摘要
蛋白精氨酸甲基转移酶(PRMTs)对组蛋白和非组蛋白的翻译后修饰在生物学过程中起到了非常重要的作用。PRMT5是精氨酸残基对称双甲基化的主要酶,已被视为肿瘤治疗的潜在靶点。在过去10年中,发现和开发PRMT5抑制剂已经成为科研人员关注的热点。本文介绍了PRMT5的结构、生化功能以及其与肿瘤的相关性,同时就目前在研的PRMT5抑制剂与其结合方式以及生物活性进行综述,并讨论了PRMT5抑制剂在肿瘤治疗中的应用潜力。
蛋白质精氨酸甲基转移酶(protein arginine methyltransferase,PRMT)催化的精氨酸胍基甲基化,是一种常见的真核生物细胞翻译后修饰,影响细胞信号传导、基因转录、mRNA翻译、DNA重组和修复等多种生物学过
PRMTs甲基化特定精氨酸胍基氮原子,由S-腺苷甲硫氨酸(S-adenosyl methionine,SAM)提供甲基,同时释放出一个分子的S-腺苷-L-同型半胱氨酸(S-adenosyl-L-homocysteine,SAH)。根据修饰形式,将PRMTs分成3类(

图1 3种PRMTs催化精氨酸甲基化反应
PRMT: 蛋白质精氨酸甲基转移酶; SAM: S-腺苷甲硫氨酸; SAH: S-腺苷-L-同型半胱氨酸; MMA: ω-
PRMT5是细胞中主要的sDMA形成酶,N端为TIM桶状结构蛋白(TIM barrel),C端是甲基转移结构域(

图2 PRMT5:MEP50复合体的总体结构(PDB:4GQB)(A)和放大的H4R3底物肽结合槽(B
在一项对线虫PRMT5的突变研究中,将底物口袋中的保守残基之一Phe379变为蛋氨酸,可以实现H4R3同时进行sDMA和aDM
PRMT5广泛存在于细胞核和胞浆中,通过调控组蛋白及多种非组蛋白的甲基化修饰影响各种细胞过程。
PRMT5通过许多核质底物的甲基化起作用,包括组蛋白H2A、H3和H
部分非组蛋白也被鉴定为PRMT5的底物,在细胞功能调控中发挥重要作
最近几年,作为一个抗肿瘤药物靶点,PRMT5越来越引起科学界的关注。它在多种肿瘤中过表达,包括胶质母细胞瘤、肺癌、前列腺癌等,并在不同肿瘤中发生机制不
例如,在临床上越来越多人胶质母细胞瘤病例的发生与PRMT5过表达相
此外,由于代谢酶5-甲硫腺苷磷酸化酶(5'-methylthioadenonine phosphorylase,MTAP)基因与人类染色体9p21上的抑癌基因CDKN2A非常接近,因此在CDKN2A基因缺失的肿瘤中经常伴随MTAP缺失,是肿瘤中突变频率最高的基因之一。MTAP缺失使其底物甲硫腺苷(methylthioadenosine,MTA)积累,MTA能选择性抑制PRMT5甲基转移酶的活性,并使其对进一步的PRMT5抑制更为敏
近年来,PRMT5作为肿瘤治疗的潜在靶点受到特别关
EPZ015666是第一个报道的具有细胞增殖抑制活性且可口服的PRMT5抑制剂,其IC50 为22 nmol/

PRMT5:MEP50-SAM-EPZ015666共晶结构显示(

图3 EPZ015666与PRMT5:MEP50和SAM的共晶结构(PDB:4X61
化合物GSK3326595保持了THIQ结构,PRMT5酶抑制活性IC50为6.2 nmol/L,该化合物分别通过调节人结肠癌细胞株SW480中p53与人套细胞淋巴瘤细胞株Z-138中p21的活性抑制SW480与Z-138增殖,诱导细胞周期阻滞。在Z-138皮下肿瘤异种移植小鼠模型中,GSK3326595有效抑制肿瘤体积(0.2~100 mg/kg,po,bid或50~200 mg/kg,po,qd)。同时该化合物呈现良好的药代动力学特性,静脉给药1 mg/kg后,AUC0-t为415 h·ng/mL,t1/2为1.42 h,分布体积为1.42 L/kg,血浆清除率为39 mL/(kg·min)。小鼠经口给予1 mg/kg,cmax为1 050 ng/mL,AUC0-t为3 450 h·ng/mL,t1/2为0.25 h,生物利用度为80.7

