更新于：2024-09-05

BRD4

更新于：2024-09-05

基本信息

别名

BRD4、bromodomain containing 4、bromodomain-containing 4

+ [8]

简介

Chromatin reader protein that recognizes and binds acetylated histones and plays a key role in transmission of epigenetic memory across cell divisions and transcription regulation. Remains associated with acetylated chromatin throughout the entire cell cycle and provides epigenetic memory for postmitotic G1 gene transcription by preserving acetylated chromatin status and maintaining high-order chromatin structure (PubMed:23589332, PubMed:23317504, PubMed:22334664). During interphase, plays a key role in regulating the transcription of signal-inducible genes by associating with the P-TEFb complex and recruiting it to promoters. Also recruits P-TEFb complex to distal enhancers, so called anti-pause enhancers in collaboration with JMJD6. BRD4 and JMJD6 are required to form the transcriptionally active P-TEFb complex by displacing negative regulators such as HEXIM1 and 7SKsnRNA complex from P-TEFb, thereby transforming it into an active form that can then phosphorylate the C-terminal domain (CTD) of RNA polymerase II (PubMed:23589332, PubMed:19596240, PubMed:16109377, PubMed:16109376, PubMed:24360279). Promotes phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II (PubMed:23086925). According to a report, directly acts as an atypical protein kinase and mediates phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II; these data however need additional evidences in vivo (PubMed:22509028). In addition to acetylated histones, also recognizes and binds acetylated RELA, leading to further recruitment of the P-TEFb complex and subsequent activation of NF-kappa-B (PubMed:19103749). Also acts as a regulator of p53/TP53-mediated transcription: following phosphorylation by CK2, recruited to p53/TP53 specific target promoters (PubMed:23317504). Acts as a chromatin insulator in the DNA damage response pathway. Inhibits DNA damage response signaling by recruiting the condensin-2 complex to acetylated histones, leading to chromatin structure remodeling, insulating the region from DNA damage response by limiting spreading of histone H2AX/H2A.x phosphorylation.

关联

项与 BRD4 相关的药物

Apabetalone

靶点

BRD4

作用机制

BRD4抑制剂 [+1]

在研机构

深圳市海普瑞药业集团股份有限公司 [+1]

原研机构

Resverlogix Corp.

在研适应症

心血管疾病 [+3]

非在研适应症

急性冠状动脉综合征 [+8]

最高研发阶段临床3期

首次获批国家/地区-

首次获批日期1800-01-20

PLX-2853

靶点

BRD4

作用机制

BRD4抑制剂

在研机构

原研机构

在研适应症

非在研适应症

最高研发阶段临床2期

首次获批国家/地区-

首次获批日期1800-01-20

RNK05047

靶点

BRD4

作用机制

BRD4抑制剂

在研机构

珃诺生物医药科技（杭州）有限公司初创企业

原研机构

珃诺生物医药科技（杭州）有限公司初创企业

在研适应症

晚期恶性实体瘤 [+3]

非在研适应症-

最高研发阶段临床1/2期

首次获批国家/地区-

首次获批日期1800-01-20

项与 BRD4 相关的临床试验

NCT03160430 / Not yet recruiting临床1/2期

A Two-Part Phase 2a Study in Patients With End-Stage Renal Disease Treated With Hemodialysis; Part A is an Open-Label Study Arm to Evaluate the Effect of Hemodialysis on the Pharmacokinetics of 100 mg RVX000222; and Part B is a Double-Blind, Randomized, Placebo-Controlled, Sequential Cross-Over Study Arm to Evaluate the Efficacy, Safety, and Pharmacokinetics of RVX000222

This is a multi-center, two-part study; Part A and Part B. Part A of the study is an open-label, single-dose pharmacokinetic (PK) evaluation of 100 mg RVX000222 on dialysis and non-dialysis days in eight (8) End Stage Renal Disease (ESRD) patients who receive hemodialysis as standard of care.
Part B of the study is a double-blind, placebo-controlled study in up to thirty six (36) ESRD patients receiving hemodialysis using a sequential cross-over design with RVX000222 at a daily oral dose of 100 mg b.i.d. (200 mg per day) or matching placebo in combination with SoC.
The primary objective of the study is to evaluate if treatment with RVX000222 in combination with standard of care (SoC) decreases plasma alkaline phosphatase in comparison to placebo and SoC.

