整合蛋白家族是一群表現在大部分細胞細胞膜上的異構穿膜雙體蛋白，並且調控著細胞與細胞之間和細胞與細胞間質間的交互作用。整合蛋白αvβ6位於表皮細胞並且會再傷口癒合、發炎、和癌症發生時會被大量誘發。整合蛋白αIIbβ3則是在止血以及血栓形成中的血小板凝集扮演重要角色。先前已有報告指出具有ARGDLXXL和KGD motif的蛋白可以分別具有專一性的結合到整合蛋白αvβ6和αIIbβ3。在本研究中，蛇毒去整合蛋白，馬來腹蛇蛇毒蛋白 (rhodostomin, Rho) 和龜殼花蛇毒蛋白 (trimucrin, Tmu) 被利用來當作骨架，以設計針對整合蛋白αvβ6和αIIbβ3的專一性拮抗劑。在設計整合蛋白αvβ6專一性拮抗劑的實驗中，我們發現48ARGDRP和48ARGDKP分別針對整合蛋白αvβ6有很好的活性，IC50值分別為24.0和36.4 nM。相反的，48ARGD(D/E)P和48ARGDLP則具有很差的活性，IC50值分別為大於5000和357.3 nM。這些結果說明了若要和整合蛋白αvβ6結合，RGD右側的胺基酸要為正電荷的胺基酸比較好，而非負電荷的胺基酸。總結目前針對整合蛋白αvβ6的拮抗蛋白活性分別為48ARGDRP > 48ARGDKP > 48ARGDWP > 48ARGDMP > 48ARGDAP > 48ARGDLP> 48ARGDDP。我們也將在Helicobacter pylori發現的序列48ARGDLAAL引入馬來腹蛇蛇毒蛋白中，但發現這個突變白的活性很低，IC50值大於2000 nM。這說明了引入相同的RGD序列到不同的骨架蛋白中可能會導致不同的構型改變。此外，52RP的衍生突變蛋白比起52LA的衍生突變蛋白有更好的活性，這說明了馬來腹蛇蛇毒蛋白中若有52RP會較容易和整合蛋白αvβ6結合。從48ARGDRP和整合蛋白αvβ6結合的docking結果，可以發現與在LAP和整合蛋白αvβ6結合的結果中有不同的交互作用，這些作用力的差異或許可以做為未來拮抗劑專一性設計的一個新方向。在優化整合蛋白αIIbβ3專一性拮抗劑的實驗中，我們發現41KKKRT-50AKGDRR、 41MKKGT-50AKGDRR和41IEEGT-50AKGDRP的突變蛋白分別具有IC50值127.1、 115.6和511.2 nM。這些結果指出龜殼花蛇毒蛋白的連接區域若有較多帶正電荷的胺基酸會較容易和整合蛋白αIIbβ3結合。本項研究的結果將能提供未來針對可用於纖維化及心肌梗塞的整合蛋白αvβ6和αIIbβ3專一性拮抗劑藥物設計的基礎。

Integrins are a family of heterodimeric receptors that are expressed on the surface of most cells, where they mediate cell-cell and cell-extracellular matrix interactions. Integrin αvβ6 is epithelial-specific and strongly induced during wound healing, inflammation and carcinogenesis. Integrin αIIbβ3 plays a critical role in platelet aggregation that is essential for hemostasis and thrombosis. It was reported that proteins with ARGDLXXL and KGD motifs can selectively bind to integrins αvβ6 and αIIbβ3, respectively. In this study, we propose to use rhodostomin (Rho) and trimucrin (Tmu), snake venom disintegrins, as scaffolds to develop integrins αvβ6 or αIIbβ3-specific antagonists. In the study on the design of integrin αvβ6-specific antagonists, we found that the 48ARGDRP and 48ARGDKP mutants exhibited high affinity with the IC50 values of 24.0 and 36.4 nM. In contrast, the 48ARGD(D/E)P and 48ARGDLP mutants exhibited low affinity with the IC50 values of > 5000 and 357.3 nM. These results suggest that the binding of integrin αvβ6 prefers the C-terminal residue adjacent to the RGD motif with positively charged residues but not with negatively charged and hydrophobic residues. In summary, we found that the relative affinity of 48ARGDXP mutants to integrin αvβ6 were 48ARGDRP > 48ARGDKP > 48ARGDWP > 48ARGDMP > 48ARGDAP > 48ARGDLP> 48ARGDDP. We also incorporated the 48ARGDLAAL amino acid sequence of CagL in Helicobacter pylori, the integrin αvβ6-binding loop, into Rho, and it exhibited low affinity with the IC50 value of about 2000 nM. These results suggested that the corporation of the RGD motif into different scaffolds may exhibit different conformations. In addition, 52RP derived mutants exhibited better activity than mutants derived from 52LA, suggesting that binding of integrins αvβ6 prefers 52RP in Rho. From the docking results of 48ARGDRP binding to integrin αvβ6, we can see the difference interactions that LAP binding to integrins αvβ6 processes, which may provide us a new perspective for future design in selectivity. In the study on the design of integrin αIIbβ3-specific antagonist, we found that 41KKKRT-50AKGDRR, 41MKKGT-50AKGDRR, and 41IEEGT-50AKGDRP mutants had the IC50 values of 127.1, 115.6, and 511.2 nM. These results indicate that the binding of integrin αIIbβ3 prefers positively charged residues in the linker region of Tmu. The results of this study will serve as the basis for the design of integrin αvβ6- or αIIbβ3-specific drugs for the treatments of fibrosis and myocardial infarction.

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