更新于：2024-11-01

MEK1

更新于：2024-11-01

基本信息

别名

Dual specificity mitogen-activated protein kinase kinase 1、ERK activator kinase 1、MAP kinase kinase 1

+ [10]

简介

Dual specificity protein kinase which acts as an essential component of the MAP kinase signal transduction pathway. Binding of extracellular ligands such as growth factors, cytokines and hormones to their cell-surface receptors activates RAS and this initiates RAF1 activation. RAF1 then further activates the dual-specificity protein kinases MAP2K1/MEK1 and MAP2K2/MEK2. Both MAP2K1/MEK1 and MAP2K2/MEK2 function specifically in the MAPK/ERK cascade, and catalyze the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade. Activates BRAF in a KSR1 or KSR2-dependent manner; by binding to KSR1 or KSR2 releases the inhibitory intramolecular interaction between KSR1 or KSR2 protein kinase and N-terminal domains which promotes KSR1 or KSR2-BRAF dimerization and BRAF activation (PubMed:29433126). Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, adhesion, survival and differentiation, predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. One target of the MAPK/ERK cascade is peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor that promotes differentiation and apoptosis. MAP2K1/MEK1 has been shown to export PPARG from the nucleus. The MAPK/ERK cascade is also involved in the regulation of endosomal dynamics, including lysosome processing and endosome cycling through the perinuclear recycling compartment (PNRC), as well as in the fragmentation of the Golgi apparatus during mitosis.

关联

项与 MEK1 相关的药物

Tunlametinib

妥拉美替尼

靶点

MEK1 x MEK2

作用机制

MEK1抑制剂 [+1]

在研机构

上海科州药物研发有限公司初创企业

原研机构

上海科州药物研发有限公司初创企业

在研适应症

NRAS突变黑色素瘤 [+9]

非在研适应症

KRAS突变非小细胞肺癌

最高研发阶段批准上市

首次获批国家/地区

中国

首次获批日期2024-03-12

Selumetinib sulfate

司美替尼

靶点

MEK1 x MEK2

作用机制

MEK1抑制剂 [+1]

在研机构

National Cancer Institute [+11]

原研机构

Array BioPharma, Inc.

在研适应症

神经纤维瘤病 [+53]

非在研适应症

急性早幼粒细胞白血病 [+65]

最高研发阶段批准上市

首次获批国家/地区

美国

首次获批日期2020-04-10

Binimetinib

贝美替尼

靶点

MEK1 x MEK2

作用机制

MEK1抑制剂 [+1]

在研机构

Pierre Fabre Médicament SAS [+10]

原研机构

Array BioPharma, Inc.

在研适应症

BRAF突变的甲状腺未分化癌 [+32]

非在研适应症

急性髓性白血病 [+33]

最高研发阶段批准上市

首次获批国家/地区

美国

首次获批日期2018-06-27

855

项与 MEK1 相关的临床试验

NCT06666348 / Not yet recruiting临床1/2期IIT

A Phase 1/2 Study of Mirdametinib and Vinblastine for Newly Diagnosed or Previously Untreated Patients With Pediatric Low-grade Glioma and Activation of the MAPK Pathway

This is a 3-part open-label study (feasibility phase, treatment phase and follow-up phase) of orally administered mirdametinib in combination with intravenous vinblastine chemotherapy in patients with PLGG with activation of MAPK pathway.
Feasibility Phase:
The maximum tolerated/recommended phase 2 dose (MTD/RP2D) of the mirdametinib plus vinblastine combination will be assessed using a modified Rolling-6 design.
Treatment Phase:
Patients will receive mirdametinib twice daily (continuously) at a fixed dose (2mg/m2 po BID up to 4 mg BID) for a total of 13 cycles (28 days cycle). Weekly intravenous vinblastine at MTD will be given for a total of 17 cycles. If adverse events occur, two dose reductions are allowed.
Follow-up Phase:
Following the end of treatment, patients will be scheduled for a follow-up visit every 6 months for 36 months to evaluate PFS, TTP and OS.

