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(fù)旦大學(xué)生命科學(xué)學(xué)院生物化學(xué)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(fù)旦大學(xué)生命科學(xué)學(xué)院生物化學(xué)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(guò)度活化而引起KP的神經(jīng)毒性產(chǎn)物喹啉酸(quinolinic acid,QUIN)的蓄積,是導(dǎo)致神經(jīng)紊亂和神經(jīng)退行性疾病的重要原因。IDO1同時(shí)又是免疫耐受酶,在誘導(dǎo)母胎免疫耐受和腫瘤免疫逃逸中均發(fā)揮重要作用,被視為新的免疫檢查點(diǎn)。IDO1與阿爾茨海默病、老年性白內(nèi)障、癌癥等多種疾病發(fā)病機(jī)制的相關(guān)性已被證實(shí),因此IDO1抑制劑作為極具潛能的藥物受到日益廣泛的關(guān)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(jīng)毒素喹啉酸(quinolinic acid,QUIN)在內(nèi)的一些代謝產(chǎn)物[1]。IDO1的過(guò)度活化及其引起的QUIN含量的增多,與神經(jīng)系統(tǒng)炎癥、抑郁、神經(jīng)退行性疾病的發(fā)病機(jī)制密切相關(guān)[2-3]。IDO1還具有免疫耐受功能,被視為新的免疫檢查點(diǎn)[4-5]。腫瘤細(xì)胞、抗原呈遞細(xì)胞如巨噬細(xì)胞、樹突狀細(xì)胞上的IDO1均可誘導(dǎo)T細(xì)胞對(duì)腫瘤抗原的免疫耐受[6]。

IDO1有2個(gè)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(xì)胞對(duì)腫瘤抗原的免疫耐受中的角色和機(jī)制還有待深入研究[7,10-13]。

IDO1已被證實(shí)是一個(gè)重要的藥物發(fā)現(xiàn)靶標(biāo),IDO1抑制劑作為具有新藥靶、新機(jī)制的藥物,可應(yīng)用于治療腫瘤、阿爾茨海默病、抑郁癥等多種疾病,社會(huì)和經(jīng)濟(jì)效益顯著,應(yīng)用前景廣闊[14-15]。

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

催化L-色氨酸分解代謝的限速酶 哺乳動(dòng)物體內(nèi)催化必需氨基酸L-Trp循KP分解代謝的首個(gè)反應(yīng)的限速酶有3個(gè),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(xì)胞內(nèi)的含血紅素的多聚體雙加氧酶[18],由4個(gè)含血紅素的相同亞基組成[19],于1936年首次被發(fā)現(xiàn)于兔肝臟中[20],主要分布在哺乳動(dòng)物的肝臟,經(jīng)過(guò)刺激后在其他部位如皮膚、胎盤和腦中也有表達(dá)[21]。外源的色氨酸、犬尿氨酸(kynurenine,Kyn)以及糖皮質(zhì)激素可誘導(dǎo)內(nèi)源TDO2基因的表達(dá)[18,22]。人TDO的編碼基因(Tdo或Tdo2)位于4號(hào)染色體上(4q32.1 )[23],全長(zhǎng)約16 kb,包含12個(gè)外顯子和6個(gè)內(nèi)含子,其單體蛋白質(zhì)產(chǎn)物分子量約46.7 kD,由406個(gè)氨基酸構(gòu)成[7,24]。 真核和原核TDO都對(duì)L-Trp具有較高催化活性,而對(duì)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(fā)現(xiàn)TDO與腫瘤免疫逃逸有關(guān)[9,28],TDO活性的增強(qiáng),造成Kyn等色氨酸代謝產(chǎn)物的含量顯著上升,進(jìn)而提高體內(nèi)調(diào)節(jié)性T細(xì)胞的水平并隨之產(chǎn)生免疫耐受[29],Kyn可以結(jié)合并激活芳香烴受體,增加腫瘤細(xì)胞存活率來(lái)幫助腫瘤細(xì)胞進(jìn)行免疫逃逸[8]。

