吲哚胺2,3-雙加氧化酶1及其抑制劑的研究

張勝男 劉 鑫 楊 青

(復旦大學生命科學學院生物化學系 上海 200438)

吲哚胺2,3-雙加氧化酶1及其抑制劑的研究

張勝男 劉 鑫 楊 青△

(復旦大學生命科學學院生物化學系 上海 200438)

吲哚胺2,3-雙加氧化酶1( indoleamine 2,3-dioxygenase 1,IDO1)是肝臟以外催化色氨酸沿著犬尿氨酸途徑(kynurenine pathway,KP)分解代謝的限速酶。IDO1過度活化而引起KP的神經毒性產物喹啉酸(quinolinic acid,QUIN)的蓄積,是導致神經紊亂和神經退行性疾病的重要原因。IDO1同時又是免疫耐受酶,在誘導母胎免疫耐受和腫瘤免疫逃逸中均發揮重要作用,被視為新的免疫檢查點。IDO1與阿爾茨海默病、老年性白內障、癌癥等多種疾病發病機制的相關性已被證實,因此IDO1抑制劑作為極具潛能的藥物受到日益廣泛的關注。本文就IDO1的生物活性及其抑制劑的研究作一綜述。

吲哚胺2,3-雙加氧酶 1; 抑制劑; 腫瘤免疫治療

吲哚胺2,3-雙加氧化酶1( indoleamine 2,3-dioxygenase 1,IDO1) 可催化L-色氨酸(L-tryptophan,L-Trp)沿著犬尿氨酸途徑(kynurenine pathway,KP)分解代謝生成包括神經毒素喹啉酸(quinolinic acid,QUIN)在內的一些代謝產物[1]。IDO1的過度活化及其引起的QUIN含量的增多,與神經系統炎癥、抑郁、神經退行性疾病的發病機制密切相關[2-3]。IDO1還具有免疫耐受功能,被視為新的免疫檢查點[4-5]。腫瘤細胞、抗原呈遞細胞如巨噬細胞、樹突狀細胞上的IDO1均可誘導T細胞對腫瘤抗原的免疫耐受[6]。

IDO1有2個同工酶,即色氨酸雙加氧酶 (tryptophan 2,3-dioxygenase,TDO)和吲哚胺2,3-雙加氧酶2(indoleamine 2,3-dioxygenase 2,IDO2)[7]。TDO在多種腫瘤的免疫逃逸中起一定作用[8-9],而IDO2在T細胞對腫瘤抗原的免疫耐受中的角色和機制還有待深入研究[7,10-13]。

IDO1已被證實是一個重要的藥物發現靶標,IDO1抑制劑作為具有新藥靶、新機制的藥物,可應用于治療腫瘤、阿爾茨海默病、抑郁癥等多種疾病,社會和經濟效益顯著,應用前景廣闊[14-15]。

本文對IDO1的生物活性及其抑制劑的研究現狀作一介紹,以促進IDO1抑制劑類藥物的研發。

催化L-色氨酸分解代謝的限速酶 哺乳動物體內催化必需氨基酸L-Trp循KP分解代謝的首個反應的限速酶有3個,即TDO、IDO1和IDO2[7,16-17](圖1)。

圖1 L-色氨酸的犬尿氨酸代謝途徑

Fig 1 The kynurenine pathway of L-tryptophan metabolism

TDO(或TDO 2,EC.1.13.11.11)是存在于細胞內的含血紅素的多聚體雙加氧酶[18],由4個含血紅素的相同亞基組成[19],于1936年首次被發現于兔肝臟中[20],主要分布在哺乳動物的肝臟,經過刺激后在其他部位如皮膚、胎盤和腦中也有表達[21]。外源的色氨酸、犬尿氨酸(kynurenine,Kyn)以及糖皮質激素可誘導內源TDO2基因的表達[18,22]。人TDO的編碼基因(Tdo或Tdo2)位于4號染色體上(4q32.1 )[23],全長約16 kb,包含12個外顯子和6個內含子,其單體蛋白質產物分子量約46.7 kD,由406個氨基酸構成[7,24]。 真核和原核TDO都對L-Trp具有較高催化活性,而對D-Trp的催化活性很低[25-26]。TDO與IDO序列相似性很低,但是TDO與IDO其含亞鐵血紅素的活性部位高度相似[27]。近年研究發現TDO與腫瘤免疫逃逸有關[9,28],TDO活性的增強,造成Kyn等色氨酸代謝產物的含量顯著上升,進而提高體內調節性T細胞的水平并隨之產生免疫耐受[29],Kyn可以結合并激活芳香烴受體,增加腫瘤細胞存活率來幫助腫瘤細胞進行免疫逃逸[8]。

