基于肝癌患者CYP2E1活性增高研究CYP2E1特异性抑制剂

Nitrosamines and hepatitis virus both are the recognized risk factors for hepatocellular carcinoma (HCC). CYP2E1 is of particular interest because of its involvement in the metabolic activation and inflammation.Our previous studies indicated CYP2E1 activity was increased in HCC patients and CYP2E1 activity was negatively correlated with overall survival, moreover, higher CYP2E1 innate activity had a causal relationship with hepatofibrogenesis and HCC induced by diethylnitrosamine in rats, which could be prevented by non-specific inhibitor of CYP2E1. Accordingly, we hypothesize that increased CYP2E1 activity might play an important role in the development and progression of HCC..This study focuses on the key basis of the discovery and application of CYP2E1 specific inhibitors. Discovery for CYP2E1 specific inhibitors is based on computer aided design, structure optimization and inhibition screening. Differences in the interaction of specific inhibitors with different CYP2E1 mutant recombinant will be investigated to determine the key amino acid sites and the affinity of the inhibitor binding. Hepatitis, hepatofibrogenesis and HCC model induced by nitrosamine in rats will be applied to study the effect of specific inhibitor on prevention and treatment. Cell model and transgenic animal will be used to explore the mechanism on prevention and treatment of specific inhibitors. Finally, preliminary safety as well application prospects of specific inhibitors will be evaluated.

肝癌发生与亚硝胺、肝炎等有关，CYP2E1具有代谢活化亚硝胺作用和致炎作用。我们前期研究证明，肝癌患者CYP2E1代谢亚硝胺活性明显增高且与生存期呈现明显负相关，大鼠CYP2E1先天活性与亚硝胺诱发的肝纤维化和肝癌具有因果关系，CYP2E1非特异性抑制剂则可抑制之。据此提出假说“CYP2E1活性增高可能是肝癌等发生发展的重要原因”。.本研究主要对CYP2E1特异性抑制剂的发现和应用的关键基础进行研究。经计算机辅助设计、抑制功能筛选、结构优化等发现CYP2E1特异性抑制剂；根据特异性抑制剂与不同CYP2E1突变型重组体相互作用差异，确定抑制剂与受体结合的关键氨基酸位点及亲和力；应用亚硝胺等所致大鼠肝炎、肝纤维化和肝癌模型，探讨特异性抑制剂的防治作用；应用细胞模型和转基因动物，探讨特异性抑制剂的防治作用机制；对特异性抑制剂进行初步安全性评价。最终发现CYP2E1特异性抑制剂，并评价其应用前景。

CYP2E1具有代谢活化亚硝胺的作用和致炎作用，与肝炎、肝纤维化和肝癌的发生、发展密切相关。我们前期研究证明，肝癌患者CYP2E1代谢亚硝胺活性明显增高且与生存期呈现明显负相关；大鼠CYP2E1先天活性与亚硝胺诱发的肝纤维化和肝癌具有因果关系，CYP2E1非特异性抑制剂可抑制之。然而目前尚无公认的CYP2E1体内外特异性抑制剂。.本研究通过计算机辅助设计、体内外抑制活性选择、结构优化筛选等发现SMI 12为CYP2E1较好的特异性小分子抑制剂，并以脓毒症、肝炎、肝纤维化和肝癌动物模型为研究对象探究了SMI 12的应用基础。采用LPS诱导大、小鼠脓毒症的发热模型，给予SMI 12干预后可改善LPS所致脓毒症模型的体温改变；采用高脂饮食诱发小鼠肝炎和肝纤维化的模型，给予SMI 12干预后可显著降低小鼠肝脏脂肪变、肝炎和纤维化的发生和发展；采用间断性腹腔注射二乙基亚硝胺（DEN）制备大鼠肝纤维化模型，给予SMI 12干预后可显著改善大鼠肝纤维化病变，并发现SMI 12除抑制CYP2E1对DEN的代谢作用外，还可能参与抑制CYP2E1其他方面的作用。Cyp2e1基因敲除可显著抑制大鼠肝脏原位移植瘤模型的肿瘤增殖，确证CYP2E1可作为肝癌的防治靶点。另通过对Cyp2e1敲除大鼠进行为期18个月的表型观察以及使用SMI 12对SD大鼠进行为期2个月的长期毒性研究发现，Cyp2e1敲除以及CYP2E1长期抑制后的安全性较好。这些发现表明CYP2E1特异性抑制剂SMI 12对脓毒症、肝炎、肝纤维化和肝癌等疾病的防治具有良好的应用前景。