- 學位論文
蛋白質酪胺酸磷酸水解酶高表現系統建構及其在抑制劑篩選上之應用
Overexpression of Protein Tyrosine Phosphatases and Their Applications in Inhibitors Screening
摘要
蛋白質酪胺酸磷酸水解酶 ( PTPs ) 在多種細胞活性方面扮演著重要之角色，特別是在訊息傳遞路徑層面。而有關於個別PTP其相對應抑制劑之探討不僅可解開謎團以闡述其所發揮之功能，更可以應用至藥物開發層面。本篇論文主要著重於PTP抑制劑掃描平台之建立，因而可分成兩個部份來詳述之。在第一部分，我們完成五種不同PTPs之過度表現與純化，分別包括PTP1B、PEST、SHP2、TCPTP和VHR。在此我們分別探索可能影響PTP活性的一些關鍵因素，其中囊括培養溫度、IPTG濃度與誘導時間，期許透過這些參數之微調可以找到最有效的PTP表現條件。在第二部分，我們以對硝基苯磷酸二鈉 ( p-nitrophenyl phosphate, pNPP )當作酵素之正常受質，進而建立一套穩定的活性測試系統。該分析平台主要使用於掃描之前在本實驗室即已合成出的一系列針對五種PTPs之含仿磷酸酪胺酸基團分子庫。經初篩後，一些表現出較佳抑制效率之化合物則進一步予以酵素動力學之探討，期許經由如此之操作可以得知更多有關於這類抑制劑與PTPs之反應機轉，進而思索後續之設計與應用性。
並列摘要
Protein tyrosine phosphatases (PTPs) play important roles in a variety of cell activities, especially in the signal transduction pathways. The research of inhibitors for individual PTP offers not only solutions to elucidate the functions but also leads for drug development. The thesis was aimed to establish a screening platform for PTP inhibitors, and was therefore divided into two parts. In the first part, we performed overexpression for five different PTPs, including PTP1B, PEST, SHP2, TCPTP, and VHR. Factors such as temperature, IPTG concentration, and induction time were studied to achieve optimal expression for the individual PTP. In the second part of the thesis, we established an activity assay system using p-nitrophenyl phosphate ( pNPP ) as the substrate. This assay platform was used to screen a phosphotyrosyl mimetic-containing library that was prepared in our laboratory for the five PTPs mentioned above. Detailed kinetic study was further performed for a few compounds that showed good inhibitory effect in the preliminary screening.