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Zhejiang Da Xue Xue Bao Yi Xue Ban. 2020 Feb 25; 49(1): 1–19. Chinese. doi: 10.3785/j.issn.1008-9292.2020.02.21PMCID: PMC8800688PMID: 32621419 Language: Chinese | English 靶向Cullin-RING E3泛素连接酶的抗肿瘤策略及相关药物研发进展Targeting Cullin-RING E3 ligases for anti-cancer therapy: efforts on drug discoveryQing YU,1,2 Xiufang XIONG,1,2 and Yi SUN1,2,*Qing YU1 浙江大学医学院附属第二医院肿瘤研究所, 浙江 杭州 310009 2 浙江大学转化医学研究院, 浙江 杭州 310029 Find articles by Qing YUXiufang XIONG1 浙江大学医学院附属第二医院肿瘤研究所, 浙江 杭州 310009 2 浙江大学转化医学研究院, 浙江 杭州 310029 Find articles by Xiufang XIONGYi SUN1 浙江大学医学院附属第二医院肿瘤研究所, 浙江 杭州 310009 2 浙江大学转化医学研究院, 浙江 杭州 310029 Find articles by Yi SUNAuthor information Article notes Copyright and License information PMC Disclaimer 1 浙江大学医学院附属第二医院肿瘤研究所, 浙江 杭州 310009 2 浙江大学转化医学研究院, 浙江 杭州 310029 nc.ude.ujz@gniquy https://orcid.org/0000-0002-4519-4940第一作者：俞卿, 男, 博士研究生, 主要从事Cullin-RINGE3泛素连接酶小分子抑制剂的高通量筛选和研发; E-mail:; 通信作者：通讯作者, email: nc.ude.ujz@nusiy Received 2019 Dec 20; Accepted 2020 Jan 16.Copyright 版权所有©《浙江大学学报(医学版)》编辑部2020Copyright ©2020 Journal of Zhejiang University(Medical Sciences). All rights reserved.AbstractCullin-RING E3泛素连接酶（CRL）是泛素-蛋白酶体系统的重要组分，参与催化蛋白质的泛素化，促进随后的蛋白质降解，从而影响细胞周期、细胞凋亡、DNA复制、信号转导等多种细胞生理活动，且在多种肿瘤细胞中异常活化。以MLN4924为代表的拟素化抑制剂的成功研发有力地证实了CRL是可行的抗肿瘤靶点，具有很好的药物研发潜力。近年来，不断有新的研究通过高通量筛选、基于计算机辅助的虚拟筛选或基于结构的药物设计技术寻找特异的CRL抑制剂，但由于CRL复合物具有多种亚单位，呈蛋白-蛋白相互作用和多变的蛋白构象，缺乏典型的小分子药物结合位点等特性，其相关药物研发仍面临巨大挑战。截至目前，CRL小分子抑制剂主要以研究最为透彻的SCF泛素连接酶复合体的底物识别亚基F-box蛋白家族为靶点。此外，也发现数个通过靶向UBE2M-DCN1相互作用，特异性阻断CRL3/CRL1拟素化，从而抑制CRL3/CRL1泛素连接酶活性的小分子化合物。另一方面，也有CRL激动剂的报道，主要见于植物生长素吲哚乙酸和免疫调节性酰亚胺类药物。此外，靶蛋白水解嵌合体（PROTAC）是一项靶向蛋白-蛋白相互作用的新技术，其通过特异性小分子抑制剂链接一个CRL E3泛素连接酶来精确降解特定促癌靶蛋白，已成为近年来利用E3泛素连接酶设计抗肿瘤靶向药物的热点。 AbstractCullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery. Keywords: Ubiquitin-protein ligases, Antineoplastic/therapeutic, Cullin-RING E3 ligases, Enzyme inhibitors, Enzyme activators, Proteolysis-targeting chimera, Review人体内每时每刻都有新的蛋白合成，参与各种生理进程、维持生命活动；同时，也不断有错误折叠的、破损的或完成使命的蛋白需要清除。成千上万种蛋白在合成与降解之间需要达到精准平衡，而泛素-蛋白酶体系统就是人体内维持蛋白稳态的主要途径 [ 1] 。泛素分子通过泛素-蛋白酶体系统共价结合到底物蛋白的特定赖氨酸残基上，这一过程称为泛素化过程。经典的泛素化过程是一个由三类酶催化完成的级联反应 [ 2] 。首先，E1泛素激活酶将泛素分子激活，并传递给E2泛素偶联酶，这一环节需要ATP水解反应的参与；接着E2泛素偶联酶将泛素传递给E3泛素连接酶，后者识别并募集特定的底物蛋白，促使泛素羧基端的甘氨酸与底物的特定赖氨酸形成异肽键，最终完成泛素到底物的传递 [ 2] 。泛素分子本身含有7个赖氨酸残基，多个泛素分子可以通过某个特定的赖氨酸残基相连形成长链。因此，随着上述泛素化反应的级联进行，底物蛋白上形成多个泛素分子相连的泛素长链，从而完成典型的泛素化修饰 [ 2] 。被多聚泛素化修饰的底物蛋白最终被26S蛋白酶体识别而降解 [ 2] 。 人类基因组编码2种E1泛素激活酶、38种E2泛素偶联酶和超过600种E3泛素连接酶 [ 3- 4] 。E3泛素连接酶可根据其结构特点和作用机制分为RING(really interesting new gene)E3、HECT(homologous to the E6AP carboxyl terminus)E3和RBR(RING-in-between-RING)E3三大类 [ 4- 6] 。