为了开发更有效的PRMT5抑制剂,研究人员将GSK3326595的2位侧链移至3位,得到了一系列活性化合物,如化合物1对PRMT5具有很高的抑制活性(IC50 = 26 nmol/L

此外,研究人员在EPZ015666的基础上发现了一种新型萘环抑制剂,经过优化后得到了DC-C01(IC50 = 2.8 µmol/L),它能够呈剂量依赖地抑制一些肿瘤细胞(Z-138、Maver-1和Jeko-1)增

PRMTs含有高度保守的辅因子SAM甲基转移结合域,而SAH和天然产物西奈芬净则是SAM的结构类似物,两者均显示出良好的PRMT5抑制活性,但对其他PRMTs缺乏选择性。A9145C是早期基于西奈芬净结构研发出的SAM竞争抑制剂,在与PRMT5:MEP50和H4组蛋白肽段的晶体结构中显示,A9145C可以和SAM结合域中的几个残基(如,Asp419、Met420、Glu392和Try324)互相作用,但是它产生相互作用的结构与SAM对应的结构一致,包括腺苷部分的主体与两个氢键、尾部的羧基,仅有C5氨基部分可能与组蛋白H4产生额外的相互作用,因此它虽然能很好地结合在SAM结合域中,但仍然缺乏对PRMT5的选择

在后续的研究中,MTA被证明是一种有效的、选择性的PRMT5抑制

JNJ64619178是第一个进入临床试验的基于SAM结构的模拟核苷类似物(NCT03573310),采用环戊烷替代了SAM中的四氢呋喃环。它对PRMT5酶的抑制活性IC50为6.3 nmol/L,并对一组肺癌细胞(A549、NCI-H441、H1048、HCC78)表现良好的增殖抑制活

LLY-283和JNJ64619178的发现激发了更多研究者对SAM竞争性抑制剂的兴趣。鉴于PRMT5的活性位点附近有一个特有的半胱氨酸C449,人们推测其可作为潜在的共价修饰位