开始日期2024-11-22

申办/合作机构

Resverlogix Corp.

CTR20233943 / Recruiting临床1期

一项评价蛋白质降解剂注射用RNK05047在晚期实体瘤和淋巴瘤受试者中的安全性、耐受性、药代动力学、药效学和疗效的开放、多中心的Ⅰ期临床试验

确定RNK05047在晚期实体瘤和淋巴瘤患者中的2期临床推荐剂量（RP2D），评价RNK05047在晚期实体瘤和淋巴瘤患者中的安全性和耐受性。评价RNK05047的药代动力学（PK）特征，抗肿瘤活性的初步证据。探索安全性和/或疗效终点与PK/PD的关系。评估与RNK05047相关的生物标志物，探索生物标志物的变化和治疗结果的关系。

开始日期2024-02-19

申办/合作机构

珃诺生物医药科技（杭州）有限公司初创企业

NCT04915300 / Not yet recruiting临床2期IIT

Apabetalone for Pulmonary Arterial Hypertension: a Phase 2 Clinical Study

Throughout the past twenty years, numerous specific pharmacologic agents targeting the endothelial dysfunction associated with PAH have emerged. Short term placebo-controlled randomized trials assessing PAH-specific monotherapy with these molecules have reported improvements in pulmonary hemodynamics and exercise capacity. A recent meta-analysis also documented a reduction in short-term mortality of about ≈40% with such therapies. Several randomized clinical trials evaluating PAH-specific combination therapy have been conducted. Our recent meta-analysis showed that combination therapy was associated with a 35% risk reduction for the occurrence of clinical worsening compared to monotherapy. Nonetheless, the investigators also showed 17% of PAH patients receiving combination therapy still experienced clinical worsening over a median exposure of 16 weeks. Moreover, long-term survival on PAH-specific also therapy remains poor in the modern era, with a yearly mortality rate of 15 % in incident idiopathic PAH. The identification of innovative therapeutic targets and validation of these complementary therapeutic interventions are thus urgently needed in PAH.
The investigators and others (K. Stenmark, University of Colorado and H. Bogaard, VU University Medical Center, Amsterdam, personal communications), have published strong evidence that BRD4 plays a key role in the pathological phenotype in PAH accounting for disease progression and showed that BRD4 inhibition can reverse PAH in several animal models. Intriguingly, coronary artery disease (CAD) and metabolic syndrome are more prevalent in PAH compared with the global population, suggesting a link between these diseases. Interestingly, BRD4 is also a trigger for calcification and remodeling processes and regulates transcription of lipoprotein and inflammatory factors, all of which are important in PAH and CAD. Apabetalone, an orally available BRD4 inhibitor, is now in a clinical development stage with a good safety profile.
The overall objective of the study is to explore the efficacy and safety of apabetalone as an add-on therapy for adult PAH patients and to inform the conduct and the design of a Phase 3 trial.
The primary objective of the study is to assess the efficacy of apabetalone as evaluated by the change in PVR over a period of 24 weeks compared to placebo in adult subjects with PAH on stable background therapy.
Secondary objectives include changes at week 24 in 6MWD, plasma NT-proBNP concentration, WHO functional class, ESC/ERS risk stratification score, health-related quality of life and additional hemodynamic data from right heart.
Exploratory objectives are to evaluate the effects of apabetalone compared to placebo in adult subjects with PAH on mortality and clinically relevant morbidity events, and on circulating levels and transcription changes in whole blood markers of metabolism, vascular calcification, inflammation, DNA damage and leucocyte expression of BMPR2.

开始日期2023-10-01

申办/合作机构

Université Laval

[+2]

100 项与 BRD4 相关的临床结果

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100 项与 BRD4 相关的转化医学

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0 项与 BRD4 相关的专利（医药）

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2,374

项与 BRD4 相关的文献（医药）

2024-04-01·Life science alliance

Epigenomic states contribute to coordinated allelic transcriptional bursting in iPSC reprogramming.

