利用 Phenyl Vinyl Sulfone 和 Afatinib 的抗體來尋找細胞中的目標蛋白及其應用

Abstract

藥物的發展一直以來都是一個重要但又令人卻步的領域，但是還是吸引了許多科學家前仆後繼的進入這個領域來研究。這幾年來，一個以活性機制為基礎的藥物目標蛋白建檔的方法出現，這是一個有邏輯性的方法去找尋具有選擇性且在生物體內是有效的酵素藥物的篩選方法。然而，這個方法受限於必需去對藥物進行進一步的修飾，才能加上一些官能基讓我們來偵測或是純化。因此我們在這裡提供且驗證了一個以抗體為基礎的方法，不需要進行進一步的化籌修飾就可以針對藥物研究提供一個新方式。首先，我們針對我們有興趣的藥物製做抗體，我們挑選了苯乙烯碸 (Phenyl Vinyl Sulfone) 和妥復克 (Afatinib) 來製作會特別辨認他們的抗體。苯乙烯碸是酪氨酸磷酸酶 (Tyrosine Phosphatase) 的抑制劑，具有一個比較簡單的結構；妥復克是一個上皮細胞生長因子受體 (EGFR) 的抑制劑，一種酪氨酸激酶 (Tyrosine Kinase)。這樣會辨認特殊藥物的抗體，在我們的實驗裡證明是可以被用在西方墨點法的針測，或是可以被用在螢光染色法上來用顯微鏡觀察。此外，我們可以進一步利用免疫沉澱法和質譜分析的方法來確認在細胞裡面這些共價藥物的目標蛋白的身份，經由這些目標蛋白身份的確認，我們也許可以提供一些藥物上的機制來說明藥物為何針對某些症狀或是某些疾病有效，甚至我們可以透過這些目標蛋白的身份來開發這個藥物的新的適應症。在這裡，我們發現苯乙烯碸和它的類似藥物在試管中或是在細胞中可以被用來抑制精氨酸甲基轉移酶一號的活性，同時，在試管中也可以被用來抑制麩胱甘肽還原酶的活性。另一方面，我們發現妥復克和它的非共價類似藥物，艾瑞莎 (Gefitinib) 和得舒緩 (Erlotinib)，不只是上皮細胞生長因子受體的抑制劑，更有潛力是核醣核苷酸還原酶的抑制劑。在這些實驗中，我們在針對這些共價藥物上的偵測有更近一步的發展，我們可以針對一個特殊有興趣被挑選蛋白來偵測它和藥物的複合體的結合，而不需要透過按鍵化學法來達成，在這裡我們舉苯乙烯碸和酪氨酸磷酸酶為例子。最後我們想強調的是，以這種以抗體為基礎的方法，提供了有力的且直接正向的方法來針對這些共價藥物的目標蛋白進行身份上的確認。

Drug development is always a vital and daunting field for many scientists to participate in one after another. Recently, activity-based protein profiling has emerged as a logical tool to discover selective and in vivo active inhibitors for enzymes. However, it has been limited by the need of further chemical modification to these covalent inhibitors with the handle for detection and enrichment. Thus, we have offered and confirmed an antibody-based approach herein named target identification by specific tagging and antibody detection (TISTA) to the chemocentric approach without chemically further modifying these covalent inhibitors. First, we have successfully raised the antisera against the phenyl vinyl sulfone (PVS) and afatinib that PVS is a covalent tyrosine phosphatase inhibitor with simple structure and afatinib is a well-known covalent receptor tyrosine kinase EGFR inhibitor with more complex structure. The data showed that the antiserum can be used in the immunoblotting and immunofluorescence staining. In addition, we can use the immunoprecipitation method to identify the in cellulo target proteins and explain the mechanisms behind the chosen inhibitor and to find a new therapeutic indication based on the candidates identified by the Mass spectrometry following the immunoprecipitation. Here, we found that the PVS and its analogs can be used to inhibit protein arginine methyltranferase 1 and glutathione reductase, and afatinib and its non-covalent analogs, gefitinib and erlotinib, are not only inhibitors of EGFR but also potential inhibitors of ribonucleotide reductase. In these experiments, we also have an advance in raising an antiserum against PVS-tagged catalytic cysteine of PTP1B to individually monitor the redox status of cysteine in the PTP1B. Finally, we must to emphasize the importance that TISTA is a powerful approach and a positive selection method to identify the target proteins of covalent drugs and their non-covalent analogs.