IDO1(或INDO,EC 1.13.11.52)是存在于細(xì)胞內(nèi)的含亞鐵血紅素的單體蛋白質(zhì)類酶,于1967年首次被發(fā)現(xiàn)于兔小腸中[30],廣泛分布于哺乳動(dòng)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(huán)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(zhǎng)約15 kb,由10個(gè)外顯子和9個(gè)內(nèi)含子組成[33], 其蛋白質(zhì)產(chǎn)物分子量約42 kD, 由403個(gè)氨基酸構(gòu)成[34]。腫瘤細(xì)胞、抗原呈遞細(xì)胞如巨噬細(xì)胞、樹突狀細(xì)胞上的IDO1均可誘導(dǎo)T細(xì)胞對(duì)腫瘤抗原的免疫耐受。首先,IDO1通過(guò)降解色氨酸,造成缺乏色氨酸的微環(huán)境,使色氨酸依賴的T細(xì)胞增殖滯留在G1期[35]。其次,KP的一些代謝產(chǎn)物對(duì)T細(xì)胞有毒性,阻礙其功能[36-37]。最后,IDO1可以誘導(dǎo)調(diào)節(jié)性T細(xì)胞的擴(kuò)增,而調(diào)節(jié)性T細(xì)胞的上調(diào)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(fā)現(xiàn)[41-43]。與IDO1的廣泛分布相比,IDO2分布范圍相對(duì)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(nèi),IDO1 和IDO2 的編碼基因均位于8號(hào)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(lián)形式排列。人的IDO2基因(Ido2,INDOL1)全長(zhǎng)約81 kb,包含11個(gè)外顯子和10個(gè)內(nèi)含子,翻譯得到的蛋白質(zhì)由420個(gè)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(xì)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(zhuǎn)由IDO1所引起的對(duì)T細(xì)胞增殖的抑制作用;與之明顯不同的是,由IDO2引起的T細(xì)胞增殖抑制作用即使補(bǔ)充高濃度色氨酸或1-MT也不能逆轉(zhuǎn)[45]。由此推測(cè)在T細(xì)胞參與的免疫調(diào)節(jié)反應(yīng)中,IDO2與IDO1可能具有不同的信號(hào)調(diào)控通路,發(fā)揮不同的作用。氨基酸序列比對(duì)發(fā)現(xiàn)在人和小鼠體內(nèi),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(zhì)水平上IDO2與IDO1的空間結(jié)構(gòu)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(fā)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(jī)制也不盡相同,在神經(jīng)系統(tǒng)疾病發(fā)病機(jī)制中的作用被確認(rèn)和認(rèn)知的程度也不相同,其中IDO1受到較早的關(guān)注以及廣泛的重視,被認(rèn)為是重要的藥物研發(fā)靶標(biāo)[47-49]。

自1967年被發(fā)現(xiàn)至2000年,IDO1抑制劑的研發(fā)主要以治療神經(jīng)紊亂和神經(jīng)退行性疾病為目的,以合成化學(xué)為手段,以IDO1的底物色氨酸為模板,在研究構(gòu)效關(guān)系的基礎(chǔ)上對(duì)其進(jìn)行結(jié)構(gòu)修飾。發(fā)表的文章和注冊(cè)的專利幾乎覆蓋了所有能修飾的基團(tuán)[50-51],但是收效甚微。兩個(gè)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(jìng)爭(zhēng)性抑制劑,Ki=3.3 μmol/L)[52]和1-MT(競(jìng)爭(zhēng)性抑制劑,Ki=34 μmol/L)[53],后者被廣泛應(yīng)用于體內(nèi)外實(shí)驗(yàn)。