Article

作者: Parichitran Ayyamperumal ; Hemant Chandru Naik ; Amlan Jyoti Naskar ; Lakshmi Sowjanya Bammidi ; Srimonta Gayen

Two alleles of a gene can be transcribed independently or coordinatedly, which can lead to temporal expression heterogeneity with potentially distinct impacts on cell fate. Here, we profiled genome-wide allelic transcriptional burst kinetics during the reprogramming of MEF to induced pluripotent stem cells. We show that the degree of coordination of allelic bursting differs among genes, and alleles of many reprogramming-related genes burst in a highly coordinated fashion. Notably, we show that the chromatin accessibility of the two alleles of highly coordinated genes is similar, unlike the semi-coordinated or independent genes, suggesting the degree of coordination of allelic bursting is linked to allelic chromatin accessibility. Consistently, we show that many transcription factors have differential binding affinity between alleles of semi-coordinated or independent genes. We show that highly coordinated genes are enriched with chromatin accessibility regulators such as H3K4me3, H3K4me1, H3K36me3, H3K27ac, histone variant H3.3, and BRD4. Finally, we demonstrate that enhancer elements are highly enriched in highly coordinated genes. Our study demonstrates that epigenomic states contribute to coordinated allelic bursting to fine-tune gene expression during induced pluripotent stem cell reprogramming.

2024-02-21·Nature

Targeted protein degradation via intramolecular bivalent glues.

Article

作者: Oliver Hsia ; Matthias Hinterndorfer ; Angus D Cowan ; Kentaro Iso ; Tasuku Ishida ; Ramasubramanian Sundaramoorthy ; Mark A Nakasone ; Hana Imrichova ; Caroline Schätz ; Andrea Rukavina ; Koraljka Husnjak ; Martin Wegner ; Alejandro Correa-Sáez ; Conner Craigon ; Ryan Casement ; Chiara Maniaci ; Andrea Testa ; Manuel Kaulich ; Ivan Dikic ; Georg E Winter ; Alessio Ciulli

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target^1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

2024-02-21·Neurochemical research

Inhibition of CK2 Diminishes Fibrotic Scar Formation and Improves Outcomes After Ischemic Stroke via Reducing BRD4 Phosphorylation.

Article

作者: Xuemei Li ; Qinghuan Yang ; Peiran Jiang ; Jun Wen ; Yue Chen ; Jiagui Huang ; Mingfen Tian ; Jiangxia Ren ; Qin Yang

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-β1 (TGF-β1), SB431542 (a TGF-β1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-β1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-β1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-β1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke.

