Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA, and North Carolina State University, Raleigh, NC, 27695, USA.
Analyst. 2016 Oct 17;141(21):6008-6017. doi: 10.1039/c6an01378c.
A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (k/K) of purified ABL kinase for the lead peptide (125 s μM) is similar to that of the starting peptide (103 s μM) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR-ABL providing a readout of BCR-ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR-ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR-ABL kinase reporter in the leukemic cells possessing the BCR-ABL mutation.
一种抗肽酶 ABL 激酶底物是通过鉴定 ABL 底物肽上的蛋白酶敏感键，并将侧翼氨基酸用非天然氨基酸取代而开发的。经过迭代设计过程，先导或设计肽 X-A 在细胞质溶酶体中的半衰期比起始肽长六倍。纯化的 ABL 激酶对先导肽的催化效率（k/K）（125 s μM）与起始肽（103 s μM）相似，表明该肽仍能作为激酶底物。当在细胞质溶酶体中孵育时，先导肽会随时间缓慢降解为 4 个片段。相比之下，当加载到完整细胞中时，肽会代谢成 5 个片段，其中只有 2 个片段与溶酶体中的片段相对应。因此，这两种环境具有不同的肽酶活性，在设计抗肽酶肽时必须考虑到这一点。在这两种环境下，BCR-ABL 对底物进行磷酸化，提供 BCR-ABL 活性的读数。尽管存在多种其他激酶，但一小部分酪氨酸激酶抑制剂在溶酶体和细胞中验证了该底物对 BCR-ABL/ABL 激酶活性的特异性。尽管存在多种其他激酶，但该设计的肽 X-A 作为一种具有 BCR-ABL 突变的白血病细胞中具有长半衰期的 BCR-ABL 激酶报告物。