RING E3家族是目前研究最多的E3泛素连接酶，而其中包含RING结构域和Cullin蛋白家族的Cullin-RING E3泛素连接酶(Cullin-RING E3 ligase, CRL)是目前已发现的最大的E3泛素连接酶家族 [ 5] 。人类细胞经泛素-蛋白酶体系统降解的蛋白中约20 %蛋白的泛素化是由CRL催化的 [ 7] 。 由于泛素化过程与几乎所有细胞生理活动相关，泛素-蛋白酶体系统的异常调控会导致肿瘤等各种疾病的发生。硼替佐米(bortezomib，商品名Velcade)是第一个被美国食品药品监督管理局批准用于治疗多发性骨髓瘤的蛋白酶体抑制剂 [ 8] 。硼替佐米及后续衍生物的研发让人们看到了泛素-蛋白酶体系统作为抗肿瘤靶点的潜力，其中CRL被认为是更为理想的抗肿瘤药物靶点 [ 3, 9] 。本文从CRL的结构和功能出发，阐述CRL可作为抗肿瘤药物靶点的依据，并对CRL为靶点的抗肿瘤药物研发进展作一综述。 1 Cullin-RING E3泛素连接酶的结构及功能CRL是一个多元蛋白复合体，通常包含Cullin家族蛋白、RING蛋白、衔接蛋白和底物识别亚基四个组分 [ 4] 。Cullin家族蛋白构成CRL的骨架，Cullin家族蛋白包含Cullin-1、Cullin-2、Cullin-3、Cullin-4A、Cullin-4B、Cullin-5、Cullin-7和Cullin-9等八个经典成员，分别构成CRL1、CRL2、CRL3、CRL4A、CRL4B、CRL5、CRL7和CRL9复合体。Cullin蛋白的羧基端通过结合RING蛋白募集和绑定E2泛素偶联酶的催化中心 [ 4] 。人类RING蛋白包括RBX1(也称ROC1)和RBX2(也称ROC2/SAG)两个成员 [ 4] 。CUL1可通过RBX1或RBX2招募E2，CUL2、CUL3、CUL4A、CUL4B、CUL7和CUL9通过RBX1招募E2，CUL5通过RBX2招募E2 [ 3, 10] 。Cullin家族成员各蛋白的氨基端通过不同的衔接蛋白/底物识别亚基完成特定底物的识别和募集 [ 5] 。目前已知人类基因组表达四种衔接蛋白，其中S期激酶相关蛋白(S-phase kinase-associated protein, SKP)1结合CUL1/CUL7，转录延伸因子B/C(transcription elongation factor B polypeptide 2/1, Tceb2/1, 也叫Elongin B/C)结合CUL2/CUL5，DNA损伤结合蛋白1(DNA damage-binding protein 1, DDB1)结合CUL4A/CUL4B [ 5] 。目前发现的底物识别亚基包括69个结合CRL1的F-box蛋白 [ 11] ，超过80个结合CRL2/CRL5的VHL-box或SOCS-box蛋白 [ 12] ，约180个结合CRL3的BTB蛋白 [ 13] ，及90个结合CRL4A/CRL4B的DCAF蛋白 [ 14] 。这种多组分、高选择性的组合方式构成了400种以上CRL，用于完成数以千计底物蛋白的泛素化和降解 [ 15] 。CRL的活性受拟素化修饰(neddylation修饰)的调控，即只有经过拟素化修饰后，CRL才具有泛素连接酶活性 [ 16] 。拟素化修饰过程与泛素化相似，也是由E1、E2和E3序贯催化的级联反应，将一个NEDD8蛋白分子传递到Cullin蛋白的特定赖氨酸残基上 [ 16] 。目前发现了一种NEDD8 E1激活酶(NAE)和两种NEDD8 E2偶联酶(UBE2M和UBE2F), 数十种E3连接酶, 包括RING蛋白RBX1和RBX2。Cullin家族蛋白是目前发现的最主要的生理性拟素化修饰底物 [ 16] ( 图 1)。 Open in a separate window图1泛素-蛋白酶体系统概况和Cullin-RING E3泛素连接酶的拟素化激活 泛素-蛋白酶体系统通过对底物的泛素化和蛋白酶体降解途径完成对底物蛋白的降解，典型的泛素化过程是一个由E1泛素激活酶、E2泛素偶联酶和Cullin-RING E3泛素连接酶(CRL)序贯催化的级联反应.CRL是选择和识别底物的关键因子，其活性受拟素化修饰调控.拟素化修饰过程与泛素化类似，也由E1、E2和E3三种酶序贯调控，Cullin家族蛋白是已知最主要的拟素化修饰底物.N8：NEDD8；SRS：底物识别亚基；Adaptor：衔接蛋白；CT：羧基末端；NT：氨基末端; P:磷酸化位点；K48:以第48位赖氨酸相连的泛素化. CRL1或称SCF(SKP1-Cullin 1-F box protein)，是目前研究最透彻的CRL [ 17- 18] 。在SCF复合体中，CUL1的氨基端结合一个衔接蛋白SKP1，后者结合一个底物识别亚基F-box蛋白的F-box结构域 [ 19] 。F-box结构域是一段由约40个氨基酸组成的保守结构域，最先发现于周期蛋白F(CCNF，也称FBXO1) [ 19] 。目前已发现的69种人类F-box蛋白，根据蛋白相互作用区域可分为10种包含WD40结构域(F-box and WD repeat domain containing proteins, FBXW)、21种包含亮氨酸串联重复结构域(F-box and leucine-rich repeat proteins, FBXL)和38种包含其他多样性的结构域(FBXO proteins)三大家族 [ 11] 。目前研究最多的F-box蛋白是SKP2、FBXW7和β-TrCP(β-transducin repeats-containing proteins)。F-box蛋白通过不同的作用区域与一种或多种不同的底物结合 [ 20] 。SCF泛素连接酶复合体的经典底物包括但不限于c-Myc、c-Jun、β-catenin、Notch等促癌蛋白，p21、p27、NF1等抑癌蛋白，周期蛋白D/E等细胞周期相关蛋白，MCL-1等凋亡相关蛋白，以及IκBα、DEPTOR等信号调节因子 [ 21- 25] 。