122

项与 BRD4 相关的新闻（医药）

2024-04-07

·精准药物

我们离理性设计分子胶还有多远

今天为大家介绍一篇工作，是二月份发表在Nature上的“分子内二价胶水”的文章。通讯作者是邓迪大学的Alessio Ciulli教授和奥地利科学院分子医学研究中心的Georg E. Winter教授。Ciulli课题组关注蛋白-蛋白相互作用（PPI）中的化学生物学、结构生物学问题，尤其关注小分子化合物对于PPI的调节。Winter课题组关注小分子调控蛋白质降解，尤其是不可成药靶点的降解。——正文——故事还要从Indisulam讲起。2017年，磺酰胺类化合物Indisulam登上Science1，它介导CUL4A/BDCAF15与RBM39的互作，导致RMB39被泛素化，最终被降解（图1）。图1. 磺胺类药物诱导RBM39与CUL4A/BDCAF15形成复合物E7820是Indisulam的类似物，它也可以诱导RBM39与CUL4A/BDCAF15形成复合物（图2）2。图2. A）Indisulam及E7820结构 B) BM39-E7820-DCAF15复合物结构一些将E7820作为E3连接酶底物受体binder进行PROTAC分子设计的尝试没有取得很好的结果，靶蛋白降解效率很低。但是， E7820与(+)-JQ1（JQ1是BRD4配体，在分子胶设计领域非常常用）通过刚性Linker连接的融合分子——IBG1，活性很强，降解BRD4的DC50达到0.15 nM(图3)。图3. IBG1结构及IBG1降解BRD4但该分子并不通过DCAF15发挥作用（图4）。图4. 敲除DCAF15并不影响IBG1发挥作用通过FACS-based CRISPR–Cas9 screens，作者发现CRL4DCAF16 复合物中的组分与IBG1给药后BRD4稳定性有较强相关性。图5. FACS-based CRISPR–Cas9 screens流程示意图作者进一步证明，敲除DCAF16、DDB1消除了IBG1降解BRD4的能力。按照一般的思路，那应该是形成了BRD4-IBG1-DCAF16三元复合物，导致BRD4泛素化，然后被降解。但是E7820衍生物1a,1b,1c,1d并没有拮抗IBG1诱导的BRD4降解（图6A），证明E7820片段不是扮演PROTAC中E3连接酶binder的角色；而JQ1的衍生物1e,1f,1g也没有降解BRD2/4的能力（图6B），说明完整的E7820结构是降解所必需的。综上所述，IBG1分子发挥功能的机制与PROTAC不同。图6. A）E7820衍生物结构 B）JQ1衍生物结构这篇文章最神奇的部分到了，在此之前先看BRD4的结构（图7）。BRD4有两个Bromodomain（BD1和BD2），而JQ1跟这两个结构域都有纳摩尔级的结合力3。以往研究Bromodomain抑制剂及其复合物结构的时候，通常只会用单个的BD 1domain或者BD2 domain。图7. 鼠BRD4结构（与人高度一致）4但这篇文章截取了BD1-NLS-BD2这一段进行研究，后文将该截短体称为BRD4Tandem。作者发现DCAF16与BRD4Tandem有着天然的亲和力，IBG1的加入增强了DCAF16-的结合，也改变了结合模式，结合由焓驱动转变为熵驱动（图8）。图8. d）DCAF16滴定BRD4Tandem a）DCAF16滴定BRD4Tandem-IBG1紧接着作者证明了BD1/2同时存在是IBG1发挥作用的基础，也解出了BRD4Tandem-IBG1-DCAF16-DDB1的复合物结构（图9）。尺寸排阻色谱证明IBG1将BRD4Tandem拉紧；电镜结构表明IBG1与BRD4Tandem互作后形成一个新的疏水界面，DCAF16主要通过疏水作用与该界面互作。这也解释了为什么在IBG1存在时，DCAF16与BRD4Tandem的互作由熵驱动。图9. 冷冻电镜结构PDB ID 8OV6同时，复合物结构也表明JQ1占据了BD2 domain，E7820片段占据了BD1 domain。在此之前也有过磺酰胺结构的Bromodomain抑制剂被报道，将以前结构中的抑制剂与E7820片段叠合，发现他们采取类似的构象(图10)。但磺胺片段与Bromodomain的结合力远弱于JQ1。图10. 其他磺胺类抑制剂晶体结构中的pose与本研究中的E7820叠合图，E7820为黄色作者猜想将两个强Bromodomain抑制剂通过类似的方式组装起来，可能会达到更好的效果（图11）。图11. 作者设计的分子胶（IBG2,IBG3）IBG3的活性确实强于原始分子IBG1，降解BRD4的EC50达到了6.7 pM，三元复合物形成的ΔG绝对值更大。但以往也有人做过类似的二价Bromodomain抑制剂（图12），抑制效果很强，但并没有降解BRD4的能力。他们与IBG3的区别主要有三点：1）连接链为柔性，2）连接链与JQ1连接的位置不同，3）连接链长度不同。具体是哪个因素更重要，还需要后续深入地探讨。图12. 二价Bromodomain抑制剂作者在专利中还发现类似的结构——IBG4（图13），该化合物也能降解BRD4，机制与IBG1类似，但是IBG4降解BRD4依赖DCAF11,而不是DCAF16，且DCAF11和16在结构上相似性较小。图13. IBG4结构——小结——这篇文章无疑是成功的，他们首次发现分子内二价胶水，拓宽了分子胶类型，并成功理性设计出皮摩尔级的分子内二价胶（IBG1→IBG3）。更难能可贵的是它也带给我们启示和思考，除了VHL/CRBN配体外，慎重选择新出现的binder，如E7820。双功能分子设计看似简单，实则不然。本篇内容就是从DCAF15→DCAF16→DCAF11，过程曲折。关于合理设计分子胶/双功能分子，有人认为蛋白与E3底物受体间的弱结合或许是分子胶必须的，有人则不这么认为，该工作的结果是倾向于前者的。最近分子胶领域的工作喜欢展示自己的分子设计策略5，但这篇文章提醒我们，要严谨的验证自己设想的机制。分子胶理性设计还是一片蓝海！参考文献：1 Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356 (2017). https://doi.org/10.1126/science.aal37552 Du, X. et al. Structural Basis and Kinetic Pathway of RBM39 Recruitment to DCAF15 by a Sulfonamide Molecular Glue E7820. Structure 27, 1625-1633.e1623 (2019). https://doi.org/10.1016/j.str.2019.10.0053 Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067-1073 (2010). https://doi.org/10.1038/nature095044 Weissman, J. D. et al. The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains. J. Biol. Chem. 297 (2021). https://doi.org/10.1016/j.jbc.2021.1013265 Gourisankar, S. et al. Rewiring cancer drivers to activate apoptosis. Nature 620, 417-425 (2023). https://doi.org/10.1038/s41586-023-06348-2本文介绍的工作：Hsia, O., Hinterndorfer, M., Cowan, A.D. et al. Targeted protein degradation via intramolecular bivalent glues. Nature 627, 204–211 (2024). https://doi.org/10.1038/s41586-024-07089-6作者：任宇浩审稿：程北溟编辑：黄志贤声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号粉丝群/投稿/授权/广告等请联系公众号助手觉得本文好看，请点这里↓