图4 乙烯基硫醚的形成过程
近年来,PRMT5抑制剂已成为抗肿瘤药物研发的热点,然而目前已报道的大多数抑制剂仍局限于类似的骨架结构。在不同肿瘤发生过程中,PRMT5 导致肿瘤发生的机制各有不同且部分尚未明确,为了更好地了解PRMT5的治疗潜力,还需要挖掘更多关于PRMT5功能的生物学信息。
对于SAM非竞争性抑制剂,诺华公司和Agios公司对该类抑制剂底物结合的模式进行了实验,发现EPZ015666能够有效地抑制许多细胞株的体外生长,但缺乏肿瘤细胞株选择性,并且它对肿瘤细胞的抑制能力与细胞中MTAP的状态无关,这就有可能在临床试验中转化为潜在的毒
总之,随着对PRMT5生化功能的进一步研究,各类化合物与PRMT5结合的共晶结构的不断被报道,安全的PRMT5抑制剂逐渐被人们所发现并最终应用于临床,使肿瘤患者获益。
References
Blanc RS,Richard S. Arginine methylation:the coming of age[J]. Mol Cell,2017,65(1):8-24. [百度学术]
Wesche J,Kühn S,Kessler BM,et al. Protein arginine methylation:a prominent modification and its demethylation[J]. Cell Mol Life Sci,2017,74(18):3305-3315. [百度学术]
Jahan S,Davie JR. Protein arginine methyltransferases (PRMTs):role in chromatin organization[J]. Adv Biol Regul,2015,57:173-184. [百度学术]
Clarke TL,Sanchez-Bailon MP,Chiang K,et al. PRMT5-dependent methylation of the TIP60 coactivator RUVBL1 is a key regulator of homologous recombination[J]. Mol Cell,2017,65(5):900-916.e7. [百度学术]
Stopa N,Krebs JE,Shechter D. The PRMT5 arginine methyltransferase:many roles in development,cancer and beyond[J]. Cell Mol Life Sci,2015,72(11):2041-2059. [百度学术]
Zurita-Lopez CI,Sandberg T,Kelly R,et al. Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues[J]. J Biol Chem,2012,287(11):7859-7870. [百度学术]
Sun LT,Wang MZ,Lv Z,et al. Structural insights into protein arginine symmetric dimethylation by PRMT5[J]. Proc Natl Acad Sci U S A,2011,108(51):20538-20543. [百度学术]
Timm DE,Bowman V,Madsen R,et al. Cryo-electron microscopy structure of a human PRMT5:MEP50 complex[J]. PLoS One,2018,13(3):e0193205. [百度学术]
Antonysamy S,Bonday Z,Campbell RM,et al. Crystal structure of the human PRMT5:MEP50 complex[J]. Proc Natl Acad Sci U S A,2012,109(44):17960-17965. [百度学术]
Zhang X,Cheng XD. Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides[J]. Structure,2003,11(5):509-520. [百度学术]
Wang YX,Hu WH,Yuan YQ. Protein arginine methyltransferase 5 (PRMT5) as an anticancer target and its inhibitor discovery[J]. J Med Chem,2018,61(21):9429-9441. [百度学术]
Pal S,Vishwanath SN,Erdjument-Bromage H,et al. Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes[J]. Mol Cell Biol,2004,24(21):9630-9645. [百度学术]
Pesiridis GS,Diamond E,van Duyne GD. Role of pICLn in methylation of Sm proteins by PRMT5[J]. J Biol Chem,2009,284(32):21347-21359. [百度学术]
Andreu-Pérez P,Esteve-Puig R,de Torre-Minguela C,et al. Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF[J]. Sci Signal,2011,4(190):ra58. [百度学术]
Bezzi M,Teo SX,Muller J,et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery[J]. Genes Dev,2013,27(17):1903-1916. [百度学术]
Richters A. Targeting protein arginine methyltransferase 5 in disease[J]. Future Med Chem,2017,9(17):2081-2098. [百度学术]
Jansson M,Durant ST,Cho EC,et al. Arginine methylation regulates the p53 response[J]. Nat Cell Biol,2008,10(12):1431-1439. [百度学术]
Mounir Z,Korn JM,Westerling T,et al. ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the androgen receptor[J]. Elife,2016,5:e13964. [百度学术]
Cho EC,Zheng SS,Munro S,et al. Arginine methylation controls growth regulation by E2F-1[J]. EMBO J,2012,31(7):1785-1797. [百度学术]
Wei H,Wang BL,Miyagi M,et al. PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB[J]. Proc Natl Acad Sci U S A,2013,110(33):13516-13521. [百度学术]
Zhou Z,Sun X,Zou Z,et al. PRMT5 regulates Golgi apparatus structure through methylation of the golgin GM130[J]. Cell Res,2010,20(9):1023-1033. [百度学术]
Ren J,Wang Y,Liang Y,et al. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis[J]. J Biol Chem,2010,285(17):12695-12705. [百度学术]
Banasavadi-Siddegowda YK,Welker AM,An M,et al. PRMT5 as a druggable target for glioblastoma therapy[J]. Neuro Oncol,2018,20(6):753-763. [百度学术]
Yan F,Alinari L,Lustberg ME,et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma[J]. Cancer Res,2014,74(6):1752-1765. [百度学术]
Wu ZY,Lin YH. Long noncoding RNA LINC00515 promotes cell proliferation and inhibits apoptosis by sponging miR-16 and activating PRMT5 expression in human glioma[J]. Oncotargets Ther,2019,12:2595-2604. [百度学术]