2000年前后,隨著IDO1的免疫抑制作用被發(fā)現(xiàn),IDO1抑制劑的研發(fā)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(fù)合物的晶體結(jié)構(gòu)被解明[54],為IDO1抑制劑的設(shè)計(jì)提供了可貴的信息。IDO1抑制劑的研發(fā)不再局限于色氨酸衍生物的設(shè)計(jì)合成,而是天然產(chǎn)物提取、結(jié)構(gòu)修飾、計(jì)算機(jī)模擬設(shè)計(jì)等多種手段并存,產(chǎn)生了許多種不同結(jié)構(gòu)骨架的IDO1抑制劑。 來(lái)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(lái)自十字花科植物的Brassinin (Ki=97.7 μmol/L)[58],吲哚衍生物MTH-trp (細(xì)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(xì)胞水平IC50達(dá)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(jié)構(gòu)的IDO1抑制劑不斷被發(fā)現(xiàn),但是許多結(jié)構(gòu)骨架在進(jìn)一步的研究中表現(xiàn)出的活性或者特異性并不確定,因此未進(jìn)行藥物研發(fā)。目前世界上還未有IDO1抑制劑藥物問(wèn)世,只有NewLink Genetics公司的NLG919和Incyte公司的INCB024360兩個(gè)化合物進(jìn)入臨床試驗(yàn)[49]。2014年9月羅氏公司以頭款1.5億美元的價(jià)格收購(gòu)了NLG919,接著在2015年4月又以頭款2 300萬(wàn)美元獲得Curadev公司處于臨床前開發(fā)階段的IDO1和TDO抑制劑[72-73],從此開啟了IDO1抑制劑研發(fā)白熱化的競(jìng)賽。

IDO1在被發(fā)現(xiàn)之后較長(zhǎng)時(shí)間里沒(méi)有商品化出售,這給建立酶活檢測(cè)體系進(jìn)行IDO1抑制劑篩選帶來(lái)一定難度,藥物學(xué)家不太容易在自己的實(shí)驗(yàn)室里進(jìn)行活性檢測(cè)指導(dǎo)下的分離提取或者設(shè)計(jì)合成。因此,IDO1抑制劑篩選工作在國(guó)內(nèi)鮮有開展[74]。

從2006年開始,本課題組運(yùn)用基因工程技術(shù),制備不同種源(人、小鼠和大鼠)的IDO1、IDO2、TDO,在體外和細(xì)胞水平上建立了酶活性檢測(cè)體系,系統(tǒng)地測(cè)定抑制類型、抑制常數(shù)Ki、酶以及細(xì)胞水平IC50,建立了國(guó)內(nèi)首個(gè)IDO1抑制劑篩選平臺(tái)。采取天然提取和化學(xué)合成的手段在數(shù)百個(gè)化合物中篩選出(E)-4-(β-溴乙烯基)苯氧?；?-苯基-1,2,3-三氮唑、黃連生物堿、吲哚生物堿等結(jié)構(gòu)骨架的數(shù)十個(gè)優(yōu)良的IDO1抑制劑;完成酶動(dòng)力學(xué)測(cè)試、細(xì)胞水平抑制活性測(cè)試及IDO1蛋白質(zhì)的三維對(duì)接試驗(yàn);運(yùn)用獲得的IDO1抑制劑進(jìn)行抗腫瘤、抗阿爾茨海默病的藥效學(xué)研究,并探索其相應(yīng)的機(jī)制[15,75-78]。申請(qǐng)了多項(xiàng)IDO1抑制劑的中國(guó)發(fā)明專利和1項(xiàng)PCT國(guó)際專利。2016年3月,PCT專利的海外權(quán)利已經(jīng)成功授權(quán)給美國(guó)滬亞生物國(guó)際有限公司(HUYA),業(yè)界評(píng)價(jià)是“中國(guó)知識(shí)產(chǎn)權(quán)成功轉(zhuǎn)化的一個(gè)案例”。

IDO1抑制劑評(píng)價(jià)方法 要完整系統(tǒng)地評(píng)價(jià)一個(gè)化合物的IDO1抑制活性,一般要測(cè)定抑制劑類型、抑制常數(shù)、酶以及細(xì)胞水平的 IC50等。

酶水平活性檢測(cè) 在Takikawa等[79]研究的基礎(chǔ)上進(jìn)行優(yōu)化。在500 μL的檢測(cè)體系中,將50 mmol/L磷酸鉀緩沖液、400 μg/mL 過(guò)氧化氫酶、40 mmol/L維生素C、20 μmol/L亞甲基藍(lán)、L-Trp以及待測(cè)樣品混合,混合液37 ℃保溫3～5 min, 再向上述混合液內(nèi)加入重組人IDO1,37 ℃反應(yīng)30 min,后加入質(zhì)量濃度為300 g/L的三氯乙酸200 μL終止反應(yīng),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(zhuǎn)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(zhì)量濃度為2 g/L的對(duì)二甲氨基苯甲醛的乙酸溶液混合,犬尿氨酸與該溶液產(chǎn)生反應(yīng),并使溶液變?yōu)辄S色,使用酶標(biāo)儀在492 nm處檢測(cè)其吸光度。