开始日期2025-01-01

申办/合作机构-

NCT06502171 / Not yet recruiting临床1期IIT

A Phase 1/1b/2 Study of Cabozantinib in Combination with Selumetinib for Plexiform Neurofibroma in Adults and Adolescents with Neurofibromatosis Type 1

Based on the clinical activity of both selumetinib and cabozantinib as monotherapies in clinical trials, the demonstrated activity of these agents in reduced doses in preclinical studies, and the non-overlapping toxicity profiles, the study will assess the tolerability and efficacy of selumetinib and cabozantinib in combination in participants with NF1 ≥16 years old with progressive and/or symptomatic PN in a phase 1/1b/2 clinical trial.
Trial Design Phase 1 This will be an open label, dose escalation phase. Dose level escalation will be determined by a rolling six design. In this design, up to 6 participants can be enrolled at a given dose level and then evaluated for dose limiting toxicity (DLT) within the DLT window. The DLT window is defined as 16 weeks in this study based on the long half-life of cabozantinib and the desire to have maximum confidence about long-term tolerability of the combination prior to proceeding to the next dose level.
Phase 1b Once the recommended phase 2 dose has been determined in phase 1, an expanded cohort of 12 participants will be enrolled in phase 1b portion of the study.
Phase 2 This will be an open label, single-arm phase using the recommended phase 2 dose.

开始日期2025-01-01

申办/合作机构

The University of Alabama at Birmingham

[+2]

NCT05253131 / Not yet recruiting临床2期IIT

A Phase 1/2 Trial of the MEK Inhibitor Selumetinib and Bromodomain Inhibitor With Durvalumab (MEDI4736), a PD-L1 Antibody for Sarcomas Including Malignant Peripheral Nerve Sheath Tumors (NF112)

A multi-institutional open-label phase 1/2 trial of selumetinib in combination with BI and durvalumab in refractory/unresectable sarcomas including MPNST. The phase 1 portion will be separated in two parts and will be open to all patients with refractory/relapsed sarcomas. The phase 2 portion will be for patients with refractory/unresectable NF1-associated MPNST.

开始日期2024-12-15

申办/合作机构

The University of Alabama at Birmingham

[+1]

100 项与 MEK1 相关的临床结果

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100 项与 MEK1 相关的转化医学

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0 项与 MEK1 相关的专利（医药）

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4,709

项与 MEK1 相关的文献（医药）

2025-02-01·Bioelectrochemistry

Low-energy electric shock ameliorates cell proliferation, morphallaxis, and regeneration via driving key regenerative proteins in earthworm and 3T3 cells

Article

作者: Selvan Christyraj, Johnson Retnaraj Samuel ; Balamurugan, Nivetha ; Vaidhyalingham, Ashwin Barath ; Nagaiah, Hari Prasath ; Rajagopalan, Kamarajan ; Selvan Christyraj, Jackson Durairaj ; Radhakrishnan, Periyasamy ; Dan, Vipin Mohan

Electric stimulation regulates many cellular processes like cell proliferation, differentiation, apoptosis and cellular migration. Despite its crucial role in regulating stem cells and regeneration, it remains underexplored in both in-vivo and in-vitro settings. In this study, Eudrilus eugeniae are subjected to electric stimulation (1.5 V) prior and after amputation and which augments regeneration up to double-time. Blocking epimorphosis using 2 M thymidine retracts regeneration kinetics to one-third but such inhibition was rescued by applying electric stimulation which propels an overactive morphallaxis pattern of regeneration. Excreting electric stimulation on control worms shows minimal impact, whereas it enhances the key regenerative proteins like VEGF, COX2, YAP, c-Myc, and Wnt3a on amputated worms. Upon blocking epimorphosis, all these key regenerative proteins are down-regulated but through electric stimulation, the cells are reprogrammed to express a triple fold of the mentioned regenerative proteins, that further promotes morphallaxis. In 3T3 cells, electric stimulation accelerates cell proliferation and migrations in 5 secs exposure and it exerts its function by overexpressing VEGF mediated by MEK1. Wnt3a expression was gradually upregulated in increasing exposure (5 and 25 secs) which aids in maintaining the stemness property. The molecular mechanism underlying regeneration capability can assist in designing novel therapeutic applications.

2024-12-01·International Immunopharmacology

Effect of oxymatrine on neutrophil function based on zebrafish inflammation model and primary neutrophil inflammatory responses

Article

作者: Long, Yingxin ; Nie, Hong ; Xiao, Yuan ; Wang, Ranjing ; Zhao, Tingting ; Kong, Shang ; Cai, Kexin

