【摘要】：目的:吲哚胺-2,3-雙加氧酶1(IDO1)是將色氨酸降解為犬尿氨酸的限速酶,代謝產(chǎn)物犬尿酸可直接抑制效應(yīng)T細(xì)胞的功能,同時(shí)微環(huán)境中色氨酸的耗竭也會抑制T細(xì)胞的增殖,從而通過多途徑發(fā)揮免疫抑制的作用。很多腫瘤細(xì)胞因?yàn)槟芨弑磉_(dá)IDO1,從而能逃避T細(xì)胞的攻擊。許多研究表明,IDO1抑制劑能夠調(diào)節(jié)腫瘤微環(huán)境中的色氨酸含量,避免腫瘤微環(huán)境中T細(xì)胞增殖受抑制,因此成為潛在的免疫治療藥物。羥脒化合物INCB024360和INCB14943(INCB024360類似物)是高度有效的IDO1抑制劑。目前,IDO1抑制劑INCB024360與Pembrolizumab聯(lián)合用藥,評估其在晚期非小細(xì)胞肺癌中的療效和安全性的Ⅰ/Ⅱ期臨床研究正在進(jìn)行中。本研究的目的是解出IDO1與羥基脒化合物INCB14943的復(fù)合物晶體結(jié)構(gòu),并詳細(xì)說明IDO1與羥基脒化合物INCB14943的結(jié)合模式,從而促進(jìn)基于結(jié)構(gòu)的新型IDO1抑制劑的設(shè)計(jì)。方法:通過構(gòu)建人源IDO1的基因,并將基因插入p ET 28a載體中,轉(zhuǎn)化至大腸桿菌中表達(dá)IDO1蛋白,用Ni親和層析柱和分子篩Superdex 75柱純化。得到高純度的IDO1蛋白后,將羥基脒化合物INCB14943與IDO1蛋白質(zhì)孵育以進(jìn)行結(jié)晶。結(jié)果:通過構(gòu)建質(zhì)粒、表達(dá)和純化,現(xiàn)在已成功得到純度大于95%的人源IDO1蛋白,經(jīng)過晶體初篩,得到了晶體,在晶體衍射數(shù)據(jù)收集后,我們成功解出了IDO1和INCB14943的共晶結(jié)構(gòu),并詳細(xì)闡明了兩者之間的結(jié)合方式。INCB14943通過肟氮與IDO1蛋白中的血紅素結(jié)合。進(jìn)一步的分析還表明,INCB024360類似物的氯原子(3-Cl)與C129的硫原子之間的鹵素相互作用顯著提高了對IDO1的抑制活性。與其他報(bào)道的抑制劑相比,肟氮和鹵素相互作用被認(rèn)為是羥基脒化合物INCB14943的獨(dú)特特征。結(jié)論:我們的課題是基于羥基脒化合物INCB14943和IDO1蛋白之間的相互作用的結(jié)構(gòu)學(xué)研究,這些結(jié)構(gòu)信息將有助于未來的IDO1抑制劑設(shè)計(jì)。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R914

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相關(guān)期刊論文 前1條

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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本文編號：2343597