抑制類型及Ki、IC50值測(cè)定 利用上述反應(yīng)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(jù)所得曲線的關(guān)系判斷抑制劑類型。抑制常數(shù) Ki 及 IC50值的測(cè)定同樣利用上述反應(yīng)體系,加入不同濃度底物 L-Trp,在一個(gè)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(jì)算出 IC50值。

細(xì)胞水平IDO 抑制活性測(cè)定 將HEK 293細(xì)胞以 2.5 × 104/孔的密度接種于96孔板中, DMEM 培養(yǎng)基培養(yǎng)(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(yǎng)箱中培養(yǎng)。24 h后使用脂質(zhì)體 Lipofectamin 2000 介導(dǎo) pcDNA3.1-hIDO 質(zhì)粒轉(zhuǎn)染。轉(zhuǎn)染24 h后加入待測(cè)藥物,孵育5 h 后,取 140 μL上清到另一 96 孔板中,加入10 μL質(zhì)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(zhì)量濃度為20 g/L的對(duì)-二甲氨基苯甲醛的乙酸溶液混合顯色,最后采用酶標(biāo)儀檢測(cè)492 nm處的吸光值。

結(jié)語(yǔ) 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(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4 (cytotoxic T lymphocyte-associated antigen-4,CTLA4)之后又一被關(guān)注的新免疫檢查點(diǎn),其成為藥物發(fā)現(xiàn)靶標(biāo)的價(jià)值和前景已經(jīng)毋庸置疑。IDO1抑制劑研發(fā)過(guò)程中由于三維結(jié)構(gòu)未明、IDO1未商品化、活性篩選體系不完善所帶來(lái)的壁壘被逐個(gè)攻破,人們已經(jīng)對(duì)IDO1抑制劑的關(guān)鍵藥效基團(tuán)有所了解和認(rèn)識(shí),并對(duì)IDO1抑制劑的作用機(jī)制不斷發(fā)掘和闡明?？梢云诖诓痪玫膶?lái)IDO1抑制劑作為藥物問(wèn)世,為世人帶來(lái)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(guó)家自然科學(xué)基金 (81373396,81573310);高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金 (20130071110037);上海市科委生物醫(yī)藥重點(diǎn)課題 (12431900204) ;復(fù)旦大學(xué)遺傳工程國(guó)家重點(diǎn)實(shí)驗(yàn)室開放課題

專家簡(jiǎn)介 楊青,復(fù)旦大學(xué)生命科學(xué)學(xué)院教授,博士生導(dǎo)師。上海市生化與分子生物學(xué)學(xué)會(huì)理事,上海市藥學(xué)會(huì)生化藥物專委會(huì)委員。先后主持國(guó)家自然基金、教育部博士點(diǎn)基金博導(dǎo)類項(xiàng)目、教育部留學(xué)回國(guó)人員基金、上海市浦江計(jì)劃、上海市生物醫(yī)藥重點(diǎn)課題等研究課題。主要進(jìn)行疾病發(fā)生的分子機(jī)制研究及相關(guān)藥物的研發(fā),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(diǎn)研究IDO代謝通路失調(diào)與疾病的關(guān)系,篩選發(fā)現(xiàn)IDO抑制劑及相應(yīng)的信號(hào)傳導(dǎo)網(wǎng)絡(luò);以及藥物轉(zhuǎn)運(yùn)蛋白 (drug transporter) 在藥物代謝中的功能及其表達(dá)、定位的調(diào)控機(jī)制。研究成果先后發(fā)表于國(guó)際知名期刊。擁有自主知識(shí)產(chǎn)權(quán)的新型IDO抑制劑的PCT專利,海外權(quán)利已成功轉(zhuǎn)化給美國(guó)企業(yè)。

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.