整合蛋白是一種穿膜蛋白，它介導細胞與細胞外間質的相互作用並影響許多重要的細胞反應。在內皮細胞的整合蛋白αvβ3以及整合蛋白α5β1都被發現在血管新生的過程中扮演重要角色，而主要表達在血小板上的整合蛋白αIIbβ3會誘導癌細胞上的整合蛋白αvβ3活化，引發侵入性突起並促進上皮-間質轉換的訊息傳遞。這些整合蛋白的抑制劑在臨床前模型中可以抑制腫瘤生長。然而，有研究發現低濃度的RGD模擬整合蛋白抑制劑的促血管生成作用可能妨礙其作為抗癌劑的效力。我們推測整合蛋白與RGD抑制劑的結合不僅抑制整合蛋白與細胞外基質的相互作用，還會誘導整合蛋白的活化。Rhodostomin及trimucrin為蛇毒蛋白分離出的去整合蛋白，含有RGD序列能與整合蛋白結合並阻斷整合蛋白與細胞外間質的相互作用以達到抑制效果。由實驗室過去的研究發現，trimucrin的突變體AKGDRR與整合蛋白的結合，不會促使血小板與纖維蛋白原的結合及使整合蛋白αIIbβ3配體誘發結合位點 (ligand-induced binding site, LIBS) 的暴露。因此我們目標設計對整合蛋白具專一性但不造成活化的去整合蛋白以治療癌症。在此研究中，我們使用龜殼花蛇毒蛋白trimucrin作為模板，藉由突變在RGD序列相鄰C端的氨基酸來設計整合蛋白αvβ3和α5β1的特異性拮抗劑。九個trimucrin ARGDXX突變體已經成功由Pichia pastoris表現並純化。由突變體的安全性指數分析作為判斷突變體是否造成整合蛋白活化的依據，結果顯示RR/AR/YR/SR/RD>NR/FR>PR>DR。而細胞貼附實驗的結果表明，突變體對活化態的整合蛋白αIIbβ3及αvβ3而非活化態的整合蛋白α5β1有較好的抑制活性。藉由分析整合蛋白LIBS的暴露來探討去整合蛋白突變體對整合蛋白構型的影響，我們發現這些trimucrin突變體隨蛋白濃度的增加LIBS的暴露會隨之增加，然而在低濃度下不會造成LIBS有明顯的暴露。而去整合蛋白的突變體相較於臨床藥物eptifibatide更能競爭整合蛋白與其受質的結合。值得一提的是，54RD突變體的抑制活性好也有很高的safety index，但54DR突變體並非如此。以上的結果將作為設計不造成整合蛋白活化的專一性去整合蛋白以治療癌症的研究基礎。

Integrins are transmembrane proteins that mediate the interactions between cells and extracellular matrices (ECM) and affect many important cellular responses. Both Integrins αvβ3 and α5β1 in endothelial cells are found to play an important role in angiogenesis. Integrin αIIbβ3, which is mainly expressed on platelets, induces integrin αvβ3 activation on cancer cells and triggers invasive processes. Many studies showed that inhibitors of these integrins can suppress tumor growth in preclinical animal models. However, it was also found that the treatment of RGD-mimetic integrin inhibitors with low concentration caused proangiogenic effect and hampered their efficacy as anticancer agents. We hypothesized that the binding of RGD inhibitors not only inhibits the interactions of integrins with ECM but also induces the activation of integrins. Disintegrins, rhodostomin and trimucrin, are snake venom proteins with an RGD motif that binds to integrin and blocks the interaction of integrin with ECM. We found a trimucrin AKGDRR mutant that did not prime the binding of platelets to fibrinogen and induce the exposure of the ligand-induced binding site (LIBS) of integrin αIIbβ3. We therefore propose to design integrins-specific disintegrins without integrin activation for future cancer therapy. In this study I used trimucrin as a scaffold to design specific antagonists of integrins αvβ3 and α5β1 by mutating the residues C-terminally adjacent to RGD motif. To date, I have successfully expressed nine trimucrin ARGDXX mutants in Pichia pastoris and purified them to homogeneity. The results of safety index analyses of ARGDXX mutants showed that RR/AR/YR/SR/RD > NR/FR > PR > DR. Cell adhesion analyses showed that these mutants exhibited higher activities in inhibiting activated integrins αIIbβ3 and αvβ3 but not α5β1. The analyses of the exposure of LIBS induced by disintegrins using flow cytometry showed that trimucrin mutants induced the exposure of integrin LIBS via a concentration-dependent manner. However, they exhibited similar effect on LIBS exposure of integrin at low concentration. Rhodostomin and trimucrin mutant, but not eptifibatide, significantly inhibited the binding of fibrinogen to integrin. Interestingly, I found that RD but not DR mutant exhibited potent activity in inhibiting integrins with high value of safety index. The results of this study will serve as the basis for design of integrins-specific disintegrins without activating integrins for cancer therapy.

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