整合蛋白α5β1是纖維黏蛋白 (簡稱Fn) 的主要受體，活化後的整合蛋白α5β1能夠調控許多細胞的行為，例如凝血 (blood cloting)、傷口癒合 (wound healing) 以及組織修復 (tissue repair) 等功能。有研究指出，阻擋整合蛋白α5β1活化，能夠有效抑制VEGF所誘導的血管新生，進一步抑制腫瘤生長。而在本研究中，我們使用維黏蛋白的第三型區域中第九和第十個模組 (9,10Fn3) 作為鷹架蛋白進行藥物的設計。9,10Fn3能和整合蛋白α5β1結合，在10Fn3上的RGD motif和9Fn3上的PHSRN motif協同區域 (synergy site) 共同參與能夠讓結合更為緊密，因此我們希望研究協同區域如何影響與整合蛋白α5β1之間的結合。在實驗室先前的研究中，使用9,10Fn3當作鷹架蛋白成功設計出對整合蛋白α5β1有專一性結合的拮抗劑9,10Fn3LPCRA，IC50為65.0 nM。同時也已經証明了其能夠有效的抑制血管內皮細胞的存活、增值、遷移和管狀形成，並且在小鼠的動物模型中，能有效的抑制腫瘤生長。為了進一步改善9,10Fn3LPCRA對整合蛋白α5β1抑制能力，我們透過定點突變 (site directed mutagenesis) 方式研究9Fn3上的1376PHSRN motif，希望增加9,10Fn3LPCRA對整合蛋白α5β1之親和力。目前已經成功透過E. coli系統表現及純化27種突變蛋白以及兩種多價蛋白。由細胞黏著的實驗結果中顯示，P1376D和R1379E突變蛋白對整合蛋白α5β1辨識能力下降接近9倍和781倍左右，先前文獻也曾指出R1379之正電荷胺基酸為辨識α5β1的關鍵，而我們認為負電荷胺基酸並不適合存在於1376PHSRN motif中。當突變1376PHSRN motif為KHSRN和KKKRT時，對整合蛋白α5β1辨識能力下降2.7倍和1.7倍。透過molecular docking 實驗，我們解釋有可能位置1376為正電荷胺基酸時，會使R1379和α5次單元上的D154鹽橋變得不穩定。此外，我們發現S1378K系列的突變蛋白對整合蛋白α5β1辨識能力上升約2到3倍左右。我們從結果中發現若位置1377和1378為正電荷胺基酸時，能大幅增加對整合蛋白α5β1的結合親和力。最後，我們突變1376PHSRN motif為YKKRN序列，發現其對整合蛋白α5β1辨識能力為最高，IC50為22.8 nM。另外一方面，我們所設計的多價蛋白9,10Fn3-9,10Fn3對整合蛋白α5β1親和力上升接迎7倍，因此我們認為多價設計能夠大幅增加整合蛋白抑制劑與整合蛋白之結合能力。本次研究將可作為對α5β1設計拮抗劑的基礎。

Integrin α5β1 is a fibronectin receptor that regulates complex biological cell behavior, such as differentiation, development, wound healing, and tumor progression. Many studies showed that the blockade of integrin α5β1 inhibited VEGF-induced angiogenesis, leading to the inhibition of tumor growth or tumor regression. In this study we used the ninth and tenth module of fibronectin type III domain (9,10Fn3) as the protein scaffold to study the effect of 1376Pro-His-Ser-Arg-Asn (PHSRN) motif, the synergy site, on its integrin α5β1 binding affinity. In our previous study we have successfully designed an integrin α5β1-specific antagonist, 910LPCRA, with the IC50 value of 65.0 nM. We now used site-directed mutagenesis to engineer the synergy site of 910LPCRA to improve its activity in inhibiting integrin α5β1. In this study I have successfully expressed and purified twenty-nine 9.10Fn3 mutants in E. coli to homogeneity. According to the results of cell adhesion assay, DHSRN and PHSEN mutants exhibited 9- and >781- fold decreases in the inhibition of integrin α5β1.The mutation of 1376PHSRN into 1376KHSRN and 1376KKKRT caused 2.9- and 1.8-fold decreases in their activity. This was consistent with the result of molecular docking experiment that a positively charged residue in the1376 position destabilized the salt bridge interaction between R1379 and D154 of integrin α5 subunit. We also found that S1378K mutants exhibited 2-3-fold increase in the inhibition of integrin α5β1. These results showed that the 1377 and 1378 positions with positively charge amino acid residues in the synergy site can increase its integrin α5β1 binding affinity. In particular, the 1376YKKRN mutant exhibited highest inhibitory activity with the IC50 value of 22.8 nM. We also found that the dimeric 910LPCRA (9,10Fn3-9,10Fn3) mutant exhibited 7-fold increase in inhibiting integrin α5β1 with the IC50 value of 9.8 nM. The results of this study will serve as the basis for the design of integrin α5β1-specific antagonist.

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