【作者】 王凯；

【导师】 路海滨；

【作者基本信息】 吉林大学 ， 药剂学， 2025， 硕士

【摘要】 TP53基因编码的肿瘤抑制蛋白p53在维持基因组稳定性及抑制肿瘤发生中发挥核心作用。急性髓系白血病（Acute myeloid leukemia,AML）作为高侵袭性血液系统恶性肿瘤,其治疗困境与p53信号通路异常密切相关。通过抑制酪蛋白激酶1α（Casein kinase 1α,CK1α）和细胞周期依赖性激酶7/9（Cyclin-Dependent Kinase 7,CDK7/9）可重新激活p53的肿瘤抑制功能来治疗AML。本研究致力于开发基于蛋白水解靶向嵌合体（Proteolysis Targeting Chimeras,PROTAC）的创新疗法,利用泛素-蛋白酶体系统（Ubiquitin-proteasome system,UPS）实现靶向蛋白降解。PROTAC技术通过双功能分子（靶蛋白配体-E3连接酶配体-Linker三元复合物）实现靶蛋白的泛素化标记及后续降解,其独特的催化降解机制可突破传统抑制剂面临的耐药性挑战。值得注意的是,PROTAC分子通过可循环作用模式持续诱导靶蛋白降解,为解决“不可成药”靶点提供了突破性技术路径。本研究基于多靶点激酶抑制剂A86（N-甲基吡唑衍生物）进行系统性结构优化,旨在开发可同时降解CK1α、CDK7/9蛋白的三靶点PROTAC分子。此外,通过分析A86与CK1α、CDK9的共晶复合物结构,确定其氟嘧啶胺的N1和N2位为关键结合位点,同时发现环己二胺基团暴露在外。因此本研究构建了含或不含环己二胺的PRORAC结构（1a和1b）,并探究其对分子活性的影响。本研究基于E3连接酶配体的多样性特征,采用模块化设计策略构建了三个系列的PROTAC分子:（1）泊马度胺型:以化合物9或A86为核心靶向模块,通过差异化Linker与泊马度胺配体偶联,制备了PROTAC 13a-g、13h及13l;（2）苯甲酰胺型:以苯甲酰胺类CRBN配体为优化方向,通过Ullmann偶联反应等一系列反应最终获得化合物13i-j;（3）VHL型:采用VHL配体28与中间体化合物18直接偶联合成化合物13k。以上系列设计通过E3配体类型、Linker结构及靶标配体结构的协同优化,系统解析了多靶点PROTAC的分子构建规律。体外药效学评估显示:含环己二胺基团的PROTACs较不含者在MV4-11和MOLM-13细胞系中表现出显著增强的抗增殖活性;PEG型Linker（13a-d）较烷基型（13e-g）具有更优的细胞毒性;CRBN配体（13h-j）相较于VHL配体（13k）展现出更高的生物活性,其中苯甲酰胺型配体（13i-j）较泊马度胺型（13h）活性提升显著。值得注意的是,化合物PROTAC 13i在MV4-11(IC₅₀=0.096±0.012μM)和MOLM-13(IC₅₀=0.072±0.014μM)细胞中表现出最好抗增殖效果。为明确PROTAC 13i的抗增殖效应与其蛋白降解功能的相关性,本研究通过Western blot分析进行机制验证:采用过量沙利度胺预处理竞争性阻断CRBN介导的泛素化通路,证实其抗增殖活性依赖于CRBN-E3连接酶介导的靶蛋白降解。PROTAC 13i化合物可诱导CK1α、CDK7及CDK9的剂量依赖性降解,并伴随p53蛋白表达的同步上调,证明靶蛋白降解可能通过激活p53信号通路增强抗肿瘤效应。基于上述结果,通过流式细胞凋亡实验与Caspase-3/7活性检测发现,PROTAC 13i可显著诱导肿瘤细胞凋亡。由于CK1α与CDK7/9的协同降解可抑制致癌转录程序。q PCR分析证实,PROTAC 13i处理后的MV4-11细胞中MYC、MDM2、BCL-2及MCL-1基因表达量均显著下调。此外,ELISA实验显示该化合物可有效抑制PBMCs细胞中促炎因子TNF-α、IL-1β和IL-6的分泌。综上所述,本研究成功构建了首个可同步降解CK1α、CDK7/9的单分子三靶点PROTAC体系,其展现的协同抗肿瘤效应为克服AML耐药性提供了创新性治疗策略,并为多靶点蛋白降解剂的开发奠定了重要理论基础。更多还原