Sophora flavescens Ait. (SFA), an extensively utilized herb for the treatment of fevers, inflammatory disorders, ulcers and skin diseases related to bur, contains oxymatrine (OMT) as its principal active constituent. OMT exerts regulatory effects over inflammation, oxidative stress and apoptosis. Neutrophils, critical regulators of the inflammation response, have not been thoroughly elucidated regarding the protective properties and underlying mechanisms of OMT-mediated anti-inflammation. This study was aim to explore the protective effect of OMT on neutrophils under inflammatory conditions and delve into its potential mechanism. Leveraging the advantages of zebrafish, an animal model with a real-time dynamic observation system, we established an in vivo caudal fin wound model and a copper sulfate induced-inflammation model in zebrafish line Tg (mpx:GFP). The result revealed that OMT significantly attenuated neutrophil migration, upregulated the mRNA expression levels of JNK, casp3, mapk14a, mapkapk2a and map2k1 damaged by zebrafish caudal fin wound model, and downregulated mRNA expression levels of JNK, casp3, mapk14a, mapkapk2a and map2k1 in the copper sulfate injury model. In vitro experiments demonstrated that OMT modulated the chemotaxis response of primary neutrophils from mice, enhanced phagocytosis, reduced oxidative stress and alleviated inflammation level. We hypothesize that the OMT may exert its anti-inflammatory effects by regulating primary neutrophils through the MAPK signaling pathway.

2024-12-01·Chemico-Biological Interactions

Oxymatrine inhibits migration and invasion of esophageal squamous cell carcinoma cell lines via the MEK1/ERK/β-catenin pathway

Article

作者: Dong, Zibo ; Dong, Jingquan ; Qiang, Jingchao ; Zhou, Mengyuan ; Qiu, Tian ; Li, Xing ; Lu, Yingzhi ; Yang, Rongrong ; Xu, Baoshi ; Yang, Yongliang

Esophageal, cancer is a prevalent malignant tumour of the digestive system in China, and esophageal squamous cell carcinoma (ESCC) accounts for 90 % of all esophageal cancer cases. Currently, the primary treatment involves surgical resection combined with postoperative radiotherapy. In this study, we used two ESCC cell lines to determine whether oxymatrine (OMT) inhibits ESCC, whether the mechanism involves the MEK1/ERK/β-catenin pathway, and how OMT modulates this pathway to affect the development of ESCC. The effects of OMT treatment were monitored with Cell Counting Kit-8 (CCK-8) assays as well as with clony formation, migration and invasion, wound healing, Hoechst 33258, and Western blot analyses. The relationship between OMT and the target was also evaluated by molecular docking and cell stability experiments. These findings suggest that ESCC development and metastasis may be inhibited by OMT and that OMT targets MEK1 through the ERK/β-catenin/EMT pathway to suppress ESCC cell migration and invasion. In addition, in vivo studies confirmed that OMT can inhibit the growth of ESCC cell lines in NOG mice without causing damage to other organs. In conclusion, in vitro experiments, revealed that OMT prevents the migration and invasiveness of ESCC cells by inhibiting the ERK/β-catenin/EMT pathway and thus targeting MAP2K1 (MEK1) in ESCC.

