Molecular Design, Synthesis, and Pharmacology of Targeted Protein Degraders for the Checkpoint Kinase ATR

检查点激酶 ATR 靶向蛋白降解剂的分子设计、合成和药理学

• 批准号: 528202295
• 负责人: Professor Dr. Oliver Holger Krämer
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/528202295?language=en
• 关键词: Molecular; Design; Synthesis; Pharmacology; Targeted

About three billion base pairs are replicated within each mammalian cell cycle. Chemotherapeutics kill tumor cells through the induction of DNA replication stress and DNA damage. Exogenous and endogenous DNA stress activate checkpoint kinases, which slow down the cell cycle and initiate DNA repair. The apical checkpoint kinase ataxia telangiectasia-and-RAD3-related (ATR) is activated by stalled DNA replication forks and single strand DNA breaks. Preclinical and clinical studies have demonstrated the efficacy of ATP-competitive ATR inhibitors in combination with chemotherapeutics. Proteolysis-targeting-chimeras (PROTACs) are modern agents that inhibit and eliminate their target proteins by the ubiquitin-proteasome system. In preliminary work, we have synthesized and tested the first PROTAC for ATR in various cell systems. We demonstrate that the cereblon-targeting PROTAC Abd110 decreases ATR dependent on the E3 ubiquitin ligase cereblon and proteasomal activity. Abd110 synergistically induces apoptosis (programmed cell death) of acute myeloid and lymphatic leukemia cells when combined with the clinically used ribonucleotide reductase inhibitor hydroxyurea. We aim to optimize our PROTACs by structure-based design (using available X-ray structures of ATR and ternary PROTAC complexes of other kinases), in vitro testing, and cellular characterization. This includes pharmacological selectivity studies, targeted protein analyses, biochemical cell fractionation, flow cytometry, and genetic overexpression and knockout strategies. Promising candidates can be selected based on in vitro testing using recombinant ATR. We want to test and molecularly understand anti-leukemic effects of ATR PROTACs in combination with chemotherapeutics in a larger panel of leukemic cells and co-culture systems. This will involve analyses of DNA replication stress and DNA damage signaling. To comprehensively reveal the specificity and targets of ATR PROTACs, we will use global proteome, phospho-proteome, and transcriptome analyses. Furthermore, we will try to set up additional targeted proteolysis concepts, such as autophagy-targeting-chimeras (AUTOTACs) and chaperone-mediated protein degraders (CHAMPs/HEMTACs) for ATR. The degraders under study will be powerful tools to identify the downstream targets and biological functions of the catalytic activities and the structural/scaffolding properties of ATR. Moreover, such compounds could prospectively become new treatment options for difficult-to-treat leukemia.

在哺乳动物的每个细胞周期中，大约有30亿个碱基对被复制。化疗通过诱导DNA复制应激和DNA损伤来杀死肿瘤细胞。外源性和内源性DNA应激激活检查点激酶，从而减缓细胞周期并启动DNA修复。顶端检查点激酶共济失调毛细血管扩张和rad3相关（ATR）被DNA复制叉和单链DNA断裂所激活。临床前和临床研究已经证明atp竞争性ATR抑制剂与化疗药物联合使用的有效性。蛋白水解靶向嵌合体（proteolysis -targeting chimeras, PROTACs）是一种通过泛素-蛋白酶体系统抑制和消除其靶蛋白的现代药物。在前期工作中，我们已经在各种细胞系统中合成并测试了第一个用于ATR的PROTAC。我们证明了靶向小脑的PROTAC Abd110降低依赖于E3泛素连接酶小脑和蛋白酶体活性的ATR。当与临床使用的核糖核苷酸还原酶抑制剂羟基脲联合使用时，Abd110协同诱导急性髓系和淋巴白血病细胞凋亡（程序性细胞死亡）。我们的目标是通过基于结构的设计（利用现有的ATR x射线结构和其他激酶的三元PROTAC复合物）、体外测试和细胞表征来优化我们的PROTACs。这包括药理学选择性研究、靶向蛋白分析、生化细胞分离、流式细胞术以及基因过表达和基因敲除策略。利用重组ATR进行体外测试，可以选择有希望的候选药物。我们希望在更大的白血病细胞和共培养系统中测试并从分子上理解ATR PROTACs与化疗药物联合的抗白血病作用。这将涉及DNA复制压力和DNA损伤信号的分析。为了全面揭示ATR PROTACs的特异性和靶点，我们将使用全局蛋白质组学、磷酸化蛋白质组学和转录组学分析。此外，我们将尝试建立额外的靶向蛋白水解概念，例如针对ATR的自噬靶向嵌合体（AUTOTACs）和伴侣介导的蛋白质降解物（CHAMPs/HEMTACs）。所研究的降解物将成为鉴定ATR的下游目标和催化活性的生物学功能以及结构/脚手架特性的有力工具。此外，这些化合物有望成为治疗难治性白血病的新选择。