Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China; Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing 210009, China.
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China.
Bioorg Med Chem. 2018 Jul 23;26(12):3429-3437. doi: 10.1016/j.bmc.2018.05.014. Epub 2018 May 14.
Polo-like kinase 1 (Plk1) is an anti-cancer target due to its critical role in mitotic progression. A growing body of evidence has documented that Peptide-Plk1 inhibitors showed high Plk1 binding affinity. However, phosphopeptides-Plk1 inhibitors showed poor cell membranes permeability, which limits their clinical applications. In current study, nine candidate phosphopeptides consisting of non-natural amino acids were rationally designed and then successfully synthesized using an Fmoc-solid phase peptide synthesis (SPPS) strategy. Moreover, the binding affinities and selectivity were evaluated via fluorescence polarization (FP) assay. The results confirmed that the most promising phosphopeptide 6 bound to Plk1 PBD with the IC of 38.99 nM, which was approximately 600-fold selectivity over Plk3 PBD (IC = 25.44 μM) and nearly no binding to Plk2 PBD. Furthermore the intracellular activities and the cell membrane permeability of phosphopeptide 6 were evalutated. Phosphopeptide 6 demonstrated appropriate cell membrane permeability and arrested HeLa cells cycle in G2/M phase by regulating CyclinB1-CDK1. Further, phosphopeptide 6 showed typical apoptotic morphology and induced caspase-dependent apoptosis. In conclusion, we expect our discovery can provide new insights into the further optimization of Plk1 PBD inhibitors.
丝氨酸/苏氨酸蛋白激酶 1（Plk1）在有丝分裂进展中发挥关键作用，是一种抗癌靶点。越来越多的证据表明，肽-Plk1 抑制剂与 Plk1 具有高结合亲和力。然而，磷酸肽-Plk1 抑制剂的细胞膜通透性较差，限制了它们的临床应用。在本研究中，设计并合理合成了九种由非天然氨基酸组成的候选磷酸肽，采用 Fmoc-固相肽合成（SPPS）策略。此外，通过荧光偏振（FP）测定评估了结合亲和力和选择性。结果证实，最有前途的磷酸肽 6 与 Plk1 PBD 的结合 IC 为 38.99 nM，对 Plk3 PBD 的选择性约为 600 倍（IC = 25.44 μM），对 Plk2 PBD 几乎没有结合。此外，还评估了磷酸肽 6 的细胞内活性和细胞膜通透性。磷酸肽 6 具有适当的细胞膜通透性，并通过调节 CyclinB1-CDK1 将 HeLa 细胞周期阻滞在 G2/M 期。进一步，磷酸肽 6 表现出典型的凋亡形态，并诱导 caspase 依赖性细胞凋亡。总之，我们希望我们的发现能为 Plk1 PBD 抑制剂的进一步优化提供新的思路。