124

项与 MEK1 相关的新闻（医药）

2024-10-26

·精准药物

一文解析MAPK 通路

MAPK（丝裂原活化蛋白激酶）通路是细胞内一种重要的信号传导途径，广泛参与调控细胞生长、分化、死亡、应激反应等多种生物学过程。MAPK通路对于细胞应对外界环境变化具有核心作用，涉及到多种疾病的发生发展，包括癌症、炎症性疾病等。一、MAPK通路的组成 MAPK通路主要由三类蛋白激酶组成，它们按照特定的顺序激活： 1.MAPKKK（MAPK激酶激酶）：最上游的激酶，响应信号分子的刺激。 2.MAPKK（MAPK激酶）：由MAPKKK激活，进一步放大信号。 3.MAPK：最终的执行者，被MAPKK激活后，转移到细胞核内，通过磷酸化特定的基因调控蛋白，影响基因表达。图：MAPK通路的基本组成（来自Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer.）二、MAPK通路的主要分支 MAPK通路可以分为几个主要分支，每个分支由不同的MAPK蛋白构成，主要包括：ERK通路，JNK通路和p38 MAPK通路。 1. ERK通路（Extracellular signal-Regulated Kinases） ERK通路，也称为经典的MAPK通路，主要响应生长因子和一些其他刺激。它通常与细胞增殖和分化过程相关联。主要组成： ●上游激酶：包括Ras小GTPase，它激活RAF激酶（如BRAF）。 ●中间级激酶：RAF激活MEK1和MEK2（MAPK/ERK激酶）。 ●下游激酶：MEK激酶进一步激活ERK1和ERK2。生物学功能： ●细胞增殖：ERK通路的激活促进细胞周期蛋白的表达，推动细胞周期进程。 ●细胞分化：在某些细胞类型中，ERK激活是细胞分化必需的。 ●生存信号：ERK可以激活抗凋亡蛋白，如Bcl-2，帮助细胞逃避程序性细胞死亡。 MAPK/Erk 信号级联放大通过生长和分化中涉及的各种不同受体进行激活，包括受体酪氨酸激酶 (RTKs)、整合素以及离子通道。由不同的刺激物激活的级联反应的特定组分差异较大，但通路的结构通常包含一组对接蛋白（Shc、GRB2、Crk 等），它们将受体连接到一个鸟苷酸交换因子上（SOS、C3G 等），信号则转导至小分子 GTP 结合蛋白 (Ras，Rap1)，后者再激活级联中的核心单位，包括一个 MAPKKK (Raf)、一个 MAPKK (MEK1/2) 和 MAPK (Erk)。激活的 Erk 二聚物可在胞质中调控靶蛋白，也可转运入胞核，在核内磷酸化多种调控基因表达的转录因子。图：生长和分化中的 MAPK/Erk（来自Cell signaling） 2. JNK通路（c-Jun N-terminal Kinases） JNK通路，也称为应激激活蛋白激酶通路，主要响应应激信号，如紫外线照射、热休克和炎症因子。主要组成： ●上游激酶：包括MKK4和MKK7。 ●下游激酶：它们激活JNK1、JNK2和JNK3。生物学功能： ●细胞应激反应：JNK通路在细胞对环境应激做出反应中发挥关键作用。 ●调节细胞死亡：JNK的活化通常与细胞凋亡相关，尤其是在病理条件下。 ●炎症反应：JNK能够调节多种炎症细胞因子的表达。应激活化蛋白激酶 (SAPK)/Jun 氨基末端激酶 (JNK) 是 MAPK 家族的成员，可由各种不同环境应激、炎症细胞因子、生长因子以及 GPCR 激动剂激活。应激反应信号经 Rho 家族（Rac、Rho、cdc42）的小分子 GTP 酶传递到这个级联。和其他 MAPK 一样，膜近端激酶是一个 MAPKKK（通常为 MEKK1–4），或是混合性谱系激酶（MLK）中一员，能磷酸化并激活 MKK4 (SEK) 或 MKK7（即 SAPK/JNK 激酶）。此外， MKK4/7 能由生发中心激酶 (GCK) 家族成员以独立于 GTP 酶的方式激活。SAPK/JNK 转运入胞核后可调节多种转录因子的活性。图：SAPK/JNK信号级联放大（来自Cell signaling） 3. p38 MAPK通路 p38 MAPK通路同样响应应激信号，包括细胞因子和环境应激，常与细胞生存和炎症反应相关。主要组成： ●上游激酶：包括MKK3、MKK4和MKK6。 ●下游激酶：p38 MAPK有四个不同的同源体：p38α、p38β、p38γ和p38δ。生物学功能： ●炎症反应：p38 MAPK是许多炎症介质表达的调节者，对免疫细胞的活化起重要作用。 ●细胞周期调控：p38在某些情况下可以抑制细胞周期的进程，从而影响细胞增殖。 ●细胞分化：特别是在骨骼肌细胞和神经细胞的分化中，p38 MAPK的激活对某些分化途径至关重要。 p38 MAPK（α、β、γ 和 δ）是 MAPK 家族的成员，各种环境应激和炎症细胞因子均可将其激活。对于其他 MAPK 级联，膜近端组分是一个 MAPKKK，其为典型的 MEKK 或混合性谱系激酶 (MLK)。