CRL2复合体由CUL2、RBX1、衔接蛋白Elongin B/C和底物识别亚基VHL组成 [ 26] 。CRL2的底物包括缺氧诱导因子1α (hypoxia inducible factor-1 alpha, HIF-1α)、β 2肾上腺素能受体(β 2-adrenergic receptor, β 2-AR)、非典型蛋白激酶λ(atypical PKCλ, aPKCλ)及RNA聚合酶Ⅱ的第7大亚基(hsRPB7)等 [ 27- 30] 。其中CRL2复合体对HIF-1α的降解对于细胞缺氧适应具有重要意义，相关通路的失调可能引起炎症或肿瘤的发生 [ 31] 。CRL3复合体由CUL3、RBX1和底物识别亚基BTB组成。CRL3的底物主要包括核因子E2相关因子(NF-E2 related factor 2，Nrf2)、核因子κB抑制蛋白激酶亚基(IKKβ)和死亡结构域相关蛋白等 [ 32- 34] 。其中转录因子Nrf2是细胞在氧化应激反应中的关键调节因子，可被含有BTB结构域的Keap1蛋白识别从而被CRL3复合体募集而降解 [ 35] 。CRL4复合体由CUL4A或CUL4B、RBX1、衔接蛋白DDB1及底物识别亚基DDB1和CUL4相关因子(DDB1- and CUL4-associated factor, DCAF)组成。CRL4的底物主要包括DNA损伤应答蛋白DNA复制因子和DNA损伤修复基因编码蛋白，以及组蛋白甲基化相关蛋白PR-Set7/Set8和WD重复域结构蛋白5等 [ 24, 36- 38] 。CRL5复合体由CUL5、RBX2、衔接蛋白Elongin B/C及底物识别亚基SOCS组成。本团队2017年发表的一项研究发现CRL5可通过非典型的第11位赖氨酸泛素化降解促凋亡蛋白NOXA，揭示了NOXA是CRL5的一种底物蛋白 [ 39] 。而在此之前，已发现的CRL5底物主要是一些抗病毒相关蛋白，因部分病毒可通过“挟持”宿主的CRL5而促进其降解特定宿主蛋白。如HIV-1蛋白Vif(viral infectivity factor)可通过CRL5降解抗病毒因子APOBEC3G [ 40] 、卡波西肉瘤相关疱疹病毒(KSHV)蛋白LANA(latency-associated nuclear antigen)可通过CRL5降解VHL和p53等 [ 41] 。CRL7复合体由CUL7、RBX1、衔接蛋白SKP1和底物识别亚基FBXW8组成。相对于CRL1~CRL5，CRL7和CRL9因具有一些特殊属性而被认为是非典型的CRL。CUL7包含1689个氨基酸，其大小是典型Cullin蛋白(即CUL1~CUL5)的两倍，含有更多的蛋白-蛋白相互作用区域，因而可能具有非蛋白水解相关活性等其他生物化学功能 [ 42] 。另外，CRL7复合体虽然从组成上与CRL1很接近，但从已知的研究来看CUL7仅对SKP1-FBXW8具有高度选择性 [ 42] ，而这其中的分子学基础尚不明确。目前已知的CRL7底物主要为胰岛素受体底物1(insulin receptor substrate 1, IRS-1)，后者是一个正向调节PI3K/AKT通路的信号分子 [ 43] 。CUL9(也称PARC，p53-associated parkin-like cytoplasmic protein)与CUL7高度同源，同时还包含类似于RBR E3 Parkin的Ring-IBR-Ring结构域 [ 15] 。早期研究发现CUL9可能参与调节p53的亚细胞定位，但其分子机制尚不明确 [ 44] 。一项独立研究表明CUL9结合RBX1并可被拟素化，且可与CUL7形成非典型的CRL复合体 [ 10] 。有关CRL9的其他组分和主要底物尚不清楚( 图 2)。 Open in a separate window图2Cullin-RING E3泛素连接酶的构成及已知底物 2 Cullin-RING E3泛素连接酶作为抗肿瘤分子靶点的依据以硼替佐米为代表的蛋白酶体抑制剂的成功上市，证明了靶向泛素-蛋白酶体系统的抗肿瘤策略的可行性 [ 8] ，但由于硼替佐米广泛抑制所有依赖于26S蛋白酶体的蛋白降解途径，不可避免地会损伤正常细胞，产生不易控制的药物不良反应 [ 45] 。泛素化过程中，处于蛋白酶体上游的CRL负责识别和招募特定底物，决定底物特异性，因此以CRL为靶点的抗肿瘤药物理论上应具有更好的选择性和更低的不良反应 [ 45] 。实际上，由于CRL在细胞周期调控、细胞凋亡、DNA复制、信号转导等多种细胞生理活动中都发挥至关重要的作用，CRL的异常表达或失调会引起肿瘤等多种疾病的发生 [ 46] 。因此，CRL作为抗肿瘤药物靶点具有理论基础和可行的治疗窗口。 CRL的一些组分被证实是促癌基因，如SKP2、Cul4A及RING蛋白RBX2都被发现在多种肿瘤中频繁扩增和过表达 [ 47- 48] ；同时，CRL所调控的底物中也包括p21、p27、神经纤维瘤蛋白1(NF1)等抑癌蛋白 [ 21- 22] 。因此，通过小分子化合物抑制CRL活性，诱导抑癌蛋白积累，理论上是一种直接而有效的抗肿瘤策略( 图 3A)。事实上，小分子抑制剂MLN4924(商品名Pevonedistat)的研发证实了上述策略的可行性 [ 7] 。MLN4924是一个特异性的NAE抑制剂，可通过共价结合NEDD8，阻断NAE催化活性，从而阻断所有Cullin蛋白的拟素化修饰。由于CRL的活性依赖于拟素化修饰，MLN4924可广泛抑制肿瘤细胞中CRL活性，引起p21、p27、DEPTOR、NOXA、BIM等底物积累，从而诱导肿瘤细胞周期阻滞，诱导凋亡。MLN4924可在体外抑制多种肿瘤细胞系的增殖，包括肺癌、卵巢癌、乳腺癌、白血病、淋巴瘤、黑色素瘤、骨髓瘤和尤因肉瘤细胞等，且在多种移植瘤和转基因小鼠模型中均表现出较好的抗肿瘤疗效 [ 9] 。目前，MLN4924已完成多项Ⅰ/Ⅱ期临床试验，并表现出一定的抗多种肿瘤的疗效 [ 9] 。