抗癌藥物afatinib作用EGFR外之其它標靶蛋白

Abstract

Afatinib (BIBW 2992, Gilotrif™, Giotrif®) 為美國Boehringer Ingelheim公司所研發出來的非小細胞肺癌 (NSCLC) 標靶藥物，能以不可逆的方式共價鍵結在ErbB家族的EGFR、HER2與HER4的酪胺酸激酶上，阻斷酪胺酸激酶的磷酸化，抑制促進癌細胞生長的下游訊息傳遞。此外，Afatinib的優勢為能克服對前一代非小細胞肺癌藥物Erlotinib與Gefitinib產生的抗藥性突變 (T790M與L858R)，藉此提昇對藥物產生抗性病人的存活時間。我們實驗室先前利用自行開發的藥物目標蛋白鑑定方法 (TISTA)，發現了Afatinib不但能以EGFR作為目標蛋白，亦能以核糖核苷酸還原酶 (RNR) 作為藥物標靶，對於開拓藥物的使用範圍與合併療法提供了一些參考。在本研究中，我們由先前鑑定出可能為Afatinib的標靶蛋白中，選出與RNR具有類似功能，在核苷酸代謝中扮演重要角色的酵素，分別為DTYMK、ADSL、Nm23-H1/H2/H3、GMPS，探討其是否真的為 Afatinib的標靶蛋白。實驗結果發現DTYMK、ADSL、Nm23-H1/H2/H3、GMPS在高濃度的Afatinib 處理下，皆產生降解的現象，在低濃度時，則產生了蛋白質累積的現象，間接表示這四個蛋白皆為Afatinib的作用標靶。將處理的時間拉長到七十二小時，Afatinib在低濃度的劑量下就對四個蛋白質產生了影響。加入蛋白質降解抑制劑後，實驗結果顯示 DTYMK 與 Nm23-H1/H2/H3 是走溶酶體降解路徑，而ADSL、GMPS則現象不明顯。在低濃度時觀察到的蛋白質累積現象，實驗結果發現DTYMK、ADSL、GMPS皆是由於蛋白質生合成路徑而增加，而Nm23-H1/H2/H3則不明顯，由以上實驗結果顯示，Afatinib的確會對這四個蛋白質造成影響，很有潛力用來治療這四個蛋白質變異所造成的疾病。

Afatinib (BIBW 2992, Gilotrif™, Giotrif®) is a drug developed by Boehringer Ingelheim to treat non-small cell lung cancer as targeted therapy. Afatinib can act as an irreversible covalent inhibitor of the tyrosine kinases in the ErbB family (EGFR, HER2, HER4), which control cell signaling for cell survival, proliferation and metastasis. The advantage of afatinib is that it can overcome drug resistance mutation (T790M and L858R) induced by the noncovalent non-small lung cancer drugs (erlotinib and gefitinib) and extend the survival time of drug-resistant patients. Our laboratory previously has developed a drug target identification method (TISTA) and found that afatinib can not only target EGFR but also inhibit ribonucleotide reductase (RNR) as a direct target. This finding can serve as a foundation for further research on drug indications and combination therapy using afatinib. In this study, from a list of potential target proteins of afatinib, we selected four enzymes that have similar functions to RNR in nucleotide metabolism. These potential target proteins are DTYMK, ADSL, Nm23-H1/H2/H3, and GMPS. We aimed to investigate whether or not they are truly target proteins for afatinib. The results showed that the degradation of thymidylate kinase (DTYMK), adenylosuccinate lyase (ADSL), nucleoside diphosphate kinase (Nm23-H1/H2/H3) and GMP synthase (GMPS) in cells occurred when the cells were treated with 10 μM afatinib. However, when the cells were treated with lower concentrations of afatinib, these proteins accumulated, due to increased protein synthesis (DTYMK, ADSL and GMPS) or decreased protein degradation (Nm23-H1/H2/H3). Using various protein degradation inhibitors, it was found that DTYMK and Nm23-H1/H2/H3 degradation was mediated by lysosomal pathways. In conclusion, afatinib does affect these four proteins and has great potential for treating diseases caused by changes in these four proteins.