Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V).
Total Chemical Synthesis and Folding of All-l and All-d Variants of Oncogenic KRas(G12V).
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DOI：
10.1021/jacs.7b02988
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发表时间：
2017-06-07
影响因子：
15
通讯作者：
Danishefsky SJ
中科院分区：
文献类型：
--
作者：
Levinson AM;McGee JH;Roberts AG;Creech GS;Wang T;Peterson MT;Hendrickson RC;Verdine GL;Danishefsky SJ
Ras蛋白是参与调节细胞增殖和存活的必需GTP酶。Ras的突变致癌形式改变效应子结合和先天性GT3活性，导致下游信号转导的失调。Ras的突变形式涉及大约30%的人类癌症。尽管数十年来一直致力于开发直接的Ras抑制剂，但由于其对GTP的高亲和力和缺乏疏水结合口袋，Ras长期以来一直被认为是“不可药用的”。在此，我们报告了致癌突变KRas（G12 V）的所有L-和所有D-氨基酸生物素化变体的全化学合成。使用基于Fmoc的固相肽合成来合成蛋白质，并使用组合的天然化学连接和异腈介导的活化策略来组装。我们证明，这两个KRas（G12 V）对映体可以成功地折叠和结合核苷酸底物和可观察到的对映体歧视的结合伙伴。通过证明与其相应的对映体核苷酸三磷酸结合的KRas的镜像形式的功能能力，本研究为使用该材料进行进一步的生物化学研究奠定了基础。特别是，这种蛋白质将使镜像酵母表面展示实验能够识别致癌KRas的全D肽配体，为寻找针对这种具有挑战性的疾病靶点的新疗法提供有用的工具。
The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered ‘undruggable’ due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all L- and all D-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-D peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.
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影响因子：
64.5
作者：
Eckert, DM;Malashkevich, VN;Kim, PS
通讯作者：
Kim, PS
影响因子：
56.9
作者：
DAWSON, PE;MUIR, TW;KENT, SBH
通讯作者：
KENT, SBH
DOI：
10.1039/c39930000369
发表时间：
1993-02-21
影响因子：
--
作者：
JOHNSON, T;QUIBELL, M;SHEPPARD, RC
通讯作者：
SHEPPARD, RC
DOI：
10.1073/pnas.1300016110
发表时间：
2013-06-18
影响因子：
11.1
作者：
Hocker, Harrison J.;Cho, Kwang-Jin;Gorfe, Alemayehu A.
通讯作者：
Gorfe, Alemayehu A.
DOI：
10.1073/pnas.0831227100
发表时间：
2003-04-29
影响因子：
11.1
作者：
Becker, CFW;Hunter, CL;Engelhard, M
通讯作者：
Engelhard, M