利用抗體辨認發現AZD-9291能造成核糖核苷酸還原酶的降解來抑制癌細胞生長

Antibody-based target identification reveals AZD-9291 repressing cancer cells via down-regulation of ribonucleotide reductase

潘岳廷(Yueh-Ting Pan)

指導教授 ： 張震東

國立臺灣大學/生命科學院/生化科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703766

摘要

AZD-9291(Osimertinib, TagrissoTM)是由美國AstraZeneca所開發的非小型細胞肺癌的口服標靶藥物，其作用機制主要為抑制表皮生長因子受體(epidermal growth factor receptor tyrosine kinase, EGFR)的活化來抑制癌細胞的生長，為第三代EGFR的抑制劑。AZD-9291主要是針對EGFR發生T790M時的結構變異所設計，此變異也是非小型細胞肺癌患者對前兩代藥物產生抗性的主因。
核糖核苷酸還原酶的功能為轉換核糖核苷酸成去氧核糖核苷酸，提供DNA的合成與修補的先驅物。由於癌細胞的去氧核醣核苷酸來源主要依靠de novo pathway中核糖核苷酸還原酶來產生，目前研究發現與正常的細胞相比癌細胞會提升其核糖核苷酸還原酶的活性或表現量。我們實驗室先前藉新開發的方法學，發現核糖核苷酸還原酶為第二代EGFR抑制劑Afatinib的目標之一，在這篇論文中，我們發現AZD-9291對於核糖核苷酸還原酶與Afatinib有相同的抑制作用，而其效果更佳。長時間對H1975細胞處理AZD-9291後，我們發現構成核糖核苷酸還原酶的兩個次單位M1及M2都有顯著的下降，經過72小時藥物的處理，只需要1 nM的藥物濃度即可看見效果。目前治療胰臟癌的用藥主要為Gemcitabine，Gemcitabine藉由阻止DNA鏈的延長與抑制核醣核苷酸的活性來殺死癌細胞，然而研究發現胰臟癌細胞往往藉由提高核糖核苷酸還原酶的表現量來產生抗性，根據我們的研究結果，我們發現AZD-9291不只可以用來治療非小型細胞肺癌，對胰臟癌細胞也有一定的效果。
我們也利用實驗室開發的新方法學Target identification by specific tagging and antibody detection (TISTA)，以抗AZD-9291的血清做為媒介來找尋其他AZD-9291可能作用的目標。此抗血清的用途很廣泛，包含免疫墨點法、免疫螢光染色和免疫沉澱都可以使用。藉由使用抗AZD-9291血清行免疫沉澱，再經由質譜儀的分析，我們將可發現AZD-9291更多潛在的作用目標。也因為如此，這個新方法學可以用來找尋共價藥物潛在的目標。

關鍵字

AZD-9291 ； Gemcitabine ； 核糖核苷酸還原酶 ； 共價藥物 ； TISTA

並列摘要

AZD-9291(Osimertinib, TagrissoTM) is an oral, third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that has been developed by AstraZeneca for the treatment of advanced non-small cell lung cancer (NSCLC). AZD-9291 has been designed to target the EGFR T790M mutation that is often present in NSCLC patients with acquired EGFR TKI resistance, while sparing wild-type EGFR. According to the previous studies from our lab, ribonucleotide reductase (RNR) is a novel target of Afatinib, a second-generation EGFR TKI of NSCLC. RNR is the enzyme responsible for the conversion of ribonucleotides to 2’-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. Nowadays, researches have indicated that cancer cells may enhance their RNR expression due to their dependence of RNR for de novo dNTP biosynthesis. Here, we found that AZD-9291 has the same action as Afatinib on RNR and its effect is more potent. Long-term AZD-9291 treatment in H1975 cell causes significant decrease in the protein levels of RNR and both M1 and M2 subunit protein levels decline in cells treated with 1 nM AZD-9291 for 72 hours. Gemcitabine has been used to treat pancreatic cancer, however, pancreatic cancer cells acquire resistance through elevating the expression of RNR. Our data suggest that AZD-9291 may not only provide therapeutics advantage in lung cancer but also in pancreatic cancer.
We have also used target identification by specific tagging and antibody detection (TISTA), to unravel other potential targets of AZD-9291 using an antiserum against AZD-9291. The antiserum can be used in general antibody-based assays such as immunoblotting, immunofluorescence staining and immunoprecipitation. Target identification through immunoprecipitation and Mass spectrometric analysis reveals potential targets of AZD-9291. Therefore, this novel approach can be used in the identification of potential targets of covalent drugs.

並列關鍵字

AZD-9291 ； Gemcitabine ； Ribonucleotide reductase ； Covalent drugs ； target identification

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