人源IDO1蛋白与小分子INCB14943的晶体结构的研究
发布时间：2018-11-19 22:17
【摘要】：目的:吲哚胺-2,3-双加氧酶1(IDO1)是将色氨酸降解为犬尿氨酸的限速酶,代谢产物犬尿酸可直接抑制效应T细胞的功能,同时微环境中色氨酸的耗竭也会抑制T细胞的增殖,从而通过多途径发挥免疫抑制的作用。很多肿瘤细胞因为能高表达IDO1,从而能逃避T细胞的攻击。许多研究表明,IDO1抑制剂能够调节肿瘤微环境中的色氨酸含量,避免肿瘤微环境中T细胞增殖受抑制,因此成为潜在的免疫治疗药物。羟脒化合物INCB024360和INCB14943(INCB024360类似物)是高度有效的IDO1抑制剂。目前,IDO1抑制剂INCB024360与Pembrolizumab联合用药,评估其在晚期非小细胞肺癌中的疗效和安全性的Ⅰ/Ⅱ期临床研究正在进行中。本研究的目的是解出IDO1与羟基脒化合物INCB14943的复合物晶体结构,并详细说明IDO1与羟基脒化合物INCB14943的结合模式,从而促进基于结构的新型IDO1抑制剂的设计。方法:通过构建人源IDO1的基因,并将基因插入p ET 28a载体中,转化至大肠杆菌中表达IDO1蛋白,用Ni亲和层析柱和分子筛Superdex 75柱纯化。得到高纯度的IDO1蛋白后,将羟基脒化合物INCB14943与IDO1蛋白质孵育以进行结晶。结果:通过构建质粒、表达和纯化,现在已成功得到纯度大于95%的人源IDO1蛋白,经过晶体初筛,得到了晶体,在晶体衍射数据收集后,我们成功解出了IDO1和INCB14943的共晶结构,并详细阐明了两者之间的结合方式。INCB14943通过肟氮与IDO1蛋白中的血红素结合。进一步的分析还表明,INCB024360类似物的氯原子(3-Cl)与C129的硫原子之间的卤素相互作用显著提高了对IDO1的抑制活性。与其他报道的抑制剂相比,肟氮和卤素相互作用被认为是羟基脒化合物INCB14943的独特特征。结论:我们的课题是基于羟基脒化合物INCB14943和IDO1蛋白之间的相互作用的结构学研究,这些结构信息将有助于未来的IDO1抑制剂设计。
[Abstract]:Aim: indoleamine-2-tri-dioxygenase-1 (IDO1) is a rate-limiting enzyme that degrades tryptophan into canine uric acid. Canine uric acid, a metabolite, can directly inhibit the function of T cells. At the same time, the depletion of tryptophan in microenvironment can also inhibit the proliferation of T cells and play an immunosuppressive role through multiple pathways. Many tumor cells can escape T-cell attacks because of their high expression of IDO1,. Many studies have shown that IDO1 inhibitors can regulate the content of tryptophan in tumor microenvironment and avoid the inhibition of T cell proliferation in tumor microenvironment, so it becomes a potential immunotherapy drug. INCB024360 and INCB14943 (INCB024360 analogues) are highly effective IDO1 inhibitors. At present, stage I / II clinical studies of IDO1 inhibitor INCB024360 combined with Pembrolizumab to evaluate its efficacy and safety in advanced non-small cell lung cancer (NSCLC) are under way. The aim of this study is to elucidate the complex crystal structure of IDO1 and hydroxyamidine compound INCB14943 and explain in detail the binding mode of IDO1 and hydroxyamidine compound INCB14943 so as to promote the design of a novel IDO1 inhibitor based on structure. Methods: the gene of human IDO1 was constructed and inserted into p ET 28a vector, then transformed into E. coli to express IDO1 protein. The protein was purified by Ni affinity chromatography and molecular sieve Superdex 75 column. After high purity IDO1 protein was obtained, INCB14943 was incubated with IDO1 protein for crystallization. Results: by constructing plasmid, expressing and purifying, the human IDO1 protein with purity of more than 95% has been successfully obtained. After the primary screening of crystals, the crystal has been obtained. After collecting the data of crystal diffraction, the eutectic structures of IDO1 and INCB14943 have been successfully solved. INCB14943 binds to heme in IDO1 protein via oxime nitrogen. Further analysis showed that the halogen interaction between the chlorine atom (3-Cl) of the INCB024360 analogue and the sulfur atom of C129 significantly increased the inhibitory activity of IDO1. Compared with other reported inhibitors, the interaction between oxime nitrogen and halogen is considered to be the unique characteristic of hydroxyamidine compound INCB14943. Conclusion: our research is based on the interaction between INCB14943 and IDO1 proteins, which will be useful for the design of IDO1 inhibitors in the future.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914
本文编号：2343597
[Abstract]:Aim: indoleamine-2-tri-dioxygenase-1 (IDO1) is a rate-limiting enzyme that degrades tryptophan into canine uric acid. Canine uric acid, a metabolite, can directly inhibit the function of T cells. At the same time, the depletion of tryptophan in microenvironment can also inhibit the proliferation of T cells and play an immunosuppressive role through multiple pathways. Many tumor cells can escape T-cell attacks because of their high expression of IDO1,. Many studies have shown that IDO1 inhibitors can regulate the content of tryptophan in tumor microenvironment and avoid the inhibition of T cell proliferation in tumor microenvironment, so it becomes a potential immunotherapy drug. INCB024360 and INCB14943 (INCB024360 analogues) are highly effective IDO1 inhibitors. At present, stage I / II clinical studies of IDO1 inhibitor INCB024360 combined with Pembrolizumab to evaluate its efficacy and safety in advanced non-small cell lung cancer (NSCLC) are under way. The aim of this study is to elucidate the complex crystal structure of IDO1 and hydroxyamidine compound INCB14943 and explain in detail the binding mode of IDO1 and hydroxyamidine compound INCB14943 so as to promote the design of a novel IDO1 inhibitor based on structure. Methods: the gene of human IDO1 was constructed and inserted into p ET 28a vector, then transformed into E. coli to express IDO1 protein. The protein was purified by Ni affinity chromatography and molecular sieve Superdex 75 column. After high purity IDO1 protein was obtained, INCB14943 was incubated with IDO1 protein for crystallization. Results: by constructing plasmid, expressing and purifying, the human IDO1 protein with purity of more than 95% has been successfully obtained. After the primary screening of crystals, the crystal has been obtained. After collecting the data of crystal diffraction, the eutectic structures of IDO1 and INCB14943 have been successfully solved. INCB14943 binds to heme in IDO1 protein via oxime nitrogen. Further analysis showed that the halogen interaction between the chlorine atom (3-Cl) of the INCB024360 analogue and the sulfur atom of C129 significantly increased the inhibitory activity of IDO1. Compared with other reported inhibitors, the interaction between oxime nitrogen and halogen is considered to be the unique characteristic of hydroxyamidine compound INCB14943. Conclusion: our research is based on the interaction between INCB14943 and IDO1 proteins, which will be useful for the design of IDO1 inhibitors in the future.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914
【参考文献】
相关期刊论文 前1条
1 Shih Ling HWANG,Nancy Pei-Yee CHUNG,Jacqueline Kwai-Yi CHAN,Chen-Lung Steve LIN;Indoleamine 2,3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines[J];Cell Research;2005年03期
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