BACKGROUND Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1. METHODS Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix. RESULTS The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity. CONCLUSION Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone.

中文翻译：

---

检查特异性配体和ATP竞争性配体对二价激酶抑制剂总体有效性的影响。

背景技术鉴定选择性激酶抑制剂仍然是主要挑战。二价抑制剂的设计为获得有效和选择性抑制剂提供了合理的策略。尽管已经成功设计了二价激酶抑制剂，但尚未对一系列二价抑制剂的亲和力和选择性进行全面评估。在这里，我们提出了针对ABL1的二价激酶抑制剂的结构活性关系的评估。方法在体外表达各种带有不同特异性配体的SNAPtag构建体。通过合成包含SNAPtag靶向部分的单个ATP竞争性激酶抑制剂来实现二价抑制剂的形成，从而使二价抑制剂能够自发自组装。将组装好的二价抑制剂与K562裂解物一起孵育，然后使用固定在琼脂糖凝胶珠上的各种ATP竞争性抑制剂进行亲和力富集。使用串联质谱标签（TMT）标记和二维液相色谱-串联质谱（2D-LC-MS / MS）分析所得洗脱液。通过计算50％的给定激酶保持与亲和基质结合的浓度来确定二价抑制剂的相对结合亲和力。结果对3种亲本ATP竞争性抑制剂和9种SNAPtag偶联物进行了分析，结果鉴定出349种激酶蛋白。在所有情况下，与仅与SNAPtag偶联的亲本化合物相比，二价抑制剂对ABL1的结合亲和力和选择性都更高。虽然可以通过考虑各个特异性配体的结合亲和力来预测结合亲和力的等级顺序，但是组装的二价抑制剂的所得亲和力是不可预测的。这项研究的结果表明，随着ATP竞争性配体的效力增加，特异性配体对二价抑制剂总体结合亲和力的贡献降低。但是，特异性成分与靶标相互作用的亲和力对于实现选择性至关重要。结论通过全面的化学蛋白质组分析，这项工作为蛋白质组范围内ATP竞争性和特异性配体与其预期目标结合的影响提供了首次见识。