阻止血管新生是治療腫瘤細胞迅速擴展的策略之一，然而，目前美國FDA 所通過大部分針對 VEGF 及 VEGFR 的抗血管新生藥物在臨床上都顯示有抗藥性。整合蛋白在血管新生上扮演了至關重要的角色，它屬於穿膜蛋白，負責調控細胞之間以及細胞與細胞外基質的交互作用，並牽涉到內皮細胞的黏附、遷移以及增生，是一個適合治療腫瘤細胞生長的標靶。實驗室先前的研究曾發現，馬來腹蛇蛇毒蛋白 Rhodostomin (Rho) 的二聚體與纖連蛋白 (fibronectin) 的多聚體抑制整合蛋白 αvβ3 的活性都有明顯的提升，而與單聚體相較起來，活性則有 2-10 倍的增加。馬來腹蛇蛇毒蛋白是一個中等大小的去整合蛋白，它包含了 48PRGDMP motif 及 65PRYH C-terminus 這兩段區域會與整合蛋白有交互作用。除此之外，先前的研究還曾以 X-ray 的方式進行結構分析，發現鄰近 RGD motif 的 C 端區域會與 Rho 的 C 端相互作用，因此這樣的一個作用力或許在辨識整合蛋白上扮演一個重要的角色。在本篇研究當中，我們利用 Rhodostomin 為模板，針對多聚體、鄰近 RGD motif 的 C 端區域去做研究，像是 58TGQSA 及 54DD。到目前為止，我們已成功表現及純化十個突變蛋白，其產率介在 1.3-24.0 mg/L。在抑制整合蛋白 αvβ3 的實驗中，Rho 及 KG (Rho 突變株) 三聚體的活性顯示出 3.4-3.5 倍的提升；而在抑制整合蛋白 α5β1 的實驗中，活性則有 1.3-3.4 倍的提升；在抑制整合蛋白 αⅡbβ3 的實驗中，Rho 三聚體的活性則有 7.3 倍的提升；最後，在抑制整合蛋白 αvβ5 的實驗中，活性則有 3.7-4.6 倍的提升，這些的結果與二聚體相較起來則有相似的活性。在抑制整合蛋白 αvβ3、α5β1 及 αvβ5 的實驗結果顯示，T58N、Q60R 及 Q60I 突變株均具相似的活性，而在抑制整合蛋白 αⅡbβ3 的活性則有 1.7-2.3 倍的下降。D55A、D55N、D55K、D55R 及 D55T 突變株在抑制整合蛋白 αvβ3、α5β1、αⅡbβ3以及 αvβ5 的活性下降了 2.0-30.1 倍，其中當第 55 個胺基酸突變成 K 時，抑制整合蛋白的活性表現是最差的，其活性則分別下降 30.1 倍、6.9 倍、9.6 及 17.4 倍。當第 55 個胺基酸突變時，大多數抑制整合蛋白的活性下降，因此，推測第 55 個殘基與 C端之間的氫鍵作用力在辨識整合蛋白上扮演一個重要的角色。本研究的結果在未來則可用來提供專一性整合蛋白藥物的設計。

Preventing angiogenesis is one of the strategies to treat the rapid expansion of tumor cells. However, most of the FDA-approved angiogenesis inhibitors targeting VEGF or VEGFR have shown resistance in cancer. Integrins are transmembrane proteins that mediate the interactions of cell and extracellular matrix and involve in endothelial cell adhesion, migration, and proliferation, which are therapeutic targets to suppress tumor growth by inhibiting angiogenesis. In our previous studies, we found that dimeric forms of rhodostomin (Rho) and the multivalent forms of fibronectin exhibited 2-10-fold increases in the inhibition of integrin αvβ3 than those of monomeric forms. According to structural analysis, we found that the C-terminal region adjacent to the RGD motif interacted with the C-terminus of Rho, suggesting that this interaction may play a vital roles in the recognition of integrins. In this study, I used Rho as a protein scaffold to study the effects of multivalent forms, the C-terminal with 58TGQSA, 54DD region adjacent to RGD motif on their inhibitory activities. To date, I have successfully expressed twelve Rho mutants in Pichia pastoris and CHO cells. I purified them to homogeneity with the yields of 1.3-24.0 mg/L. The results of cell adhesion analyses showed that Rho3-tandem and KG3-tandem, a pan-integrins disintegrin mutant, exhibited 3.4-3.5-fold, 1.3-3.4-fold and 3.7-4.6-fold increases in the inhibition of integrins αvβ3, α5β1 and αvβ5, respectively. Moreover, Rho3-tandem exhibited 7.3-fold increase activity on integrin αⅡbβ3 than monomer. Dimeric and trimeric forms of Rho and KG, however, exhibited similar activities. T58N, Q60R and Q60I mutants of C-terminus also showed similar activities on inhibition of integrins αvβ3, α5β1 and αv5. In contrast, they exhibited 1.7-2.3-fold decreases on integrin αⅡbβ3. D55A, D55N, D55K, D55R, D55T mutants exhibited 2.0-30.1-fold decreases activities on integrin αvβ3, αⅡbβ3, α5β1 and αvβ5. In particular, the D55K mutant exhibited the lowest activity with 30.1-, 6.9-, 9.6-, and 17.4-fold decreases. The results of this study may provide new insights into the design of potent integrin-specific drugs.

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