蛋白降解靶向嵌合体基因疗法

2024-04-07

·GoDesign

【分子设计】我们离理性设计分子胶还有多远

今天为大家介绍一篇工作，是二月份发表在Nature上的“分子内二价胶水”的文章。通讯作者是邓迪大学的Alessio Ciulli教授和奥地利科学院分子医学研究中心的Georg E. Winter教授。Ciulli课题组关注蛋白-蛋白相互作用（PPI）中的化学生物学、结构生物学问题，尤其关注小分子化合物对于PPI的调节。Winter课题组关注小分子调控蛋白质降解，尤其是不可成药靶点的降解。——正文——故事还要从Indisulam讲起。2017年，磺酰胺类化合物Indisulam登上Science1，它介导CUL4A/BDCAF15与RBM39的互作，导致RMB39被泛素化，最终被降解（图1）。图1. 磺胺类药物诱导RBM39与CUL4A/BDCAF15形成复合物E7820是Indisulam的类似物，它也可以诱导RBM39与CUL4A/BDCAF15形成复合物（图2）2。图2. A）Indisulam及E7820结构 B) BM39-E7820-DCAF15复合物结构一些将E7820作为E3连接酶底物受体binder进行PROTAC分子设计的尝试没有取得很好的结果，靶蛋白降解效率很低。但是， E7820与(+)-JQ1（JQ1是BRD4配体，在分子胶设计领域非常常用）通过刚性Linker连接的融合分子——IBG1，活性很强，降解BRD4的DC50达到0.15 nM(图3)。图3. IBG1结构及IBG1降解BRD4但该分子并不通过DCAF15发挥作用（图4）。图4. 敲除DCAF15并不影响IBG1发挥作用通过FACS-based CRISPR–Cas9 screens，作者发现CRL4DCAF16 复合物中的组分与IBG1给药后BRD4稳定性有较强相关性。图5. FACS-based CRISPR–Cas9 screens流程示意图作者进一步证明，敲除DCAF16、DDB1消除了IBG1降解BRD4的能力。按照一般的思路，那应该是形成了BRD4-IBG1-DCAF16三元复合物，导致BRD4泛素化，然后被降解。但是E7820衍生物1a,1b,1c,1d并没有拮抗IBG1诱导的BRD4降解（图6A），证明E7820片段不是扮演PROTAC中E3连接酶binder的角色；而JQ1的衍生物1e,1f,1g也没有降解BRD2/4的能力（图6B），说明完整的E7820结构是降解所必需的。综上所述，IBG1分子发挥功能的机制与PROTAC不同。图6. A）E7820衍生物结构 B）JQ1衍生物结构这篇文章最神奇的部分到了，在此之前先看BRD4的结构（图7）。BRD4有两个Bromodomain（BD1和BD2），而JQ1跟这两个结构域都有纳摩尔级的结合力3。以往研究Bromodomain抑制剂及其复合物结构的时候，通常只会用单个的BD 1domain或者BD2 domain。图7. 鼠BRD4结构（与人高度一致）4但这篇文章截取了BD1-NLS-BD2这一段进行研究，后文将该截短体称为BRD4Tandem。作者发现DCAF16与BRD4Tandem有着天然的亲和力，IBG1的加入增强了DCAF16-的结合，也改变了结合模式，结合由焓驱动转变为熵驱动（图8）。图8. d）DCAF16滴定BRD4Tandem a）DCAF16滴定BRD4Tandem-IBG1紧接着作者证明了BD1/2同时存在是IBG1发挥作用的基础，也解出了BRD4Tandem-IBG1-DCAF16-DDB1的复合物结构（图9）。尺寸排阻色谱证明IBG1将BRD4Tandem拉紧；电镜结构表明IBG1与BRD4Tandem互作后形成一个新的疏水界面，DCAF16主要通过疏水作用与该界面互作。这也解释了为什么在IBG1存在时，DCAF16与BRD4Tandem的互作由熵驱动。图9. 冷冻电镜结构PDB ID 8OV6同时，复合物结构也表明JQ1占据了BD2 domain，E7820片段占据了BD1 domain。在此之前也有过磺酰胺结构的Bromodomain抑制剂被报道，将以前结构中的抑制剂与E7820片段叠合，发现他们采取类似的构象(图10)。