整合蛋白是由α次單元及β次單元所組成的穿膜蛋白，整合蛋白參與了許多生理機制，包括凝血作用、生長、血管新生與感染。先前的研究中也指出整合蛋白與許多疾病息息相關，包括癌症。說明了整合蛋白作為癌症治療中藥物設計目標的可能。去整合蛋白指的是一群在蛇毒中被發現由47到84個胺基酸及4到7對雙硫鍵所組成的蛋白，具有與整合蛋白結合能力的拮抗物。這群去整合蛋白主要是經由RGD所形成的結構與整合蛋白進行結合的，除了RGD motif外，RGD鄰近的部分與其C端均指出對於整合蛋白的辨認上具有重要性。在本篇研究當中，將去整蛋白Rhodostomin (Rho)作為藥物設計的架構，Rho源自於馬來蝮蛇具有68個胺基酸、6對雙硫鍵及RGD構型，我將使用Rhodostomin作為基本架構，設計出對於整合蛋白αvβx與α5β1具專一性的拮抗物，希望可發展出抵抗癌症的前驅藥物。除此之外，為了得到更多未來在設計拮抗物的資訊，本篇論文中也將對RGD鄰近的N端及C端進行研究。我已成功表現並純化出17個Rhodostomin突變物，發現了其中一個稱為39KKARTICARGRGDNP-YH68(簡稱為KG)的Rhodostomin 突變物對於整合蛋白αvβx與α5β1同時具結合能力，此蛋白與整合蛋白αvβ3、αvβ5、αvβ6、α5β1與αIIbβ3的IC50分別為22.2、73.3、62.5、10.6與31782.8 nM。另外在RGD motif鄰近N端部位Rho 46XXPRGDMP53的探討，發現突變為負電胺基酸46DDPRGDMP53和46EEPRGDMP53後，發現對於αIIbβ3和α5β1的結合能力有顯著變差。接著在RGD motif鄰近C端部位Rho 48PRGDMPXX55的探討，將54、55位置的氨基酸分別突變為各類胺基酸A、K、R、N與Y，大部分的突變對整合蛋白的辨識沒有影響，最有趣的部份是Rho PRGDMP54YY55 ，對於αvβ3、α5β1與αIIbβ3合能力的IC50為4.9、69.6和58.0 nM顯示了對於整合蛋白α5β1的結合能力有 3.7倍的上升，更細部的探討中顯示Rho D54Y才是對於整合蛋白α5β1結合能力上升的主因(IC50為81.7 nM)。研究結果中最有意義的蛋白39KKARTICARGRGDNP-YH68，可辨認專一性整合蛋白αvβx與α5β1，而在黑色素瘤的發展過程中，整合蛋白αvβ3、αvβ5與α5β1表現量會逐漸上升，於是將KG進行In Vitro實驗，發現KG可抑制黑色素瘤細胞A375的生長、移動、侵入及附著，在使用HUVEC進行血管新生的實驗中也發現KG可有效抑制血管新生。在In vivo的實驗中，證明了KG可有效抑制B16F10(小鼠黑色素瘤細胞)在B6老鼠體內生長，更進一步的使用NOD SCID免疫缺失小鼠移植A375人類黑色素瘤細胞後使用KG進行治療，發現可顯著抑制A375在NOD SCID免疫缺失小鼠上生長。這些結果說明了KG對於黑色素瘤的治療上，將會是一個很有潛力的藥物候選。

Integrins are α/β heterodimeric transmembrane proteins. With their involvement in many biological functions, such as thrombosis, growth, angiogenesis, and inflammation, integrins are discovered to associate with a broad range of diseases, including cancer and immunodeficiency disorders. Thus, integrins could serve as potential therapeutic targets in treating many diseases. Disintegrins are a family of potent integrin inhibitors found in snake venoms that contain 47 to 84 amino acids with 4-7 disulfide bonds. It has been shown that the RGD motif, the regions adjacent to the RGD motif, and C-terminal region of disintegrins are important for their activity and selectivity. In this study I used rhodostomin (Rho) as protein scaffold to design dual integrins αVβx and α5β1-specific disintegrin and to study the effect of the regions adjacent to the RGD motif. Rho is a snake venom protein isolated from Calloselasma rhodostoma and contains 68 amino acid residues with a PRGDMP motif. I have expressed seventeen Rho mutant proteins and purified them to homogeneity. I designed a Rho mutant containing 39KKARTICARGRGDNP53 amino acid sequence, which can selectively inhibit integrins αvβx and α5β1. It inhibited integrins αvβ3, αvβ5, αvβ6, α5β1, and αIIbβ3 with the IC50 values of 22.2, 73.3, 62.5, 10.6, and 31782.8 nM, suggesting that it is a dual integrins αVβx and α5β1-specific mutant. According to the cell adhesion analysis of Rho 46XXPRGDMP53mutants, such as 46DDPRGDMP and 46EEPRGDMP, it showed that the N-terminal negatively residues adjacent to the RGD motif decreased their activity to integrins αIIbβ3 and α5β1. In contrast, the C-terminal charge residues adjacent to the RGD motif caused little effect on their activity based on the cell adhesion and platelet aggregation analyses of Rho PRGDMP53XX54 mutants. Interestingly, Rho PRGDMP54YY55 mutant inhibited integrins αvβ3, α5β1, and αIIbβ3 with the IC50 values of 4.9, 33.6, and 58.0 nM, showing a 7.6-fold increase in inhibiting integrin α5β1. We also found that Rho PRGDMP54DY55mutant exhibited higher integrin α5β1 binding affinity. It is known that the progression of melanoma is associated with the expression of integrins αvβ3 and α5β1. We found that integrins αvβx and α5β1-specific mutant and Rho inhibited the adhesion, migration, and invasion of human A375 melanoma cells. It also significantly suppressed tumor growth in C57BL/6 mice implanted with B16F10 melanoma cells and in NOD SCID mice implanted with A375 human melanoma cells. The results of this study will serve as the basis to design potent integrins-specific antagonists for melanoma.

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