RAS-G12C標靶藥物ARS1620之脫靶標的鑑定

Identification the off-targets of ARS1620, a RAS-G12C targeted drug

鄭人豪(Jen-Hao Cheng)

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(2021年)

https://doi.org/10.6342/NTU202102382

摘要

ARS1620為Yi Liu及其同事們所開發的小分子化合物，藉由改進最早由Shokat等人所開發的第12號化合物，增強了藥物的效價和藥理特性。ARS1620主要針對KRAS-G12C發生的突變，藉由結合到RAS的switch-II pocket，阻止GDP交換成GTP，而使RAS保持在不活化狀態，抑制下游訊息的過度傳遞。本次實驗利用實驗室開發的TISTA方法，以抗ARS1620的血清進行免疫沉澱尋找與ARS1620作用的其他蛋白，並利用質譜分析來鑑定這些蛋白。本次實驗我們從質譜鑑定出的可能標靶蛋白中挑選幾個蛋白進行觀察，其中發現經過ARS1620處理二十四小時後，在PC-9、Huh-7及HeLa細胞均發現p53含量增加，另外利用MDM2抗體進行免疫沉澱發現，經過10 μM 的ARS1620處理二十四小時後，造成MDM2與p53結合能力的下降，進一步觀察p53活性是否受到影響，發現分別經過100 nM、1 μM 和10 μM 的ARS1620處理二十四小時後，p53與已知的DNA結合序列的結合能力並未受到影響，因此我們認為ARS1620結合到p53能夠降低p53與MDM2的結合，減少p53被蛋白酶體降解，使細胞內p53含量增加，並且p53活性並未受到影響，仍然具有與已知DNA序列(5'-RRRCWWGYYY-3'，R = A or G, Y = C or T, W = A or T)結合的能力，提升p53活性可能有助於ARS1620毒殺KRAS-G12C癌細胞。

關鍵字

KRAS-G12C抑制劑 ； ARS1620 ； TISTA ； 共價藥物

並列摘要

ARS1620 is a small-molecule compound developed by Yi Liu and colleagues in 2018, based on the No. 12 compound developed by Shokat et al. ARS1620 mainly targets the KRAS-G12C mutant molecule instead of wild-type KRAS. It can prevent GDP from being exchanged for GTP, by binding to the switch-II pocket of RAS, thus keeping RAS in an inactive state and suppressing excessive transmission of downstream signalling. In this study, I used the TISTA method developed earlier in our laboratory to unravel novel targets of AS1620. I used anti-ARS1620 serum for immunoprecipitation to fish out proteins that interact with ARS1620, and used mass spectrometry to identify these proteins. In this research, we selected several candidates from the possible target proteins and examined the effects of ARS1620 in cells. It was found that after 24 hours of ARS1620 treatment, p53 content increased in PC-9, Huh-7 and HeLa cells. In addition, immunoprecipitation experiments showed that after 24 hours of treatment with 10 μM ARS1620, the binding between MDM2 and p53 was decreased. In addition, I found that after 24 hours of treatment with 100 nM, 1 μM, and 10 μM ARS1620, the ability of p53 to bind known p53 binding sequences was not affected. Therefore, ARS1620 binding to p53 can reduce the binding of p53 to MDM2 thus reducing the degradation of p53 by the proteasome and increasing the intracellular p53 content. Furthermore, the p53 still has the binding ability to the known DNA sequence (5'-RRRCWWGYYY- 3', R = A or G, Y = C or T, W = A or T). It is possible that increased p53 activity may underlie in part the therapeutic effects of ARS1620 in cancer cells.

並列關鍵字

KRAS-G12C inhibitor ； ARS1620 ； TISTA ； covalent drugs

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