然而，由于MLN4924广泛抑制所有Cullin的拟素化修饰，大量CRL底物都会受到MLN4924的影响，同时MLN4924对非Cullin蛋白的拟素化修饰的潜在影响尚不清楚，因此MLN4924也不可避免地会产生较多的不良反应 [ 9, 49] 。此外，已有研究者观察到肿瘤细胞对MLN4924发生耐药的现象 [ 50- 51] 及其他拟素化非依赖性的活性 [ 52] 。因此，靶向特定CRL的小分子抑制剂可能会有更好的选择性和更少的不良反应。有意思的是，抑制CRL并不适用于所有肿瘤。在已知CRL底物蛋白中，除了上述典型的抑癌蛋白，也有c-Myc、c-Jun、β-TrCP、Notch等促癌蛋白。另一方面，研究表明部分CRL的组分(如FBXW7或VHL)在许多细胞中发挥抑癌作用，且在许多肿瘤中出现突变或失活 [ 53] 。因此，对于特定的肿瘤细胞，以CRL为靶点的抗肿瘤药物也可以是CRL的激动剂，即通过上调特定CRL的活性，或通过促进特定CRL与特定底物蛋白的结合，诱导促癌蛋白降解( 图 3B)。近年来，靶蛋白水解嵌合体(proteolysis-targeting chimera，PROTAC)技术的发明和应用证明了上述策略的可行性和潜在的应用价值。 表 1总结了目前已报道的CRL抑制剂和激动剂的靶蛋白、作用位点、对细胞底物蛋白的影响及研发途径等相关信息，供读者概览。以下将分别介绍Cullin-RING E3泛素连接酶抑制剂和激动剂。 Open in a separate window图3抑制和激活Cullin-RING E3泛素连接酶(CRL)的抗肿瘤策略 抑制CRL的抗肿瘤策略:通过小分子抑制剂抑制特定CRL活性或阻断其与底物结合，引起抑癌蛋白积累，发挥抗肿瘤作用; 激活CRL的抗肿瘤策略：通过CRL激活剂激活特定CRL，增强其与底物的结合，促进特定底物如促癌蛋白的降解，达到抗肿瘤的目的. 表1 Cullin-RING E3泛素连接酶抑制剂和激动剂 Table 1 Reported inhibitors and activators of Cullin-RING E3 ligases 名称 靶蛋白 影响的PPI 对细胞中主要底物的影响 研发途径 其他说明 参考文献 CRL E3抑制剂 Compound A SKP2 SKP2:SKP1 p27、p21、p53蛋白积累 高通量筛选 在多发性骨髓瘤细胞中可增加细胞对硼替佐米敏感性 [ 54] C1、C2、C16、C20 SKP2 SKP2:Cks1 p27蛋白积累 基于配体分子的虚拟筛选 首次通过虚拟筛选获得阻断SKP2-底物相互作用的小分子 [ 55] Compound #25 SKP2 SKP2:SKP1 p27、p21、Notch1蛋白积累 基于受体结构的虚拟筛选 在前列腺癌细胞系PC3中可降低肿瘤干细胞比例 [ 56] NSC689857、NSC681152 SKP2 SKP2:Cks1 p27蛋白积累 基于AlphaScreen的高通量筛选 同时具有络氨酸激酶抑制活性 [ 57] Eroflorin β-TrCP β-TrCP:PDCD4 PDCD4蛋白积累 基于荧光素酶报告系统的高通量筛选 天然产物 [ 58] SCF-I2 Cdc4(酵母FBXW7) Cdc4:Sic1 Sic1蛋白泛素化水平降低 基于荧光偏振的高通量筛选(酵母) 第一个特异性靶向WD40结构域的小分子变构抑制剂 [ 59] Suramin CUL1 CUL1:CDC34 p27、CDT1蛋白积累 基于FRET的高通量筛选 天然产物；基于FRET的K48双泛素化报告系统 [ 60] SMER3 Met30(酵母F-box) Met30:Skp1 Met4蛋白泛素化水平降低 基于化学遗传学的高通量筛选(酵母) 小分子雷帕霉素增强剂 [ 61] NAcM-HIT、NAcM-OPT等 DCN1 DCN1:UBE2M p27、Nrf2蛋白积累 基于TR-FRET的高通量筛选 特异性抑制CRL3/CRL1活性 [ 62- 65] DI-591、DI-404 DCN1 DCN1:UBE2M Nrf2蛋白积累 基于结构的药物设计 特异性抑制CRL3活性 [ 66- 67] WS-383 DCN1 DCN1:UBE2M p21、p27、Nrf2蛋白积累 基于HTRF的高通量筛选 特异性抑制CRL3/CRL1活性 [ 68] DC-2、DC-1 DCN1 DCN1:UBE2M Nrf2蛋白积累 基于HTRF和荧光偏振的高通量筛选 特异性抑制CRL3活性 [ 69] CRL E3激动剂 Auxin TIR1(植物) TIR1:Aux/IAA 促进Aux/IAA降解 非创新药研发 — [ 70- 71] Thalidomide、Lenalidomide、Pomalidomide CRL4-CRBN CRBN:IKZF1/3 促进IKZF1和IKZF3降解 非创新药研发 — [ 72- 75] NRX-1532、NRX-252262 β-catenin/ β-TrCP β-catenin:β-TrCP 促进S37A突变β-catenin降解 基于荧光偏振的高通量筛选 — [ 76] Oridonin FBXW7 尚不明确 促进c-Myc降解 非创新药研发 — [ 77] Open in a separate window“—”无相关资料.PPI：蛋白-蛋白相互作用; FRET：荧光共振能量转移; TR-FRET：时间分辨荧光共振能量转移; HTRF：均相时间分辨荧光. 3 Cullin-RING E3泛素连接酶抑制剂近年来，不断有新的研究通过高通量筛选、基于计算机辅助的虚拟筛选或基于结构的药物设计技术寻找特异的CRL抑制剂。截至目前，相关小分子抑制剂主要以研究最为透彻的SCF泛素连接酶复合体的底物识别亚基F-box蛋白家族为靶点(如SKP2、β-TrCP和FBXW7等)。此外，近年研究发现，通过靶向UBE2M-DCN1相互作用可特异性阻断CRL3/CRL1拟素化，从而抑制CRL3/CRL1泛素连接酶活性。 3.1 SKP2抑制剂SKP2是目前研究最为深入的F-box蛋白之一。