K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology
K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology
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DOI：
10.1016/j.bbrc.2017.01.147
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发表时间：
2017-03-11
影响因子：
3.1
通讯作者：
Tani, Akiyoshi
中科院分区：
文献类型：
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作者：
Sakamoto, Kotaro;Kamada, Yusuke;Tani, Akiyoshi
V-Ki-ras2 Kirsten鼠肉瘤病毒癌基因同源基因(K-RAS)的Gly(12)(如G12D、GI2V、G12C)氨基酸突变是多种癌症的主要生长驱动因素。虽然在大多数癌症患者中发现这些突变已经过去了30多年，但有效的突变K-RAS抑制剂还没有上市。在这里，我们报道了新的对K-RAS(G12D)有选择性的抑制多肽。我们以纯化的重组K-RAS(G12D)为底物，筛选T7噬菌体展示的随机多肽文库，并与野生型K-RAS结合，得到KRpep-2(Ac-RRCPLYISYDPVCRR-NH2)作为共识序列。在SPR分析和GDP/GTP交换酶实验中，KRpep-2对K-RAS(G12D)具有10倍以上的结合和抑制选择性。K-D和IC50分别为51和8.9 nm。经过后续的序列优化，我们成功地合成了KRpep-2d(Ac-RRRCPLYISDPVCRRRR-NH2)，它能抑制K-RAS(G12D)的酶活性(IC50=1.6 nM)，并显著抑制K-RAS(G12D)下游的ERK磷酸化，以及30 mU M肽浓度下的A427癌细胞的增殖。据我们所知，这是一种K-RAS(G12D)选择性抑制剂的首次报道，有助于K-RAS(G12D)靶向药物的开发和研究。(C)2017 Elsevier Inc.保留所有权利。
Amino-acid mutations of Gly(12) (e.g. G12D, GI2V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K-D and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 mu M peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. (C) 2017 Elsevier Inc. All rights reserved.