但磺胺片段与Bromodomain的结合力远弱于JQ1。图10. 其他磺胺类抑制剂晶体结构中的pose与本研究中的E7820叠合图，E7820为黄色作者猜想将两个强Bromodomain抑制剂通过类似的方式组装起来，可能会达到更好的效果（图11）。图11. 作者设计的分子胶（IBG2,IBG3）IBG3的活性确实强于原始分子IBG1，降解BRD4的EC50达到了6.7 pM，三元复合物形成的ΔG绝对值更大。但以往也有人做过类似的二价Bromodomain抑制剂（图12），抑制效果很强，但并没有降解BRD4的能力。他们与IBG3的区别主要有三点：1）连接链为柔性，2）连接链与JQ1连接的位置不同，3）连接链长度不同。具体是哪个因素更重要，还需要后续深入地探讨。图12. 二价Bromodomain抑制剂作者在专利中还发现类似的结构——IBG4（图13），该化合物也能降解BRD4，机制与IBG1类似，但是IBG4降解BRD4依赖DCAF11,而不是DCAF16，且DCAF11和16在结构上相似性较小。图13. IBG4结构——小结——这篇文章无疑是成功的，他们首次发现分子内二价胶水，拓宽了分子胶类型，并成功理性设计出皮摩尔级的分子内二价胶（IBG1→IBG3）。更难能可贵的是它也带给我们启示和思考，除了VHL/CRBN配体外，慎重选择新出现的binder，如E7820。双功能分子设计看似简单，实则不然。本篇内容就是从DCAF15→DCAF16→DCAF11，过程曲折。关于合理设计分子胶/双功能分子，有人认为蛋白与E3底物受体间的弱结合或许是分子胶必须的，有人则不这么认为，该工作的结果是倾向于前者的。最近分子胶领域的工作喜欢展示自己的分子设计策略5，但这篇文章提醒我们，要严谨的验证自己设想的机制。分子胶理性设计还是一片蓝海！参考文献：1 Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356 (2017). https://doi.org/10.1126/science.aal37552 Du, X. et al. Structural Basis and Kinetic Pathway of RBM39 Recruitment to DCAF15 by a Sulfonamide Molecular Glue E7820. Structure 27, 1625-1633.e1623 (2019). https://doi.org/10.1016/j.str.2019.10.0053 Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067-1073 (2010). https://doi.org/10.1038/nature095044 Weissman, J. D. et al. The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains. J. Biol. Chem. 297 (2021). https://doi.org/10.1016/j.jbc.2021.1013265 Gourisankar, S. et al. Rewiring cancer drivers to activate apoptosis. Nature 620, 417-425 (2023). https://doi.org/10.1038/s41586-023-06348-2本文介绍的工作：Hsia, O., Hinterndorfer, M., Cowan, A.D. et al. Targeted protein degradation via intramolecular bivalent glues. Nature 627, 204–211 (2024). https://doi.org/10.1038/s41586-024-07089-6作者：任宇浩审稿：程北溟编辑：黄志贤GoDesignID：Molecular_Design_Lab（扫描下方二维码可以订阅哦！）

