Potent antagonists of the SH2 domain of Grb2:: Optimization of the X+1 position of 3-Amino-Z-Tyr(PO3H2)-X+1-Asn-NH2
Potent antagonists of the SH2 domain of Grb2:: Optimization of the X+1 position of 3-Amino-Z-Tyr(PO3H2)-X+1-Asn-NH2
复制标题
DOI：
10.1021/jm970856n
复制
发表时间：
1998-05-21
影响因子：
7.3
通讯作者：
Caravatti, G
中科院分区：
文献类型：
--
作者：
García-Echeverría, C;Furet, P;Caravatti, G
在影响细胞生长和转化的细胞内信号传导事件中鉴定出的越来越多的元件是通过结构域或特定识别的氨基酸序列彼此物理相互作用的蛋白质。这些细胞内蛋白质相互作用是肿瘤学中特别有吸引力的靶点，因为它们参与控制癌细胞中激活或失调的有丝分裂信号转导途径。 1-3 这是涉及 Ras 信号转导途径中 Grb24 的 Src 同源 2 (SH2) 结构域的蛋白质-蛋白质相互作用的情况。 5-7 在哺乳动物细胞中，Grb2 接头蛋白将生长因子激活的酪氨酸激酶受体连接到 Sos，Sos 是一种鸟嘌呤核苷酸交换因子，在受体结合并将 Grb2-Sos 复合物易位到质膜后，将非活性 Ras ‚GDP 转化为活性 Ras ‚GTP。 8 激活的 Ras 会触发 MAP 激酶级联，这对于细胞生长和分化至关重要。 9 激活的酪氨酸激酶受体与 Grb2 之间的相互作用由信号蛋白的 Src 同源 2 结构域介导。特异性破坏这种蛋白质-蛋白质相互作用的药物可能会关闭 Ras 通路，并为阻止人类恶性肿瘤提供一个干预点。从 Grb2 的 SH2 结构域的最小识别基序开始，10 我们启动了一项药物化学计划，以鉴定特异性破坏激活的酪氨酸激酶受体与 Grb2-SH2 结构域之间相互作用的化合物。我们最近报道了 N 端基团的设计，该基团可以赋予 Grb2-SH2 结构域识别的最小三肽序列高亲和力。 11 在本通讯中，我们报告了通过优化 X+ 1 位置，进一步提高了 3-氨基-Z-Tyr-(PO3H2)-X+ 1-Asn-NH2 磷酸肽的结合亲和力。 12 简并磷酸酪氨酰肽文库表明，磷酸酪氨酰肽的 SH2 结构域的序列特异性位于紧邻磷酸酪氨酸残基羧基末端的肽部分。 13, 14 对于 Grb2 的 SH2 结构域，共有序列是 Tyr (PO3H2)-X+ 1-Asn-X+ 3，决定特异性的残基是天冬酰胺。 13, 15 合成16 和噬菌体展示库17 方法已用于鉴定磷酸酪氨酸羧基末端的最佳残基。尽管这些研究一致认为 X+ 2 位点对天冬酰胺具有排他性选择性，但在 X+ 1 位点和 X+ 3 位点观察到两种技术之间的差异。对于 X+ 1 位点，从合成肽文库中选择谷氨酰胺、谷氨酸、异亮氨酸、酪氨酸和缬氨酸 16，而对谷氨酸和蛋氨酸残基的偏好则通过噬菌体展示来确定。 17 然而，在 X+ 1 位点选择谷氨酸、谷氨酰胺、蛋氨酸或缬氨酸不会改变磷酸酪氨酰肽与 Grb2-SH2 结构域的结合亲和力。 17 上述结果似乎表明该职位的严格程度较低。利用配体结合的 Grb2-SH218 的独特结构特征来优化 3-氨基-Z-Tyr (PO3H2)-X+ 1-Asn-NH2 的 X+ 1 位置，这是最近报道的 Grb2-SH2 结构域 (X+ 1) Ile) 的有效拮抗剂。 11
A growing number of the elements identified in intracellular signaling events that affect cell growth and transformation are proteins that physically interact with each other via domains or specifically recognized amino acid sequences. These intracellular proteinprotein interactions are particularly attractive targets in oncology due to their participation in controlling mitogenic signal transduction pathways that are activated or deregulated in cancer cells. 1-3 This is the case for the protein-protein interactions involving the Src homology 2 (SH2) domain of Grb24 in the Ras signal transduction pathway. 5-7 In mammalian cells, the Grb2 adaptor protein links the tyrosine kinase receptors activated by growth factors to Sos, a guanine nucleotide exchange factor that, upon receptor binding and translocation of the Grb2-Sos complex to the plasma membrane, converts the inactive Ras ‚GDP to active Ras ‚GTP. 8 Activated Ras triggers the MAP kinase cascade that is essential for cell growth and differentation. 9 The interaction between the activated tyrosine kinase receptors and Grb2 is mediated by the Src homology 2 domain of the signaling protein. Agents that specifically disrupt this protein-protein interaction could potentially shut down the Ras pathway and present an intervention point for blocking human malignancy. Starting with the minimal recognition motif of the SH2 domain of Grb2, 10 we have initiated a medicinal chemistry program to identify compounds that specifically disrupt the interaction between activated tyrosine kinase receptors and the Grb2-SH2 domain. We recently reported the design of an N-terminal group that can impart high affinity to the minimal tripeptide sequence recognized by the Grb2-SH2 domain. 11 In the present communication, we report a further improvement in the binding affinity of the 3-amino-Z-Tyr-(PO3H2)-X+ 1-Asn-NH2 phosphopeptide by optimizing the X+ 1 position. 12Degenerate phosphotyrosyl peptide libraries have shown that the sequence specificities of SH2 domains for phosphotyrosyl peptides lay in the portion of the peptide immediately carboxy-terminal of the phosphotyrosine residue. 13, 14 For the SH2 domain of Grb2, the consensus sequence is Tyr (PO3H2)-X+ 1-Asn-X+ 3 and the residue that determines specificity is asparagine. 13, 15 Synthetic16 and phage display library17 approaches have been used to identify optimal residues carboxy-terminal to phosphotyrosine. Although these studies agree on the exclusive selectivity for asparagine at the X+ 2 position, differences between the two techniques were observed for positions X+ 1 and X+ 3. For position X+ 1, glutamine, glutamic acid, isoleucine, tyrosine, and valine were selected from synthetic peptide libraries, 16 while a preference for glutamic acid and methionine residues was identified by phage display. 17 However, selection of glutamic acid, glutamine, methionine, or valine at the X+ 1 position did not alter the binding affinity of phosphotyrosyl peptides for the Grb2-SH2 domain. 17 The above results seemed to indicate a low stringency for this position. The unique structural features of the ligand-bound Grb2-SH218 were exploited to optimize the X+ 1 position of 3-amino-Z-Tyr (PO3H2)-X+ 1-Asn-NH2, a recently reported potent antagonist of the Grb2-SH2 domain (X+ 1) Ile). 11