新型分子胶LTL-04-061通过CRBN介导的泛素-蛋白酶体通路靶向降解OTULIN并显著抑制胃癌细胞增殖OA
Novel molecular glue LTL-04-061 targets OTULIN for degradation via the CRBN-mediated ubiquitin-proteasome pathway and significantly suppresses gastric cancer cell proliferation
目的 筛选靶向卵巢肿瘤结构域线性链接特异性去泛素化酶(ovarian tumor domain deubiquitinase with linear linkage specificity,OTULIN)的新型分子胶,为胃癌治疗提供新策略.方法 以高表达OTULIN的AGS-NC、SNU601-NC胃癌细胞及OTULIN基因敲除细胞(AGS-sgOTULIN、SNU601-sgOTULIN)为研究材料,设对照组(DMSO)、LTL-04-061浓度梯度组(0.1、0.3、1.0、3.0、5.0、7.0、10.0 µmol/L)及蛋白酶体抑制剂干预组(MG-132/MLN4924联合5 µmol/L LTL-04-061),采用Western blot检测OTULIN蛋白表达水平,运用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)检测OTULIN的mRNA水平;利用免疫共沉淀(co-immunoprecipitation,Co-IP)验证蛋白相互作用;结合分子对接与100 ns分子动力学模拟(molecular dynamics simulation,MD)分析复合物构象;通过CCK-8法、细胞增殖实验、克隆形成实验评估分子胶对胃癌细胞恶性增殖的抑制作用.结果 分子胶LTL-04-061在0.1～10.0 µmol/L浓度梯度下呈浓度依赖性降解OTULIN蛋白,5 µmol/L浓度时降解效果显著,且该浓度下OTULIN mRNA相对表达水平无显著变化(P>0.05);蛋白酶体抑制剂MG-132与MLN4924可阻断其降解作用,结合Co-IP实验结果提示该过程依赖CRBN介导的泛素-蛋白酶体通路;同时LTL-04-061可介导OTULIN与CRBN形成三元稳定复合物,并与关键残基HIS-339(OTULIN)、TRP-378/HIS-380(CRBN)存在强相互作用,MD模拟显示50 ns后复合物均方根偏差波动小于0.1 nm,80～100 ns时氢键数量维持较高水平,回旋半径数值保持稳定,无明显波动,表明复合物整体紧凑性未发生显著变化;溶剂可及表面积在此时间段内亦无明显波动,进一步证实复合物构象稳定,未出现明显的结构舒展或塌陷;LTL-04-061呈浓度依赖性抑制胃癌细胞的增殖与克隆形成;CCK-8实验结果显示,LTL-04-061对AGS细胞和SNU601细胞的半数抑制浓度分别为8.95 µmol/L和11.70 µmol/L,并且OTULIN敲除后其抑制效果显著减弱(P<0.01).结论 OTULIN在胃癌中发挥促癌作用,分子胶LTL-04-061通过CRBN依赖的泛素-蛋白酶体通路,靶向降解OTULIN蛋白,进而抑制肿瘤细胞恶性增殖,为胃癌分子靶向治疗提供新的候选前体药物.
Objective To screen novel molecular glues targeting ovarian tumor domain deubiquitinase with linear linkage specificity(OTULIN)and provide a new therapeutic strategy for gastric cancer.Methods Gastric cancer cell lines with high OTULIN expression(AGS-NC,SNU601-NC)and OTULIN-knockout cells(AGS-sgOTULIN,SNU601-sgOTULIN)were used.Experimental groups included:control(DMSO),LTL-04-061 concentration gradients(0.1,0.3,1.0,3.0,5.0,7.0,10.0 µmol/L),and proteasome inhibitor intervention(MG-132/MLN4924+5 µmol/L LTL-04-061).OTULIN protein expression was analyzed by Western blotting,while mRNA levels were quantified by qPCR.Protein interactions were validated by co-immunoprecipitation(Co-IP).Molecular docking and 100 ns molecular dynamics(MD)simulations were performed to analyze complex conformations.Anti-proliferative effects were assessed using CCK-8,cell proliferation,and colony formation assays.Results LTL-04-061 degraded OTULIN protein in a concentration-dependent manner(0.1 to 10.0 µmol/L),with significant degradation at 5 µmol/L.At this concentration,OTULIN mRNA levels remained unchanged(P>0.05).Degradation was blocked by MG-132/MLN4924,indicating dependence on the CRBN-mediated ubiquitin-proteasome pathway.Co-IP confirmed LTL-04-061-induced OTULIN-CRBN ternary complex formation.Key interactions involved OTULIN HIS339 and CRBN TRP378/HIS380.MD simulations showed stable complex conformation after 50 ns(RMSD fluctuation<0.1 nm),sustained hydrogen bonds(80 to 100 ns),and stable radius of gyration(Rg)and solvent-accessible surface area(SASA),indicating no structural expansion/collapse.LTL-04-061 concentration-dependently suppressed gastric cancer cell proliferation and colony formation.CCK-8 assays revealed IC50 values of 8.95 µmol/L(AGS)and 11.70 µmol/L(SNU601).OTULIN knockout significantly attenuated this inhibition(P<0.01).Conclusion OTULIN exerts pro-oncogenic effects in gastric cancer.The molecular glue LTL-04-061 targets OTULIN for CRBN-dependent ubiquitin-proteasomal degradation,thereby suppressing malignant proliferation.This identifies LTL-04-061 as a promising precursor for molecularly targeted gastric cancer therapy.
刘振昆;彭紫依;向俊宇;王斌;蒋白山;邱秋;李玉红
陆军军医大学(第三军医大学)基础医学院细胞生物学教研室,重庆||陆军军医大学(第三军医大学)大坪医院消化内科,消化系统肿瘤精准防治重庆市重点实验室,重庆陆军军医大学(第三军医大学)大坪医院消化内科,消化系统肿瘤精准防治重庆市重点实验室,重庆陆军军医大学(第三军医大学)大坪医院消化内科,消化系统肿瘤精准防治重庆市重点实验室,重庆陆军军医大学(第三军医大学)大坪医院消化内科,消化系统肿瘤精准防治重庆市重点实验室,重庆武汉大学药学院,武汉大学免疫与代谢前沿科学中心,湖北武汉重庆市合川区人民医院消化内科,重庆陆军军医大学(第三军医大学)基础医学院细胞生物学教研室,重庆
医药卫生
胃癌OTULIN分子胶LTL-04-061靶向治疗
gastric cancerOTULINmolecular glueLTL-04-061targeted therapy
《陆军军医大学学报》 2026 (3)
251-260,10
国家重点研发计划项目(2023YFC3402102)重庆市科卫联合医学科研项目(2020FYYX028)合川人医发(2024)109号邱秋登峰人才项目 Supported by the National Key Research and Development Program of China(2023YFC3402102),the Joint Medical Research Project of Chongqing Science and Technology Commission and Chongqing Health Commission(2020FYYX028),and the Qiu Qiu Dengfeng Talent Project(2024-109)of Hechuan People's Hospital of Chongqing.
评论