大量研究表明 SKP2 是一个促癌基因，通过介导多种抑癌底物蛋白的泛素化降解过程，在多种肿瘤的发生和发展过程中发挥重要作用 [ 78- 80] 。已知的SKP2底物包括p27、p21、p57、TOB1、RASSF1、FOXO1和RBL2等 [ 81] 。淋巴瘤、前列腺癌和乳腺癌等多种肿瘤细胞中均发现SKP2异常高表达 [ 82- 84] 。因此SKP2是一个极具潜力的抗肿瘤靶点。 Chen等 [ 54] 通过基于体外p27泛素化反应的高通量筛选模型，发现了一个小分子化合物Compound A( 图 4A)。Compound A可特异性地引起p27、p21和p53等底物的积累，导致G 1/S期阻滞，诱导细胞凋亡。免疫共沉淀实验显示Compound A可阻断SKP2与SCF复合体的结合，提示SKP2可能是Compound A的作用靶点。Wu等 [ 55] 根据已发表的SKP2-Cks1-p27晶体结构上的一个蛋白-蛋白相互作用区域，通过计算机辅助虚拟筛选技术筛选到了一类可干扰SKP2-Cks1与底物p27结合的小分子抑制剂。这类化合物(C1、C2、C16和C20)能在多种肿瘤细胞中诱导p27蛋白积累，引起细胞周期阻滞( 图 4B)。这类化合物的发现证明了基于结构设计化合物来阻断E3和底物相互作用的可行性 [ 55] 。Chan等 [ 56] 以SCF SKP2复合体晶体结构(数据库编号:2AST)上的SKP2-SKP1相互作用区域为靶点，通过计算机辅助高通量虚拟筛选技术找到了一系列潜在的SKP2抑制剂。其中，化合物Compound-25可结合SKP2，阻断SKP2-SKP1相互作用，抑制SCF SKP2泛素连接酶的活性，并在一些肿瘤细胞系及荷瘤小鼠模型中表现出抗细胞增殖作用( 图 4A)。Compound-25还能通过抑制SKP2活性，降低肿瘤干细胞的比例，减弱肿瘤干细胞的成球能力，提示SKP2可能与维持肿瘤干细胞的干性有关 [ 56] 。Dana等 [ 57] 基于AlphaScreen技术设计了一个检测体外Cks1-SKP2相互作用的高通量筛选模型，通过对筛选所得化合物进行验证、构效关系研究和药物化学优化，得到两个小分子化合物NSC689857和NSC681152。这两个小分子可干扰重组蛋白Cks1-SKP2的相互作用，IC 50分别为(36±6.2)μmol/L和(75.6±6.8)μmol/L，均可在体外阻断p27 Kip1的泛素化(IC 50分别约为30 μmol/L和80 μmol/L)。尽管这类化合物在肿瘤细胞中的靶点尚未证实，该研究为寻找SKP2小分子抑制剂提供了一种可行的高通量筛选方法( 图 4B)。Elizabeth等 [ 85] 以前列腺癌LNCaP细胞的内源性核内p27蛋白水平为表型，通过高通量筛选在7000多个化合物中找到SMIP001和SMIP004这两个阳性化合物。其中，SMIP004可降低细胞内SKP2水平，促使p27积累，有效抑制前列腺癌LNCaP细胞的增殖(半数致死浓度为1.09 μmol/L)。该团队在后续研究中发现SMIP004可能通过诱导线粒体功能异常所致的氧化应激，间接引起SKP2的表达水平下调，其具体机制仍有待进一步阐明 [ 86] 。Misook等 [ 87] 拟通过基于亲和力的微珠筛选系统，采用自主合成的类肽分子库，筛选阻断SKP2-SKP1相互作用的化合物，却意外地找到了一个可特异性结合SKP2而非SKP1的化合物M1。结果显示，M1并未结合到SKP2-SKP1相互作用区域，而是特异性地干扰了SKP2与p300(一个具有乙酰转移酶活性的转录辅助激活因子)的结合，从而促进p300介导的p53蛋白乙酰化，促进p53积累，诱导p53介导的细胞凋亡，从而发挥抗肿瘤作用 [ 87] 。尽管化合物M1不是直接阻断SKP2泛素连接酶活性的抑制剂，但该研究提供了以下重要信息：①该团队采用自主合成的一系列含大环结构的类肽作为潜在配体，这类较大的分子可以更好地覆盖蛋白-蛋白相互作用区域，类似方法可用于筛选其他CRL抑制剂；②M1-SKP2的晶体结构解析可能为高选择性SKP2抑制剂的设计提供线索；③SKP2的多重作用机制或可被用于设计新的单药或多药联用的抗肿瘤治疗方案。 Open in a separate window图4靶向Cullin-RING E3泛素连接酶的小分子抑制剂 A：靶向SKP1-SKP2相互作用的小分子抑制剂；B：靶向SKP2-Cks1-p27的小分子抑制剂；C：靶向β-TrCP的小分子抑制剂；D：靶向WD40结构域的小分子抑制剂；E：靶向CUL1的抑制剂；F：靶向Met30的小分子抑制剂. 3.2 β-TrCP抑制剂目前已发现的β-TrCP(包括β-TrCP1和β-TrCP2)的底物涉及细胞各种生理过程，其中包括Cdc25A、Emi1(也称FBXO5)、周期蛋白D1等细胞周期相关蛋白，Snail、ECMFn、Twist等细胞迁移相关蛋白，及MCL-1、PDCD4(programmed cell death 4)、Pro-caspase-3等细胞凋亡相关蛋白 [ 88] 。由于这些底物中既有β-catenin和CDC25A等促癌蛋白，也有IκB和PDCD4等抑癌蛋白 [ 47] ，因此靶向β-TrCP的抑制剂在不同组织来源的肿瘤细胞、不同的遗传背景等条件下可能会有不同的结局。 Johanna等 [ 58] 基于荧光素酶的高通量筛选模型，从近13.5万个天然产物中找到一个阳性化合物密花棉毛叶菊素醋酸酯(erioflorin)( 图 4C)。免疫共沉淀结果表明，erioflorin可影响β-TrCP和其底物PCDC4蛋白的结合，阻断PCDC4的降解。PDCD4蛋白是一个肿瘤抑制因子，在多种肿瘤细胞中表现为异常低表达 [ 89] 。Erioflorin促进了PCDC4蛋白的积累，同时还导致肿瘤相关转录因子AP-1和NF-κB下调。2.5~5 μmol/L的erioflorin可明显抑制结肠癌RKO细胞、乳腺癌MCF7细胞和宫颈癌Hela细胞增殖。 3.3 FBXW7抑制剂大量研究认为FBXW7是一个肿瘤抑制因子，参与降解c-Myc、c-Jun、周期蛋白E、mTOR等多种关键促癌蛋白 [ 90] 。