【Abstract】 The tumor suppressor protein p53 encoded by the TP53 gene plays a core role in maintaining genomic stability and inhibiting tumorigenesis.Acute myeloid leukemia(AML)is a highly invasive hematologic malignant tumor,and its treatment difficulties are closely related to the abnormal p53 signaling pathway.AML can be treated by inhibiting casein kinase 1α(Caseinkinase1α,CK1α)and cell cycle-dependent kinase 7/9(Cyclin-Dependent Kinase7,CDK7/9).This study is committed to developing innovative therapies based on proteolysis-targeting Chimeras(PROTAC),which uses the Ubiquitin-proteasome system(UPS)to achieve targeted protein degradation.PROTAC technology achieves ubiquitination marking and subsequent degradation of target proteins through bifunctional molecules(target protein ligand-E3 ligase ligand-Linker ternary complex).Its unique catalytic degradation mechanism can break through the drug resistance challenges faced by traditional inhibitors.It is worth noting that PROTAC molecules continuously induce target protein degradation through a cyclable mode of action,providing a breakthrough technical path to solve"unprepared"targets.This study is based on the multi-target kinase inhibitor A86(N-methylpyrazole derivative),which aims to develop three-target PROTAC molecules that can simultaneously degrade CK1αand CDK7/9 proteins.In addition,by analyzing the structure of eutectic complexes of A86 CK1αand CDK9,it was determined that the N1 and N2 positions of its fluoropyrimidinamine were key binding sites,and it was also found that the cyclohexane diamine group was exposed.Therefore,this study constructed PRORAC structures(1a and 1b)with or without cyclohexanediamine and explored their effects on molecular activity.Based on the diversity characteristics of E3 ligase ligands,three series of PROTAC molecules were constructed using a modular design strategy:(1)Pomalidomide type:Compound 9or A86 as the core targeting module,and PROTAC13a-g,13h and 13l were prepared by differentiated Linker coupling;(2)Benzamide type:Compound 13i-j was finally obtained by taking benzamide CRBN ligands as the optimization direction,and a series of reactions such as Ullmann coupling reactions were finally obtained;(3)VHL type:VHL ligand 28 was used to directly couple with intermediate compound18 to form compound 13k.The above series of designs systematically analyze the molecular construction rules of multi-target PROTAC through the coordinated optimization of E3 ligand type,Linker structure and target ligand structure.In vitro pharmacodynamic evaluation showed that PROTACs containing cyclohexanediamine groups showed significantly enhanced antiproliferative activity in MV4-11 and MOLM-13 cell lines than those with lesser PROTACs;PEG-type Linker(13a-d)had better cytotoxicity than alkyl(13e-g);CRBN ligand(13h-j)showed higher biological activity compared with VHL ligand(13k),and the benzamide ligand(13i-j)increased significantly compared with pomalidomide(13h).It is worth noting that the compound PROTAC13i showed the best antiproliferative effect in MV4-11(IC50=0.096±0.012μM)and MOLM-13(IC50=0.072±0.014μM)cells.To clarify the correlation between the anti-proliferative effect of PROTAC13i and its protein degradation function,this study was subject to mechanism verification through Western blot analysis:excessive thalidomide pretreatment competitively blocked the CRBN-mediated ubiquitination pathway,confirming that its anti-proliferative activity is dependent on the CRBN-E3 ligase-mediated target protein degradation.The PROTAC13i compound can induce dose-dependent degradation of CK1α,CDK7,and CDK9,and is accompanied by synchronous upregulation of p53 protein expression,demonstrating that target protein degradation may enhance the anti-tumor effect by activating the p53 signaling pathway.Based on the above results,it was found through flow cytometry apoptosis experiment and Caspase-3/7 activity detection that PROTAC13i can significantly induce tumor cell apoptosis.Because the co-degradation of CK1αand CDK7/9 can inhibit oncogenic transcription programs.q PCR analysis confirmed that the expression of MYC,MDM2,BCL-2 and MCL-1 genes in MV4-11 cells treated with PROTAC13i were significantly downregulated.In addition,ELISA experiments showed that this compound can effectively inhibit the secretion of pro-inflammatory factors TNF-α,IL-1β,and IL-6 in PBMCs cells.To sum up,this study successfully constructed the first single-molecular three-target PROTAC system that can synchronously degrade CK1αand CDK7/9.The synergistic antitumor effect it demonstrated provides an innovative therapeutic strategy for overcoming AML drug resistance and lays an important theoretical foundation for the development of multi-target protein degradants.更多还原