IDO1(或INDO,EC 1.13.11.52)是存在于細胞內的含亞鐵血紅素的單體蛋白質類酶,于1967年首次被發現于兔小腸中[30],廣泛分布于哺乳動物肝臟以外的多種組織,尤其是淋巴組織和胎盤[31]。IDO1是肝臟外可催化色氨酸分子中吲哚環氧化裂解,使其循KP分解代謝的限速酶[32]。人IDO1的編碼基因(Ido1,INDO)為單拷貝,全長約15 kb,由10個外顯子和9個內含子組成[33], 其蛋白質產物分子量約42 kD, 由403個氨基酸構成[34]。腫瘤細胞、抗原呈遞細胞如巨噬細胞、樹突狀細胞上的IDO1均可誘導T細胞對腫瘤抗原的免疫耐受。首先,IDO1通過降解色氨酸,造成缺乏色氨酸的微環境,使色氨酸依賴的T細胞增殖滯留在G1期[35]。其次,KP的一些代謝產物對T細胞有毒性,阻礙其功能[36-37]。最后,IDO1可以誘導調節性T細胞的擴增,而調節性T細胞的上調是腫瘤免疫治療的重要阻礙[38-40]。

IDO2(EC 1.13.11)于2007年首次被發現[41-43]。與IDO1的廣泛分布相比,IDO2分布范圍相對較小,如在小鼠的器官中,腎臟的IDO2 含量最高, 其次是副睪、肝臟[42-43]。與IDO1相比,IDO2催化L-Trp降解的酶活性很弱[44]。在人類和小鼠體內,IDO1 和IDO2 的編碼基因均位于8號染色體上(8p11.21),IDO2的基因位于IDO1的下游,二者呈頭-尾相連的串聯形式排列。人的IDO2基因(Ido2,INDOL1)全長約81 kb,包含11個外顯子和10個內含子,翻譯得到的蛋白質由420個氨基酸組成,分子量略大于46 kD[41-42,13]。研究表明IDO2也具有抑制T細胞增殖的作用[12,45]。提高色氨酸濃度或加入抑制劑1-甲基色氨酸(1-methyl-tryptophan,1-MT)可以逆轉由IDO1所引起的對T細胞增殖的抑制作用;與之明顯不同的是,由IDO2引起的T細胞增殖抑制作用即使補充高濃度色氨酸或1-MT也不能逆轉[45]。由此推測在T細胞參與的免疫調節反應中,IDO2與IDO1可能具有不同的信號調控通路,發揮不同的作用。氨基酸序列比對發現在人和小鼠體內,IDO1和 IDO2具有43%的序列同源性,但與TDO的同源序列均很少;蛋白質水平上IDO2與IDO1的空間結構類似,核心催化殘基相同,并且二者在功能上是保守的[42,46]。

IDO1抑制劑研發歷程 盡管TDO、IDO1和IDO2都具有催化色氨酸循KP代謝的功能,但是其組織分布以及催化底物能力有所不同,參與腫瘤免疫逃逸的作用及機制也不盡相同,在神經系統疾病發病機制中的作用被確認和認知的程度也不相同,其中IDO1受到較早的關注以及廣泛的重視,被認為是重要的藥物研發靶標[47-49]。

自1967年被發現至2000年,IDO1抑制劑的研發主要以治療神經紊亂和神經退行性疾病為目的,以合成化學為手段,以IDO1的底物色氨酸為模板,在研究構效關系的基礎上對其進行結構修飾。發表的文章和注冊的專利幾乎覆蓋了所有能修飾的基團[50-51],但是收效甚微。兩個比較有代表性的IDO1抑制劑分別是3-丁基-β咔啉(3-butyl-β-carboline,非競爭性抑制劑,Ki=3.3 μmol/L)[52]和1-MT(競爭性抑制劑,Ki=34 μmol/L)[53],后者被廣泛應用于體內外實驗。