在许多肿瘤中存在 FBXW7基因丢失或突变 [ 91- 92] 。在一些骨髓瘤细胞中，FBXW7通过降解p100(后者可抑制NF-κB信号通路)，从而促进肿瘤细胞增殖 [ 93] 。此外，有研究表明下调FBXW7可增强肿瘤干细胞对化疗的敏感性 [ 9] 。因此，在特定条件下，通过抑制FBXW7可发挥抗肿瘤作用。 Stephen等 [ 59] 通过高通量筛选得双平面二元羧酸化合物SCF-I2，可有效抑制酵母来源的F-box蛋白Cdc4(对应人FBXW7蛋白)，阻断SCF Cdc4介导的底物泛素化( 图 4D)。共结晶结构解析揭示SCF-I2可结合Cdc4的WD40结构域，干扰Cdc4与底物的结合。SCF-I2因而成为第一个特异性靶向WD40结构域的小分子抑制剂。染料木素(genistein)是从豆科植物根部提取的一种异黄酮类天然化合物，对多种人肿瘤细胞表现出抗肿瘤活性 [ 94] 。Ma等 [ 94] 发现在人前列腺癌细胞中genistein可通过抑制miR-223，下调FBXW7的表达，从而抑制细胞增殖。 3.4 其他SCF抑制剂笔者参与的一项研究通过自主建立基于荧光共振能量转移(FRET)的体外K48泛素化报告系统，高通量筛选以RBX1/CUL1 E3泛素连接酶为靶点的小分子抑制剂 [ 60] 。在预筛选中，天然产物Suramin表现出了显著的抑制作用( 图 4E)。Suramin最初被用于治疗非洲锥虫病、盘尾丝虫病等，且其抗肿瘤效果已有相关报道，但作用机制尚不明确 [ 95] 。该研究通过免疫共沉淀实验进一步证实Suramin可阻断CUL1与泛素偶联酶Cdc34的相互作用。计算机模拟对接实验提示Suramin可能通过竞争性结合CUL1羧基端的一个保守区域而发挥抑制作用 [ 60] 。Mariam等 [ 61] 通过基于酵母系统的高通量筛选模型发现了一个特异性的SCF Met30小分子抑制剂SMER3( 图 4F)。Met30是酵母的一种F-box蛋白。SMER3通过直接结合Met30，阻断Met30与Skp1的相互作用，干扰SCF复合体的形成，从而抑制SCF Met30的泛素连接酶活性。Maryna等 [ 96] 巧妙设计了一系列泛素分子的突变体(ubiquitin variants，UBv)，这些UBv可以特异性地结合到SKP1-F-box蛋白的相互作用区域，干扰SKP1-F-box-CUL1 E3连接酶复合体的装配，从而抑制SCF的连接酶活性。此外，在真核细胞中过表达这类UBv可有效抑制细胞中相应SCF的连接酶活性 [ 96] 。虽然这些UBv并不能直接作为药物应用，但可以作为一种检测潜在小分子抑制剂的工具，也可为设计特异性的小分子抑制剂提供结构基础和新的思路。 3.5 靶向UBE2M-DCN1相互作用的特异性CRL3/CRL1抑制剂DCN1(defective in cullin neddylation 1)是最早在线虫和酿酒酵母细胞中发现的一种保守的拟素化相关蛋白 [ 97] 。研究发现DCN1可同时结合NEDD8 E2偶联酶和cullin，稳定CRL复合体的催化中心，从而促进cullin的拟素化修饰，是一种协同E3连接酶(Co-E3) [ 98] ( 图 5)。哺乳动物细胞表达五种DCN同源蛋白(DCN1-like proteins, DCNL)，即DCNL1~DCNL5。这些DCNL蛋白在羧基端都有一段保守的拟素化增强域 [ 62] 。目前有关人DCNL调控的下游通路尚未完全阐明，但已发现 DCN1 基因在多种鳞状细胞癌中高表达，且与病因特异性生存率呈负相关 [ 99- 100] ，因此DCN1/DCNL本身即是一个潜在的抗肿瘤靶点。近年来，结构生物学研究揭示了DCN1与NEDD8 E2偶联酶UBE2M和CRL1复合体相互作用的结构基础，发现DCN1与UBE2M的乙酰化氨基端紧密结合是发挥其“协同E3”作用的必要前提 [ 98] 。而UBE2M-DCN1的相互作用区域具有潜在的小分子化合物的结合口袋 [ 62] 。2017年，Guy和Schulman团队首先通过基于时间分辨荧光共振能量转移技术(TR-FRET)的高通量筛选，从60万个小分子化合物库中筛选并优化得到一类靶向UBE2M-DCN1相互作用的哌啶尿素类小分子抑制剂NAcM-HIT和NAcM-OPT [ 62] ( 图 5A)。在细胞和动物实验中，这类哌啶尿素类小分子均能有效阻断UBE2M-DCN1相互作用，且特异性抑制CUL1和CUL3拟素化，导致相应底物p27和Nrf2积累 [ 62- 64] 。同年，Wang和我们团队基于UBE2M-DCN1的结构设计了特异性靶向蛋白相互作用区域的小分子化合物DI-591 [ 66] ( 图 5B)。DI-591及衍生物DI-404 [ 67] ( 图 5B)不仅特异性地阻断UBE2M-DCN1相互作用，且在细胞水平特异性抑制CUL3的拟素化，诱导Nrf2积累。最新的几项研究又相继报道了数种不同类型的UBE2M-DCN1抑制剂，包括特异性抑制CUL3/CUL1的WS-383 [ 68] 和一系列吡唑-吡啶类化合物Compound 1和Compound 27 [ 65] ( 图 5A)，以及特异性抑制CUL3的DC-2及其衍生物DC-1 [ 69] ( 图 5B)。上述抑制剂的发现建立了以靶向UBE2M-DCN1相互作用来特异性抑制CUL3/CUL1拟素化的新策略。此外，进一步探究DCN1/DCNL家族蛋白与不同cullin蛋白成员之间的联系，将有可能为设计靶向不同CRL复合体的小分子抑制剂提供新的思路和线索。 Open in a separate window图5靶向UBE2M-DCN1抑制CRL3/CRL1的小分子抑制剂 A：特异性抑制CUL3和CUL1拟素化修饰，引起CRL3和CRL1失活的小分子化合物；B：特异性抑制CUL3拟素化修饰引起CRL3失活的小分子化合物.AcN：乙酰化修饰的氨基端. 4 Cullin-RING E3泛素连接酶激动剂4.1 植物生长素近年研究发现植物系统中有一类天然的E3泛素连接酶激活剂。