間各別交互作用，並利用HADDOCK軟體計算蛋白質錯合物結構及WST1 assay解析對細胞生理活性的影響。
在本實驗中，我們希望藉由核磁共振技術來了解hFGF1-FGFR2 D2 domain與hFGF1-Suramin的反應，利用HSQC找出殘基作用的位置，再利用實驗所得的距離、氫鍵限制條件和雙面角等，經由HADDOCK計算出結構，搭配PyMOL軟體顯示出三維結構並找出其作用力；最後藉由WST1 assay，進而推測出hFGF1-Suramin-FGFR2 D2 domain三者間的生物活性。
在本篇研究能夠幫助我們更加了解hFGF1-Suramin-FGFR2 D2 domain三者之間的反應、三維結構和生物活性，並希望此篇研究對於抗癌藥物有進一步的幫助，且發展出與suramin相關的衍生物和新型具有生物活性的拮抗劑。

關鍵字

母纖維生長因子 ； 母纖維生長因子受體 ； 蘇拉明 ； 蛋白質純化 ； 核磁共振儀水溶液結構 ； 蛋白質-配體作用 ； HADDOCK ； WST-1 分析

並列摘要

The human fibroblast growth factor (hFGF) family has a high degree of structural similarity. hFGFs have been classified into 22 subcategories. FGFs are β-sheet proteins which has extensive of pathological and physiological activity of the proteins. The extracellular portion of hFGF1 interacts with FGFR2 D2, generating three common downstream signaling cascades that ultimately affects mitosis and differentiation. Suramin is an antiparasitic drug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, blocking the interaction between hFGF1 and FGFR2 D2. In this study, we used Varian 700 MHz NMR to titrate hFGF1 with FGFR2 D2 and suramin to elucidate their interactions and binding constant. From HSQC and ITC data, we can know the residues of protein-protein binding regions and the interaction between protein-protein and protein-drug respectively. We further use HADDOCK to calculate protein-protein and protein-drug complex model. Then docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed and further analyzed. We used the PyMOL software to prove the electrostatic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity.
Based on our findings, the results will be useful for synthesis the derivatives of drug and the development of new antimitogenic activity drugs.

並列關鍵字

fibroblast growth factor (FGF) ； fibroblast growth factor receptor (FGFR) ； Suramin ； protein purification ； NMR solution structure ； Protein-ligand interaction ； HADDOCK ； WST-1 assay

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