2000年前后,隨著IDO1的免疫抑制作用被發現,IDO1抑制劑的研發掀起高潮。2006年,人IDO1與配體抑制劑4-苯基咪唑(4-phenylimidazole,4-PI )的復合物的晶體結構被解明[54],為IDO1抑制劑的設計提供了可貴的信息。IDO1抑制劑的研發不再局限于色氨酸衍生物的設計合成,而是天然產物提取、結構修飾、計算機模擬設計等多種手段并存,產生了許多種不同結構骨架的IDO1抑制劑。 來自海洋生物的Exiguamine A (Ki=0.21 μmol/L)[55]和Annulin C (Ki=0.14 μmol/L)[56-57],來自十字花科植物的Brassinin (Ki=97.7 μmol/L)[58],吲哚衍生物MTH-trp (細胞水平IC50=0.5 μmol/L)[47],苯醌衍生物Quinone (Ki=0.58 μmol/L)[48,57],抑制效率提高了10倍的4-PI的衍生物[59], 咪唑類化合物NLG919 (IC50=38 nmol/L)[60-62],三氮唑衍生物MMG-0358(IC50=0.33 μmol/L)[63],N-羥基瞇類INCB024360(IC50=72 nmol/L)[64-67],Amg-1(IC50=3 μmol/L)[46],細胞水平IC50達到pmol/L的2-氨基苯脲類化合物[68-71]等是比較典型的代表。

盡管不同結構的IDO1抑制劑不斷被發現,但是許多結構骨架在進一步的研究中表現出的活性或者特異性并不確定,因此未進行藥物研發。目前世界上還未有IDO1抑制劑藥物問世,只有NewLink Genetics公司的NLG919和Incyte公司的INCB024360兩個化合物進入臨床試驗[49]。2014年9月羅氏公司以頭款1.5億美元的價格收購了NLG919,接著在2015年4月又以頭款2 300萬美元獲得Curadev公司處于臨床前開發階段的IDO1和TDO抑制劑[72-73],從此開啟了IDO1抑制劑研發白熱化的競賽。

IDO1在被發現之后較長時間里沒有商品化出售,這給建立酶活檢測體系進行IDO1抑制劑篩選帶來一定難度,藥物學家不太容易在自己的實驗室里進行活性檢測指導下的分離提取或者設計合成。因此,IDO1抑制劑篩選工作在國內鮮有開展[74]。

從2006年開始,本課題組運用基因工程技術,制備不同種源(人、小鼠和大鼠)的IDO1、IDO2、TDO,在體外和細胞水平上建立了酶活性檢測體系,系統地測定抑制類型、抑制常數Ki、酶以及細胞水平IC50,建立了國內首個IDO1抑制劑篩選平臺。采取天然提取和化學合成的手段在數百個化合物中篩選出(E)-4-(β-溴乙烯基)苯氧酰基、4-苯基-1,2,3-三氮唑、黃連生物堿、吲哚生物堿等結構骨架的數十個優良的IDO1抑制劑;完成酶動力學測試、細胞水平抑制活性測試及IDO1蛋白質的三維對接試驗;運用獲得的IDO1抑制劑進行抗腫瘤、抗阿爾茨海默病的藥效學研究,并探索其相應的機制[15,75-78]。申請了多項IDO1抑制劑的中國發明專利和1項PCT國際專利。2016年3月,PCT專利的海外權利已經成功授權給美國滬亞生物國際有限公司(HUYA),業界評價是“中國知識產權成功轉化的一個案例”。

IDO1抑制劑評價方法 要完整系統地評價一個化合物的IDO1抑制活性,一般要測定抑制劑類型、抑制常數、酶以及細胞水平的 IC50等。

酶水平活性檢測 在Takikawa等[79]研究的基礎上進行優化。在500 μL的檢測體系中,將50 mmol/L磷酸鉀緩沖液、400 μg/mL 過氧化氫酶、40 mmol/L維生素C、20 μmol/L亞甲基藍、L-Trp以及待測樣品混合,混合液37 ℃保溫3～5 min, 再向上述混合液內加入重組人IDO1,37 ℃反應30 min,后加入質量濃度為300 g/L的三氯乙酸200 μL終止反應,并在65 ℃水浴鍋中加熱15 min,使之完成從N-甲酰犬尿氨酸到犬尿氨酸的轉化,然后12 000 r/min離心10 min(離心半徑為5.5 cm)。吸取上清100 μL與等體積的質量濃度為2 g/L的對二甲氨基苯甲醛的乙酸溶液混合,犬尿氨酸與該溶液產生反應,并使溶液變為黃色,使用酶標儀在492 nm處檢測其吸光度。

抑制類型及Ki、IC50值測定 利用上述反應體系,加入50 μmol/L 抑制劑和不同濃度的IDO1 酶,其他處理方法同上,最后以v ～ [E]作圖,根據所得曲線的關系判斷抑制劑類型。抑制常數 Ki 及 IC50值的測定同樣利用上述反應體系,加入不同濃度底物 L-Trp,在一個底物濃度下,加入不同濃度的抑制劑,其他處理方法同上,最后以 Dixon 作圖法得到Ki值[71],利用改良寇氏法計算出 IC50值。