植物生长素吲哚乙酸是广泛存在于植物中的一种重要的生长激素，参与植物细胞的各种生理活动 [ 5] 。研究发现一种植物细胞的F-box蛋白转运抑制反应蛋白1(transport inhibitor response 1，TIR1)是吲哚乙酸的受体分子 [ 70] 。吲哚乙酸可促进SCF TIR1泛素连接酶对转录抑制因子生长素响应蛋白(Aux/IAA)家族的泛素化和降解，从而调节下游通路 [ 101] 。结构生物学研究发现，吲哚乙酸通过物理性地占据TIR1和底物蛋白之间的特定空缺区域，像“分子胶水”一样加强了TIR1与底物蛋白之间的相互作用，从而在几乎不改变整体构象的情况下，上调TIR1的活性，促进了底物蛋白的降解 [ 71] ( 图 6A)。除了吲哚乙酸以外，目前还发现茉莉酮酸酯、赤霉素、水杨酸和独角金内酯这四种植物激素，与CRL复合体均相关 [ 5] 。这类天然泛素连接酶激动剂的发现极大拓宽了人们对CRL调控真核细胞生理活动的认识，也为研发CRL激动剂提供了新的思路。 Open in a separate window图6Cullin-RING E3泛素连接酶激活剂的“分子胶水”作用原理图 A：植物系统中以吲哚乙酶(auxin)为代表的天然CRL E3激活剂的作用模式图；B：以CRL4-DDB1 E3泛素连接酶复合体的底物识别亚基CRBN为靶点的免疫调节性酰亚胺类药物(IMiD)作用原理图.Aux/IAA:转录抑制因子生长素响应蛋白；DDB1:DNA损伤结合蛋白1；IKZF1/3:Ikaros家族锌指蛋白1/3. 4.2 免疫调节性酰亚胺类药物随着相关机制的不断阐明，免疫调节性酰亚胺类药物(immunomodulatory imide drugs, IMiD)沙利度胺及其衍生物来那度胺和泊马度胺成为靶向CRL激动剂的一类代表药物。沙利度胺曾因严重的致畸作用而被停用，后被重新用于治疗多发性骨髓瘤和麻风病，但其作用靶点一直未被阐明 [ 102] 。Ito等 [ 72] 发现沙利度胺的主要靶点为CUL4-DDB1 E3泛素连接酶复合体的一个底物识别亚基cereblon(CRBN)。后续研究发现沙利度胺可促进CRBN招募和降解新的底物Ikaros家族锌指蛋白1和3(Ikaros family zinc finger protein 1/3, IKZF1/3) [ 73] 。免疫调节性酰亚胺类药物都有一个戊二酰亚胺环，连着一个可变的邻苯二甲酰亚胺。结构生物学研究表明，免疫调节性酰亚胺类药物可通过戊二酰亚胺环与CRBN紧密结合，而通过邻苯二甲酰亚胺结构上的微小差异，与不同的底物相结合 [ 74- 75] 。与吲哚乙酸非常相似，免疫调节性酰亚胺类药物从空间上弥补了CRL和底物之间的空隙，延展了蛋白与蛋白的相互作用区域，从而像“分子胶水”一样促进CRBN与底物之间的相互作用，底物被降解( 图 6B)。免疫调节性酰亚胺类药物的发现为研发新型CRL激动剂提供了可供参考的分子基础。 4.3 靶向β-catenin Ser37突变的CRL激动剂β-catenin是Wnt信号通路里的经典效应蛋白，其降解主要由SCF β-TrCP介导，但肿瘤细胞中β-catenin的E3结合位点上高频出现磷酸化位点(Ser37)的突变，可能导致β-catenin在多种肿瘤中异常积累 [ 103] 。美国Nurix公司的一项最新研究通过基于荧光偏振技术设计的高通量筛选模型，寻找能增强β-TrCP/SKP1复合体和Ser37突变的β-catenin之间相互作用的小分子化合物 [ 76] 。通过35万个化合物的高通量筛选后，他们得到了以NRX-1532为代表的一类具有“分子胶水”性质的小分子化合物。晶体结构解析证实这类化合物可从空间上填补β-catenin因Ser37突变而产生的空隙，从而模拟Ser37磷酸化的β-catenin，促进β-catenin和β-TrCP/SKP1的结合。研究者基于结构的药物设计和药物化学优化得到NRX-252262，较NRX-1532的活性提高了近10 000倍, 半数有效浓度(50 % effective concentration，EC 50)约为3.8 nmol/L。由于Ser37突变可能会影响β-catenin的另一个重要位点——Ser33的磷酸化状态进而影响β-catenin的泛素化水平，这类化合物在含有β-catenin S37A纯合子突变的卵巢癌细胞系TOV-112D中的活性不如预期 [ 76] 。但这一研究仍为设计新型的CRL激动剂提供了可行的筛选和优化方法。 4.4 二萜类天然化合物冬凌草甲素(oridonin)是一种二萜类天然化合物，具有多种抗肿瘤活性 [ 104] 。研究发现，在人髓性白血病K562细胞系裸鼠移植瘤模型上，冬凌草甲素可上调FBXW7的表达和GSK-3的活性，降解过剩的c-Myc，从而诱导肿瘤细胞凋亡，抑制肿瘤生长 [ 77] 。 5 利用Cullin-RING E3泛素连接酶的靶蛋白水解嵌合体技术近年来，PROTAC技术成为利用E3泛素连接酶活性设计抗肿瘤靶向药物的热点技术 [ 105] 。PROTAC的原理是人为设计一个小分子嵌合体，嵌合体一端包含一个可被CRL识别的配体基团，另一端包含一个可特异性结合某个靶蛋白分子的配体基团，两者中间是一个特别设计的连接子(linker)。嵌合体化合物进入细胞后与靶蛋白分子结合，将后者呈现给CRL，然后靶蛋白分子被泛素化、最终被降解 [ 106] ( 图 7)。该技术最早由Craig和Raymond团队报道于2001年 [ 106] 。他们设计的第一个PROTAC化合物，即PROTAC-1，一端连接一个可共价结合底物蛋白MetAp-2(methionine aminopeptidase-2)的小分子ovalicin，另一段连接了一段可被SCF β-TrCP识别的IκB多肽( 表 2)。当PROTAC-1进入细胞后，MetAp-2会被募集到SCF β-TrCP，随后被泛素化而降解 [ 106] 。可见，PROTAC技术可通过“劫持”一个特定的E3泛素连接酶来精确降解“非靶向”的靶蛋白 [ 105] 。