瑞香素及噁唑类小分子抑制吲哚胺-2,3-双加氧酶1(IDO1)的研究

Daphnetin and An Oxazole Compound Inhibit Indoleamine 2,3-Dioxygenase 1

吲哚胺-2,3-双加氧酶1(indoleamine 2,3-dioxygenase 1,IDO1)在肿瘤免疫中发挥了重要作用,为了获得新型的IDO1小分子抑制剂,本研究利用He La细胞系建立了IDO1抑制剂筛选模型,筛选抑制IDO1活性的天然小分子化合物。将He La细胞接种于48孔板中,加入干扰素-γ(IFN-γ)诱导He La细胞中IDO1的表达,检测HeLa细胞分泌IDO1的酶代谢活性。对化合物库筛选后发现瑞香素(Daphnetin)和一个噁唑类小分子ZH-26能够抑制IDO1酶活性,采用Graph Pad Prism计算瑞香素和ZH-26的IC50值,结果显示瑞香素的IC50为16. 50±0. 33μM,ZH-26的IC50为4. 68±0. 21μM。进一步在HEK-293A细胞中过表达IDO1,不同浓度瑞香素和ZH-26处理细胞后也表现出对IDO1活性的抑制作用。采用Western blot方法发现瑞香素显著下调IFN-γ诱导的IDO1蛋白表达,而ZH-26则对IFN-γ诱导的IDO1的表达没有影响。综上,瑞香素和ZH-26在He La细胞内没有发现明显的细胞毒作用。本实验首次发现了瑞香素和ZH-26具有抑制IDO1的活性,不但为了解瑞香素这一天然来源临床药物的抗肿瘤机制提供新的视角,也为开发新的靶向IDO1的肿瘤免疫治疗候选药物奠定了基础。

Indoleamine2,3-dioxygenase1(IDO1)plays a key role in cancer immunity,the aim of this study was to discover new IDO1inhibitors.The inhibitor screening model of indoleamine2,3-dioxygenase1(IDO1)was established by using HeLa cell line,and two small molecule compounds inhibit IDO1activity were screened out.Interferon-γ(IFN-γ)was added to induce the expression of IDO1in HeLa cells seeded in48-well plates,to reflect the enzymatic activity of IDO1secreted by HeLa cells.Screening of the compound library revealed that daphnetin and an oxazole compound ZH-26inhibited IDO1activity,and the IC50values of daphnetin and ZH-26were analyzed using GraphPad Prism software.The IC50of daphnetin was16.50±0.33μM,and the IC50of ZH-26was4.68±0.21μM.In addition,the inhibitory effects on IDO1activity which was overexpressed in HEK-293A cells were also observed after treatment with different concentrations of daphnetin and ZH-26.Western blot assay showed that daphnetin significantly down-regulated the expression of IDO1induced by IFN-γwhereas ZH-26had no such effect.Daphnetin and ZH-26did not show obvious cytotoxic effect in HeLa cells.In conclusion,daphnetin and ZH-26were firstly found to inhibit IDO1activity in this study,which not only provided new insight into the understanding of anti-tumor effect of daphnetin,but also warranted further development of both compounds as new drug candidates for tumor immunotherapy by targeting IDO1.