蛋白降解靶向嵌合体基因疗法

2024-03-30

·精准药物

利用Cullin E3连接酶接头蛋白SKP1进行靶向蛋白降解

研究背景由与E3泛素连接酶配体连接蛋白质靶向配体形成的蛋白水解靶向嵌合体（PROTAC）的靶向蛋白质降解已成为一种通过蛋白酶体介导的降解致病蛋白质的强大治疗方式。虽然有超过600个E3连接酶，但是大多数PROTAC只利用了Cullin-RING E3泛素连接酶的底物受体——cereblon和VHL。近年来，还发现了许多其他针对其他E3连接酶的募集蛋白，包括cIAP、MDM2、DCAF16、DCAF11、RNF114、RNF4、FEM1B、KEAP1和DCAF1。并不是所有的E3连接酶都对癌细胞活力至关重要，因此，癌细胞中可能会出现耐药机制，从而损害E3连接酶募集并使PROTAC无效。开发针对泛素-蛋白酶体机制核心成分的募集剂，例如针对对细胞活力至关重要的CUL1-CUL7 E3连接酶中的Cullin-RING E3连接酶复合接头蛋白（如DDB1、SKP1 或ELOB/ELOC）的募集剂，可以避免PROTAC可能产生的潜在未来耐药机制。加州大学伯克利分校Nomura课题组研究了CUL1-RING E3连接酶接头蛋白SKP1是否可以被募集用于PROTAC介导的新底物降解。研究内容该课题组在基于凝胶的基于活性的蛋白质分析（ABPP）中筛选了1284个半胱氨酸反应性共价配体，筛选了针对人SKP1-FBXO7-CUL1-RBX1复合物（图1）。该筛选产生的性能最良好的化合物是EN884（图2）。EN884显示了SKP1-FBXO7-CUL1-RBX1复合物中人SKP1蛋白的半胱氨酸反应性探针的剂量反应置换（图3）。图1.半胱氨酸反应性共价配体库筛选结果（针对SKP1-FBXO7-CUL1-RBX1复合物）图2.EN884结构图3.EN884与半胱氨酸探针反应该课题组对与 EN884 一起孵育的SKP1-FBXO7-CUL1-RBX1复合物进行了串联质谱（MS/MS）分析，以确定修饰位点（图4），发现了一个被修饰的位点——SKP1上的C160（图5），该位点由于特定的F-Box结构域，表现出独特的构象。因此，该课题组假设EN884可能会利用SKP1和CUL1底物受体之间的集体界面并结合。图4.串联质谱分析图5.结合位点位置接下来，该课题组合成了两个基于EN884的炔烃功能化探针SJH1-37-m、SJH1-37-p（图6）。基于凝胶的ABPP评估这些探针与SKP1复合物的结合，与对位相比，间位结合能力更佳（图7）。图6.探针结构图7.探针结合能力值得注意的是，由于C160位置特殊，EN884和SJH1-37m不与单体SKP1结合（图8）。这表明，CUL1复合物的其余部分，或至少是底物受体，是配体与SKP1结合所必需的。图8.探针与单体SKP1结合该课题组进一步证明，SJH1-37m通过SKP1的下拉在细胞中结合SKP1，而不是不相关的蛋白质，如GAPDH（图9）。图9.SJH1-37m探针的SKP1下拉接下来，该课题组进行了基于质谱的ABPP，以使用先前建立的同位素脱硫生物素-ABPP（isoDTB-ABPP）鉴定HEK293T细胞中EN884参与SKP1的半胱氨酸。该课题组发现EN884在SKP1上与C160有7%的最小参与（图10）。这种最小参与与已应用在PROTAC（包括DCAF16、DCAF11和RNF114）中的其他共价E3连接酶募集剂观察到的最小参与一致。图10.HEK293T细胞中EN884的isoDTB-ABPP分析为了证明共价SKP1配体可用于PROTAC应用，该课题组合成了四种不同的PROTAC，通过C2、C4、C5或PEG3连接子将EN884的衍生物与BET家族抑制剂JQ1连接起来，分别生成SJH1-51A、SJH1-51B、SJH1-51C和SJH1-51D（图11）。