然而，初代的PROTAC分子存在一些缺陷，如相对分子质量过大导致口服生物利用率和药代动力学属性较差、高浓度时因底物饱和容易产生“钩状效应”而失去疗效等 [ 45] 。实际上，由于可供选择的E3泛素连接酶和各种不同类型的配体超过600种，PROTAC技术具有超高的可塑性。而随着更多更好配体的发现，以及药物化学、合成化学的发展，上述缺陷也在被逐渐克服。如Craig团队早在2004年就通过对E3连接子羧基端的特殊修饰(poly-D-Arg序列)显著提高了PROTAC的细胞通透性，从而设计出第一个可以进入细胞的PROTAC [ 107] ( 表 2)。而一些PROTAC的EC 50已低至纳摩尔级别 [ 111- 112] 。此外，还有一些PROTAC则成功利用了SCF以外的CRL，如CRL2 VHL等 [ 113- 115] 。 Open in a separate window图7靶蛋白水解嵌合体的基本原理图 PROTAC一端包含一个可被Cullin-RING E3泛素连接酶(CRL)识别的配体基团，另一端包含一个可特异性结合某个靶蛋白分子的配体基团，两者中间是一个特别设计的连接子(linker).嵌合体化合物进入细胞后与底物蛋白结合，将后者呈现给CRL，底物蛋白被泛素化，最终被降解.PROTAC：靶蛋白水解嵌合体. 表2 具有代表性的靶蛋白水解嵌合体(PROTAC)及相关衍生技术 Table 2 Representative proteolysis-targeting chimeras (PROTACs) and related technologies 名称 结构 识别的E3 靶蛋白 其他说明 参考文献 PROTAC-1 SCF β-TrCP MetAp-2 第一个PROTAC [ 106] PROTAC-4 CRL2 VHL FKBP12 F36V 第一个可进入细胞的PROTAC [ 107] TrkAPPF RS2α CRL2 VHL FRS2α 第一个磷酸化的PROTAC [ 108] SARM-nutlin PROTAC MDM2 雄激素受体 第一个全小分子的PROTAC [ 109] ARV-110 暂无 暂无 雄激素受体 第一个进入临床试验的PROTAC [ 110] Open in a separate window最近几年，PROTAC技术巧妙而易于操作的设计思路也催生了一大批基于PROTAC的衍生技术：①含有受体酪氨酸激酶(receptor tyrosine kinase, RTK)磷酸化序列的磷酸化PROTAC(phosphoPROTAC) [ 108] ( 表 2)；②E3连接端和底物蛋白连接端均使用小分子化合物的全小分子PROTAC(all-small-molecule PROTAC) [ 109] ( 表 2)；③利用点击化学(click chemistry)在细胞内自发合成PROTAC的CLIPTAC(in-cell click-formed PROTAC) [ 116] ；④基于受体介导的内吞溶酶体通路的靶向内体嵌合体(endosome targeting chimeras, ENDTAC) [ 117] 等。上述技术充分拓展了PROTAC技术的应用范围，也进一步推进了具有高选择性、低毒性、良好生物利用率的新型抗肿瘤PROTAC的研发。值得一提的是，美国Arvinas公司研发的靶向雄激素受体的PROTAC药物ARV-110已于2019年3月开始在转移性去势抵抗性前列腺癌(mCRPC)患者中开展Ⅰ期临床试验({"type":"clinical-trial","attrs":{"text":"NCT03888612","term_id":"NCT03888612"}}NCT03888612)，成为目前第一个也是唯一一个进入临床试验的PROTAC [ 110] ( 表 2)。 6 结语从20世纪80年代泛素-蛋白酶体系统的初步发现，到2003年第一个蛋白酶体抑制剂硼替佐米的上市，再到2004年Ciechanover、Hershko和Rose因发现泛素介导的蛋白质降解共同获得诺贝尔化学奖，泛素-蛋白酶体系统对人体的重要性及其在治疗人类疾病上的应用价值被不断发掘。近年来，随着结构生物学研究的深入，人们对CRL复合体结构和功能的了解不断加深。拟素化抑制剂MLN4924的出现有力地证实了靶向CRL用于治疗肿瘤的可行性，并进一步推动了以CRL为靶点的抗肿瘤药物研发。 然而，尽管具有很好的成药潜力和不断扩大的市场，研发以CRL为靶点的抗肿瘤药物仍面临挑战。目前报道的相关化合物主要靶向SCF泛素连接酶，且靶点主要集中在SKP2-SKP1的蛋白-蛋白相互作用区域，而靶向其他CRL(如CRL5)或其他CRL的组分(如RBX1/RBX2)的小分子抑制剂仍鲜有报道。这可能是因为SCF E3(SKP2-SKP1)的研究较为透彻，有较为明确的结构特点，更易于表达纯化和设计体外相互作用模型。事实上，所有CRL均为多亚基蛋白质复合物，缺少明确的、典型的小分子药物结合位点。而且，泛素化反应是一个动态的、快速的催化反应，依赖于大量瞬时的蛋白-蛋白相互作用，而这些蛋白-蛋白相互作用区域往往比较平坦而宽泛，且伴随着蛋白空间构象的不断改变，导致很多小分子化合物无法轻易结合上去。另外，不同的泛素连接酶活性区域之间具有差异，因此无法基于一个通用的蛋白骨架模型去设计特异性的小分子化合物。上述挑战一定程度上限制了相关药物的研发进程。 因此，解析更多CRL及其组分的蛋白晶体结构，明确CRL与底物、CRL各组分之间的相互作用机制，寻找更多潜在的药物结合位点，将是解决上述难点的重要助推力。此外，高通量筛选技术、计算机辅助的虚拟筛选技术以及基于结构的药物设计等技术的结合将有助于研发更多高特异性的CRL抑制剂/激动剂。而PROTAC等新技术的不断发展，也为基于CRL的相关药物研发提供了新的可能。 Funding Statement国家重点研发计划(2016YFA0501800) References1. 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