Computer-aided design and development of isoform selective inhibitors of Casein Kinase 1

项目摘要 蛋白激酶对细胞功能和发育至关重要，组成性激活的激酶 在癌症治疗的发展中被广泛地作为目标。选择性是激酶的关键问题 治疗发现和生物医学研究，特别是在脱靶效应方面。由于ATP结合 位点在激酶组中高度保守，大多数ATP竞争性激酶抑制剂具有特异性 问题，特异性激酶抑制剂的开发仍然是现代药物治疗的重要瓶颈。 发现和生物医学研究。 酪蛋白激酶1（CK 1）参与多种细胞信号转导途径，包括Wnt/β- 连环蛋白、Hedgehog和Hippo信号通路，及其错误调节导致各种人类疾病。 在人类中存在六种CK 1同种型，例如CK 1 α、CK 1 γ1、CK 1 γ2、CK 1 γ3、CK 1 δ和CK 1 ε，并且每种CK 1同种型都具有相同的结构。 同种型在细胞信号转导中显示出各自的生理作用。目前，CK 1亚型选择性 抑制剂很少开发，大多数已知的CK 1特异性药物已被发现是多靶点的 或泛激酶抑制剂。因此，这将是一个引人注目的未来发展方向 CK 1亚型选择性抑制剂，用于研究CK 1亚型的特异性信号传导机制， 病理生理条件。本研究的目的是开发CK 1亚型选择性抑制剂 通过计算分析、合成化学、生化分析和结构生物学。 将进行CK 1亚型的分子动力学（MD）模拟，以确定CK 1亚型的不同特征。 每种酶的ATP结合口袋，用于设计选择性抑制剂。此外，计算 技术和分析方法将被应用于解释负责选择性抑制的机制 每一种异构体。CK 1和我们新的特异性抑制剂的X射线共晶结构将被解决，以帮助 进一步的设计工作和验证通过计算分析和分子建模产生的假设。 小分子的多步合成和表征将主要用于优化 我们的起始试剂和设计的化合物的效力和特异性。ADP-Glo生物化学测定和 高分辨率质谱分析将用于研究CK 1激酶活性的抑制 同种型。将进行一系列基于细胞的增殖和功能测定，以评估细胞效价 选择性抑制剂如MTT或CellTiter-Glo细胞活力测定和4 E-BP 1-和LRP 6-磷酸化 分析。

Project Summary Protein kinases are essential to cellular function and development, and constitutively activated kinases have been widely targeted in the development of cancer therapies. Selectivity is a critical issue in kinase therapeutic discovery and biomedical research, especially in regard to off-target effects. Since the ATP binding site are highly conserved across the kinome, most ATP-competitive kinase inhibitors suffer from specificity issues, and the development of specific kinase inhibitors remains a significant bottleneck in modern drug discovery and biomedical research. Casein kinase 1 (CK1) is involved in various cellular signal transduction pathways including Wnt/β- catenin, Hedgehog, and Hippo signaling pathways, and its mis-regulation results in various human diseases. There are six CK1 isoforms in humans such as CK1α, CK1γ1, CK1γ2, CK1γ3, CK1δ, and CK1ε, and each isoform displays individual physiological roles in cellular signal transduction. Currently, CK1 isoform-selective inhibitors are rarely developed, and most of the known CK1-specific agents have been revealed as multi-target or pan-kinase inhibitors upon further investigation. Thus, it will be a compelling future direction to develop isoform selective inhibitors of CK1 for the investigation of specific signaling mechanism of CK1 isoforms in pathophysiological conditions. The objective of this research is to develop isoform selective inhibitors of CK1 via computational analysis, synthetic chemistry, biochemical assays, and structural biology. Molecular dynamics (MD) simulations of CK1 isoforms will be performed to identify distinct features in the ATP-binding pocket of each enzyme for the design of selective inhibitors. In addition, the computational techniques and analysis methods will be applied to explain the mechanisms responsible for selective inhibition of each isoform. X-ray co-crystal structures of CK1 and our new specific inhibitors will be solved to assist further design efforts and validate hypotheses generated by computational analysis and molecular modeling. Multi-step synthesis and characterization of small molecules will be mainly conducted for the optimization of the potency and specificity of our starting agents and designed compounds. ADP-Glo biochemical assays and high-resolution Mass Spectrometry analysis will be used to study the inhibition of kinase activity of CK1 isoforms. A series of cell-based proliferation and functional assays will be performed to assess cellular potency of selective inhibitors such as MTT or CellTiter-Glo cell viability assay and 4E-BP1- and LRP6-phosphorylation analyses.