Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.
Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.
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DOI：
10.1021/jm800523u
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发表时间：
2008-12-11
影响因子：
7.3
通讯作者：
Bottaro DP
中科院分区：
文献类型：
--
作者：
Giubellino A;Shi ZD;Jenkins LM;Worthy KM;Bindu LK;Athauda G;Peruzzi B;Fisher RJ;Appella E;Burke TR;Bottaro DP
我们以前已经证明，一种有效的Grb2 SH2结构域结合的合成拮抗剂(1)在培养细胞模型中阻断生长因子刺激的运动、侵袭和血管生成，以及在动物中的肿瘤转移。为了表征1对Grb2的SH2结构域的选择性，我们合成了一种生物素化的衍生物(3)，它保持了高亲和力的Grb2 SH2结构域结合和强大的生物活性。为了研究1和3对Grb2的选择性，用生物素化的拮抗剂3从细胞提取液中固定化目标蛋白，然后用质谱仪进行鉴定。使用缺少单个关键结合决定簇的生物素化类似物平行鉴定非特异性结合。用免疫沉淀、免疫印迹和光学显微镜进一步表征了该拮抗剂的作用机制。这种确定蛋白质结合拮抗剂选择性和作用分子基础的方法应该在药物开发中得到广泛应用。
We have shown previously that a potent synthetic antagonist of Grb2 SH2 domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analog that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.
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影响因子：
5.6
作者：
MIKOL, V;BAUMANN, G;HOMMEL, U
通讯作者：
HOMMEL, U
影响因子：
4.8
作者：
Singh, RR;Song, CY;Kumar, R
通讯作者：
Kumar, R
DOI：
10.1007/s10989-006-9014-7
发表时间：
2006-03
影响因子：
2.5
作者：
Burke, TR
通讯作者：
Burke, TR
影响因子：
5.3
作者：
SONGYANG, Z;SHOELSON, SE;CANTLEY, LC
通讯作者：
CANTLEY, LC
影响因子：
11.2
作者：
Giubellino, Alessio;Gao, Yang;Bottaro, Donald P.
通讯作者：
Bottaro, Donald P.