Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Int J Biochem Cell Biol. 2013 Aug;45(8):1939-50. doi: 10.1016/j.biocel.2013.06.006. Epub 2013 Jun 19.
A novel 18 amino acid peptide PYC98 was demonstrated to inhibit JNK1 activity toward c-Jun. We observed a 5-fold increase in the potency of the retro-inverso form, D-PYC98 (a D-amino acid peptide in the reversed sequence) when compared with the inhibition achieved by L-PYC98, prompting our further evaluation of the D-PYC98 inhibitory mechanism. In vitro assays revealed that, in addition to the inhibition of c-Jun phosphorylation, D-PYC98 inhibited the JNK1-mediated phosphorylation of an EGFR-derived peptide, the ATF2 transcription factor, and the microtubule-regulatory protein DCX. JNK2 and JNK3 activities toward c-Jun were also inhibited, and surface plasmon resonance analysis confirmed the direct interaction of D-PYC98 and JNK1. Further kinetics analyses revealed the non-ATP competitive mechanism of action of D-PYC98 as a JNK1 inhibitor. The targeting of the JNK1 common docking site by D-PYC98 was confirmed by the competition of binding by TIJIP. However, as mutations of JNK1 R127 and E329 within the common docking domain did not impact on the affinity of the interaction with D-PYC98 measured by surface plasmon resonance analysis, other residues in the common docking site appear to contribute to the JNK1 interaction with D-PYC98. Furthermore, we found that D-PYC98 inhibited the related kinase p38 MAPK, suggesting a broader interest in developing D-PYC98 for possible therapeutic applications. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cells, we confirmed that the cell-permeable D-PYC98-TAT inhibited c-Jun Ser63 phosphorylation during hyperosmotic stress. Thus, D-PYC98-TAT is a novel cell-permeable JNK inhibitor.
一种新型的 18 个氨基酸肽 PYC98 被证明可以抑制 JNK1 对 c-Jun 的活性。与 L-PYC98 相比，我们观察到其反式异构体 D-PYC98（反向序列中的 D-氨基酸肽）的效力增加了 5 倍，这促使我们进一步评估 D-PYC98 的抑制机制。体外实验表明，除了抑制 c-Jun 磷酸化外，D-PYC98 还抑制了 JNK1 介导的 EGFR 衍生肽、ATF2 转录因子和微管调节蛋白 DCX 的磷酸化。JNK2 和 JNK3 对 c-Jun 的活性也被抑制，表面等离子体共振分析证实了 D-PYC98 与 JNK1 的直接相互作用。进一步的动力学分析表明，D-PYC98 作为 JNK1 抑制剂的作用机制是非 ATP 竞争性的。通过 TIJIP 的结合竞争，证实了 D-PYC98 靶向 JNK1 的共同 docking 位点。然而，由于 JNK1 共同 docking 结构域内的 R127 和 E329 突变不影响表面等离子体共振分析测量的与 D-PYC98 的相互作用亲和力，因此共同 docking 位点中的其他残基似乎有助于 JNK1 与 D-PYC98 的相互作用。此外，我们发现 D-PYC98 抑制了相关激酶 p38 MAPK，这表明开发 D-PYC98 用于可能的治疗应用具有更广泛的兴趣。最后，在评估该肽作为细胞中底物竞争性抑制剂的功效时，我们证实可渗透细胞的 D-PYC98-TAT 在高渗应激期间抑制 c-Jun Ser63 磷酸化。因此，D-PYC98-TAT 是一种新型的可渗透细胞的 JNK 抑制剂。
