去泛素化酶抑制剂克服肿瘤硼替佐米耐药的机制研究

The amplification and point mutation of 20S proteasome β5 subunit is the main reason for multiple myeloma and other cancers resistant to targeted therapeutic drug bortezomib. The existing second generation of proteasome inhibitors selectively inhibit the 20S proteasome and show the limitation in overcoming bortezomib resistance. The deubiquitinases inhibitor b-AP15 can inhibit the growth of bortezomib-resistant cells. Therefore, focus on the deubiquitinases in the upstream of 20S proteasome, finding the specific deubiquitinases inhibitors will be a new approach to overcome bortezomib resistance. Our previous studies demonstrate the metal complexes CuPT inhibits deubiquitinases activity and induces apoptosis of bortezomib-resistant cells. Further molecular docking study shows CuPT could combine with 19S proteasomal deubiquitinases. We thus propose deubiquitinases may be the main target for CuPT in overcoming bortezomib resistance. In this project, we would use a series of technologies such as dynamic enzyme activity detection, ubiquitin chain dissociation experiment and deubiquitinases labeling techniques to proceed a study in vitro and in vivo as well as in drug resistant patients primary cells. We will discuss the relationship between deubiquitinases inhibition and drug resistance overcoming, and investigate the molecular mechanisms of overcoming the drug resistance effect mediated by CuPT. The results of this project would provide reliable evidence and new ideas to overcome bortezomib resistance by targeting deubiquitinases, and it also provides the experimental data for CuPT to overcome bortezomib resistance in clinical translational research .

20S蛋白酶体β5亚基的扩增与点突变是多发性骨髓瘤等对靶向治疗药物硼替佐米耐药的主要原因。第二代蛋白酶体抑制剂选择作用于20S蛋白酶体，在克服硼替佐米耐药中显示出局限性。研究表明去泛素化酶抑制剂b-AP15能抑制耐药细胞生长。因此，关注20S蛋白酶体上游的去泛素化酶，寻找靶点明确的去泛素化酶抑制剂将是克服硼替佐米耐药的新途径。我们前期研究证明金属络合物CuPT抑制去泛素化酶活性，诱导耐药细胞凋亡，进一步分子对接显示其与19S蛋白酶体去泛素化酶结合。由此我们提出去泛素化酶可能是CuPT克服硼替佐米耐药的主要靶点。本项目拟采用动态酶活性检测、泛素链解离实验、去泛素化酶标签法等技术进行体内外及耐药病人半在体研究，从多角度探讨CuPT抑制去泛素化酶与抗耐药的关系和分子机制。本项目将获得CuPT靶向去泛素化酶克服耐药的可靠证据，为克服硼替佐米耐药提供新思路，也为其解决耐药的临床转化研究提供实验数据。

20S蛋白酶体β5亚基的扩增与点突变是肿瘤对靶向治疗药物硼替佐米耐药的主要原因。第二代蛋白酶体抑制剂选择作用于20S蛋白酶体，在克服硼替佐米耐药中显示出局限性。已有研究表明去泛素化酶抑制剂能抑制耐药细胞生长。因此，关注20S蛋白酶体上游的去泛素化酶，寻找靶点明确的去泛素化酶抑制剂将是克服硼替佐米及其他传统靶向治疗药物耐药的新途径。该项目中，我们设计合成了系列吡啶硫酮金属络合物，通过体内外实验及病人半在体实验，证实了系列吡啶硫酮金属络合物通过抑制19s蛋白酶体相关的去泛素化酶USP14和UCHL5的活性，引起细胞内泛素化蛋白的大量聚集，从而激活细胞内的内质网应激反应，通过调节钙离子的浓度，介导了线粒体依赖的细胞凋亡途径，通过激活的caspase3对敏感和耐药的肿瘤细胞中的Bcr-Abl进行切割，下调Bcr-Abl及其下游信号转导通路蛋白的表达与活性，或通过抑制蛋白酶体活性阻止耐药细胞中NF-κB的激活，从而诱导敏感和耐药肿瘤细胞发生凋亡。此外，我们论证了其他新型的去泛素化酶抑制剂b-AP15抑制去泛素化酶活性、克服肿瘤对传统靶向治疗药物耐药的分子学机制。相关研究成果以第一/通讯作者发表于Journal of Hematology & Oncology，Cell Death & Disease，Scientific Reports，oncotarget 和Medical chemistry杂志。另外，吡啶硫酮金属络合物PtPT的应用专利已被授权。本项目通过深入探讨系列吡啶硫酮金属络合物和新型的去泛素化酶抑制剂介导的去泛素化酶功能抑制与克服肿瘤耐药作用之间的关系，明确了其分子学机制，获得系列吡啶硫酮金属络合物直接调控耐药细胞去泛素化酶的可靠证据。本项目一定程度上为确立19S蛋白酶体去泛素化酶作为克服肿瘤耐药的新靶点提供更充分的科学依据，为进一步解决肿瘤耐药的临床科学研究提供新的切入点和新的思路。