Zhang S,Ma Y,Hu X,et al. Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth[J]. J Cell Mol Med,2019,23(2):1333-1342. [百度学术]
Deng X,Shao G,Zhang HT,et al. Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth[J]. Oncogene,2017,36(9):1223-1231. [百度学术]
Mavrakis KJ,3rdMcDonald ER,Schlabach MR,et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5[J]. Science,2016,351(6278):1208-1213. [百度学术]
Kaniskan HÜ,Martini ML,Jin J. Inhibitors of protein methyltransferases and demethylases[J]. Chem Rev,2018,118(3):989-1068. [百度学术]
Kaniskan HÜ,Konze KD,Jin J. Selective inhibitors of protein methyltransferases[J]. J Med Chem,2015,58(4):1596-1629. [百度学术]
Luo MK. Inhibitors of protein methyltransferases as chemical tools[J]. Epigenomics,2015,7(8):1327-1338. [百度学术]
Kaniskan HÜ,Jin J. Recent progress in developing selective inhibitors of protein methyltransferases[J]. Curr Opin Chem Biol,2017,39:100-108. [百度学术]
Hu H,Qian K,Ho MC,et al. Small molecule inhibitors of protein arginine methyltransferases[J]. Expert Opin Investig Drugs,2016,25(3):335-358. [百度学术]
Lin H,Luengo JI. Nucleoside protein arginine methyltransferase 5 (PRMT5) inhibitors[J]. Bioorg Med Chem Lett,2019,29(11):1264-1269. [百度学术]
Chan-Penebre E,Kuplast KG,Majer CR,et al. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models[J]. Nat Chem Biol,2015,11(6):432-437. [百度学术]
Duncan KW,Rioux N,Boriack-Sjodin PA,et al. Structure and property guided design in the identification of PRMT5 tool compound EPZ015666[J]. ACS Med Chem Lett,2016,7(2):162-166. [百度学术]
Chikkanna D,Panigrahi SK,Rajagopalan S,et al.Abstract A174:Novel inhibitors of protein arginine methyltransferase 5 (PRMT5) for the treatment of solid tumors[C]// AACR-NCI-EORTC International Conference:Molecular Targets and Cancer Therapeutics.Philadelphia,2018. [百度学术]
Bergman YE,Camerino MA,Stupple PA.Benzopiperidine derivatives and their use in the treatment of cancer and hemoglobinopathies:WO2017153520 A1[P/OL].2016-08-25[2020-01-20].http://www.freepatentsonline.com/y2020/0010451.htmL. [百度学术]
Michelle R,David J,Hale M,et al.PRMT5 inhibitors:WO2019094311[P/OL].2019-05-16[2020-01-20].https://worldwide.espacenet.com/patent/search/family/066438946/publication/WO2019094311A1?q=WO2019094311&called_by=epo.org. [百度学术]
Nicholas LT,Rebecca M,Andrew M.Tricyclic compounds for use in treatment of proliferative disorders:WO2018167276A1[P/OL].2018-09-20[2020-01-20].https://worldwide.espacenet.com/patent/search/family/058688220/publication/WO2018167276A1?called_by=epo.org&q=WO2018167276A1. [百度学术]
Ye Y,Zhang BD,Mao RF,et al. Discovery and optimization of selective inhibitors of protein arginine methyltransferase 5 by docking-based virtual screening[J]. Org Biomol Chem,2017,15(17):3648-3661. [百度学术]
Bonday ZQ,Cortez GS,Grogan MJ,et al. LLY-283,a potent and selective inhibitor of arginine methyltransferase 5,PRMT5,with antitumor activity[J]. ACS Med Chem Lett,2018,9(7):612-617. [百度学术]
Tatlock J H,Mcalpine I J,Tran-Dube M B,et al.Substituted nucleoside derivatives useful as anticancer agents:WO2016135582A1[P/OL].2016-09-01[2020-01-30].https://worldwide.espacenet.com/patent/search/family/055451515/publication/WO2016135582A1?called_by=epo.org&q=WO2016135582A1. [百度学术]
Wu T J,Brehmer D B,Beke L B,et al.Novel 6-6 bicyclic aromatic ring substituted nucleoside analogues for use as PRMT5 inhibitors: US2018243328A1[P/OL].2018-08-30[2020-01-30].http://www.freepatentsonline.com/y2018/0243328.html. [百度学术]
Wu T,Millar H,Gaffney D,et al.Abstract 4859:JNJ-64619178,a selective and pseudo-irreversible PRMT5 inhibitor with potent in vitro and in vivo activity,demonstrated in several lung cancer models[J].Cancer Res,2018,78(13S):4859-4859. [百度学术]
Lin H,Wang M,Zhang YW,et al. Discovery of potent and selective covalent protein arginine methyltransferase 5 (PRMT5) inhibitors[J]. ACS Med Chem Lett,2019,10(7):1033-1038. [百度学术]
Marjon K,Cameron MJ,Quang P,et al. MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis[J]. Cell Rep,2016,15(3):574-587. [百度学术]