細胞水平IDO 抑制活性測定 將HEK 293細胞以 2.5 × 104/孔的密度接種于96孔板中, DMEM 培養基培養(含10% 胎牛血清、50 U/mL青霉素、50 mg/mL 鏈霉素),置于37 ℃、濕度 95%、5% CO2的培養箱中培養。24 h后使用脂質體 Lipofectamin 2000 介導 pcDNA3.1-hIDO 質粒轉染。轉染24 h后加入待測藥物,孵育5 h 后,取 140 μL上清到另一 96 孔板中,加入10 μL質量濃度為300 g/L的三氯乙酸,在65 ℃加熱15 min,然后12 000 r/min離心10 min(離心半徑為5.5 cm),取等體積質量濃度為20 g/L的對-二甲氨基苯甲醛的乙酸溶液混合顯色,最后采用酶標儀檢測492 nm處的吸光值。

結語 在腫瘤免疫治療被日漸重視的今天,IDO1作為繼程序性死亡蛋白1 (programmed death protein-1,PD-1)和細胞毒性T淋巴細胞相關抗原4 (cytotoxic T lymphocyte-associated antigen-4,CTLA4)之后又一被關注的新免疫檢查點,其成為藥物發現靶標的價值和前景已經毋庸置疑。IDO1抑制劑研發過程中由于三維結構未明、IDO1未商品化、活性篩選體系不完善所帶來的壁壘被逐個攻破,人們已經對IDO1抑制劑的關鍵藥效基團有所了解和認識,并對IDO1抑制劑的作用機制不斷發掘和闡明?？梢云诖诓痪玫膶鞩DO1抑制劑作為藥物問世,為世人帶來健康的福音。

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The study of indoleamine 2,3-dioxygenase 1 and its inhibitors

ZHANG Sheng-nan, LIU Xin, YANG Qing△

(DepartmentofBiochemistry,SchoolofLifeSciences,FudanUniversity,Shanghai200438,China)

Indoleamine 2,3-dioxygenase 1 (IDO1) is an extrahepatic enzyme that catalyzes the first and rate-limiting step in the catabolism of the essential amino acid tryptophan (Trp) along the kynurenine pathway (KP).The overexpression or overactivation of IDO1 leads to the accumulation of downstream neurotoxic metabolite,quinolinic acid (QUIN),which is the main reason of nervous disorder and neurodegenerative disease.As an immunotolerant enzyme,IDO1 is regarded as a new immune checkpoint due to vital role in both the induction of maternal-fetal immune tolerance and tumor immune escape.Furthermore,the correlation between IDO1 and the pathogenesis of Alzheimer′s disease (AD),age-related cataracts and cancer has been confirmed,which brings IDO1 inhibitors increasingly widespread attention as promising drugs.This paper reviews the biological activity and development of IDO1 inhibitors.

indoleamine 2,3-dioxygenase 1; inhibitor; cancer immunotherapy

國家自然科學基金 (81373396,81573310);高等學校博士學科點專項科研基金 (20130071110037);上海市科委生物醫藥重點課題 (12431900204) ;復旦大學遺傳工程國家重點實驗室開放課題

專家簡介 楊青,復旦大學生命科學學院教授,博士生導師。上海市生化與分子生物學學會理事,上海市藥學會生化藥物專委會委員。先后主持國家自然基金、教育部博士點基金博導類項目、教育部留學回國人員基金、上海市浦江計劃、上海市生物醫藥重點課題等研究課題。主要進行疾病發生的分子機制研究及相關藥物的研發,2006年起從事吲哚胺2,3-雙加氧化酶(IDO)抑制劑的研究工作。重點研究IDO代謝通路失調與疾病的關系,篩選發現IDO抑制劑及相應的信號傳導網絡;以及藥物轉運蛋白 (drug transporter) 在藥物代謝中的功能及其表達、定位的調控機制。研究成果先后發表于國際知名期刊。擁有自主知識產權的新型IDO抑制劑的PCT專利,海外權利已成功轉化給美國企業。

R730.3

B

10.3969/j.issn.1672-8467.2017.01.001

2016-08-10;編輯:張秀峰)

△Corresponding author E-mail:yangqing68@fudan.edu.cn

*This work was supported by the National Natural Science Foundation of China (81373396,81573310),the Research Fund for the Doctoral Program of Higher Education in China (20130071110037),the Key Medical Project of Shanghai Science and Technology Committee (12431900204) and the Research Fund for the State Key Laboratory of Genetic Engineering,Fudan University.