所有四种PROTAC都降解了HEK293T细胞中BRD4的短亚型（图12），其中使用C4烷基接头的SJH1-51B观察到的降解最强。图11.四种PROTAC结构图12.四种PROTAC降解效果并且，基于串联质量标记（TMT）的MDA-MB-231细胞中SJH1-51B的定量蛋白质组学分析显示BRD4中等选择性降解，其中16种蛋白质的水平显着降低>50%（图13）。图13.SJH1-51B的定量蛋白质组学与蛋白酶体介导的降解一致，SJH1-51B介导的BRD4降解通过用蛋白酶体抑制剂硼替佐米预处理而减弱（图14）。它还显示出对活性Cullin RING E3连接酶所需的NEDD化的依赖性，NEDD化抑制剂MLN4924可减弱BRD4的降解（图15）。最重要的是，该课题组证明SKP1敲低完全减弱了SJH1-51B在HEK293T细胞中对BRD4的降解（图16）。图14.硼替佐米预处理结果图15.MLN4924预处理结果图16.SKP1敲低细胞结果BRD4是一种相对容易被PROTAC降解的蛋白质。因此，该课题组接下来测试了共价SKP1募集剂是否可用于降解另一种新底物蛋白——雄激素受体（AR）。该课题组合成了4种AR PROTAC，将共价SKP1募集剂与来自ARV-110的Arvinas AR PROTAC靶向配体连接起来，没有连接子或C4、C6或C7烷基连接子，分别产生SJH1-63、SJH1-62C、SJH1-62D和SJH1-62B（图17）。该课题组观察到所有4种PROTAC的AR降解，其中带有C7烷基接头的SJH1-62B在LNCaP前列腺癌细胞中显示出最有效和最强大的AR降解（图17）。图17.四种AR PROTAC结构与降解效果该课题组进一步证明，SJH1-62B介导的AR降解在蛋白酶体抑制剂预处理后减弱（图18）。定量蛋白质组学分析还表明，AR与其他11种蛋白质的选择性降解在5000多种定量蛋白质中也显著降低（图19）。图18.SJH1-62B的硼替佐米预处理结果图19.SJH1-62B的定量蛋白质组学研究总结该课题组发现了一种SKP1募集物，它共价靶向位于SKP1和CUL1 F-box底物受体界面的SKP1的固有无序C160。这一观察结果为发现仅在含有IDR的蛋白质与其相互作用的蛋白质形成复合物时出现的可连接位点提供了概念证明。他们进一步证明，这种共价SKP1配体可用于PROTAC应用，以降解细胞中的新底物，如BRD4和AR。鉴于SKP1的重要性，SKP1的共价募集可能潜在地克服在癌症治疗中利用非本质E3连接酶的PROTAC的耐药性机制。虽然该课题组证明了在PROTAC中使用Cullin RING E3连接酶衔接蛋白的概念，但SKP1配体仍处于早期阶段，需要进行重大的药物化学优化，以提高效力、选择性和类药物特性。该课题组的研究结果还强调了使用共价化学蛋白质组学方法来识别泛素-蛋白酶体系统中蛋白质的补充物的实用性，该系统可用于靶向蛋白质降解应用。作者：潘相奕校审：周金明编辑：郑一榕时间：2024年3月29日参考文献Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation ACS Chemical Biology 2024 19 (2), 442-450. DOI: 10.1021/acschembio.3c00642 声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号粉丝群/投稿/授权/广告等请联系公众号助手觉得本文好看，请点这里↓

蛋白降解靶向嵌合体