A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1.
A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1.
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配体可以选择性地与具有单氨基酸点突变的蛋白质结合，提供了检测或治疗野生型存在的异常蛋白质的潜力。然而，如果点突变与可寻址位置（如结合袋）不相关，则很难开发选择性配体。在这里，我们报告了一种全化学合成的表位靶向策略，我们使用该策略发现了一个5聚肽，该肽对Akt1癌蛋白Pleckstrin同源结构域的E17K转化点突变具有选择性。合成了一个含有E17K突变和I19[丙氨酸]取代的Akt1片段，形成了一个可寻址的合成表位。通过原位筛选，从库中选择共价点击该炔呈递表位的叠氮化物呈递肽。一种肽对肿瘤蛋白的体外选择性相对于野生型为10:1，在细胞中具有类似的选择性。这个5聚肽被扩展成一个更大的配体，选择性地阻断E17K Akt1与其PIP3底物的相互作用。
Ligands that can selectively bind to proteins with single amino acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wildtype. However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical, synthetic epitope-targeting strategy which we used to discover a 5-mer peptide with selectivity for the E17K transforming point mutation in the Pleckstrin Homology Domain of the Akt1 oncoprotein. A fragment of Akt1 containing the E17K mutation and a I19[Propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that covalently clicked onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to wildtype, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 substrate.