MAPKKK 磷酸化并激活 MKK3/6，后者即是 p38 MAPK 激酶。MKK3/6 也可在凋亡因素的刺激下直接由 ASK1 激活。p38 MAPK 参与调节 HSP27、MAPKAPK-2 (MK2)、MAPKAPK-3 (MK3) 和其他几种转录因子，包括 ATF-2、Stat1、Max/Myc 复合体、MEF-2 和 Elk-1，而 CREB 通过激活 MSK1 间接调控。图：p38 MAPK信号转导相互作用通路（来自Cell signaling）三、MAPK通路的激活机制 MAPK通路通常是通过细胞表面的受体如受体酪氨酸激酶（RTKs）或G蛋白偶联受体（GPCRs）接收信号，触发一系列下游事件： 1.受体激活：受到外界信号分子（如生长因子）的结合后，受体自身活化或聚合，激活与其相连的适配蛋白。 2.Ras蛋白的激活：在ERK通路中，适配蛋白进一步激活小G蛋白Ras。 3.MAPKKK的激活：Ras激活RAF家族蛋白（MAPKKK的一种），后者激活MEK（MAPKK）。 4.MAPK的激活：MEK进一步激活ERK，ERK磷酸化核内的靶蛋白，如转录因子，从而改变基因表达。四、MAPK通路的生理与病理意义 MAPK通路在维持正常细胞功能和发展中起着关键作用，但当这些通路异常激活或抑制时，可能导致多种疾病的发生： ●癌症 MAPK通路的持续激活常见于多种肿瘤，促进肿瘤细胞的增殖和存活。 MAPK信号通路与细胞增殖和分化密切相关。在许多恶性肿瘤中发现MAPK信号的持续激活。细胞增殖在很大程度上有助于肿瘤发生。肿瘤转移和侵袭是致命原因。在哺乳动物MAPK通路中，ERK通路是研究最充分的，在大约三分之一的人类癌症中失调。 ERK1/2通路在肿瘤细胞的增殖、分化、转移和侵袭中起重要作用。温等人发现乙型肝炎病毒x蛋白通过p38/MAPK通路使p53基因失活，从而诱导原发性肝癌（PLC）。Kim MS等人证明，p38 MAPK是Ras诱导的乳腺癌细胞侵袭和转移的关键信号分子。许多研究表明，p38 MAPK通路在细胞外刺激产生的MMP（基质金属蛋白酶）中发挥重要的调节功能。MMP是一种与肿瘤侵袭和迁移密切相关的蛋白水解酶。此外，p38 MAPK通路促进维持肿瘤细胞生长所必需的肿瘤血管生成。癌症经常暴露于各种压力条件下，例如缺氧和炎症。许多MAPK通路都参与应激信号传导。因此，一些激酶在癌症中被应激激活，以响应炎症、DNA 损伤和细胞凋亡。缺氧状态在实体瘤中广泛存在。缺氧信号刺激MKP-1的激活。MKP-1 进一步激活 SAPK/JNK，从而激活 c-Jun。活性 c-Jun 促进多个下游基因转录，这对癌细胞的增殖和存活很重要。 MAPK信号转导的畸变会影响大多数致癌过程，对癌症的发展和进展至关重要。大多数癌症相关病变，如受体酪氨酸激酶的过表达、受体酪氨酸激酶的激活突变、激活配体的持续自分泌或旁分泌产生、Ras 突变和 B-Raf 突变导致 ERK 信号转导的组成型激活。据报道，c-Jun 的 JNK 活性和磷酸化在 Ras 诱导的肿瘤发生中起关键作用。 ●炎症性疾病在炎症性疾病中，MAPK通路通过影响细胞因子、生长因子和应激反应等因素的表达和活性，起到了至关重要的调控作用。 MAPK通路参与调控包括肿瘤坏死因子α（TNF-α）、白细胞介素（如IL-1, IL-6）等多种炎症性细胞因子的产生。这些细胞因子是炎症反应中的关键介质，能够激活更多的免疫细胞和促进炎症反应的发展。MAPK通路通过调节各种趋化因子和黏附分子的表达，影响免疫细胞（如巨噬细胞、T细胞）的活化、迁移和入侵到炎症部位。在炎症和其他应激条件下，JNK和p38 MAPK的激活促进细胞应对损伤和恢复平衡。由于MAPK通路在炎症反应中的核心角色，研究者开发了多种针对该路径的抑制剂，以治疗如类风湿性关节炎、银屑病等炎症性疾病。例如，p38 MAPK抑制剂已被证明可以减轻炎症反应，通过减少炎症性细胞因子的产生。 ●神经退行性疾病在神经退行性疾病的研究中，MAPK通路显示出其在神经保护和神经损伤中的双重作用，这些疾病包括阿尔茨海默病（AD）、帕金森病（PD）、亨廷顿病（HD）和肌萎缩侧索硬化症（ALS）等。 AD中，异常的tau蛋白磷酸化与MAPK通路的过度活化有关。特别是JNK和p38 MAPK在tau磷酸化和神经纤维缠结的形成中起着重要作用。PD中，JNK和p38 MAPK的活化与多巴胺神经元的死亡相关。MAPK通路参与了氧化应激和炎症反应的调控，这些都是PD的关键病理过程。在HD中，JNK的激活与疾病相关的神经毒性密切相关。JNK抑制剂显示出在预防神经退行方面的潜力。五、MAPK通路的治疗应用由于MAPK通路在多种疾病中的核心作用，该通路的组分成为了药物开发的重要靶点。目前已有多种针对MAPK通路的抑制剂被开发用于治疗特定类型的癌症（如BRAF抑制剂在黑色素瘤中的应用）和其他疾病。此外，针对JNK和p38 MAPK的抑制剂也在被研究中，以期治疗炎症和神经退行性疾病。六、总结 MAPK通路是一条复杂的信号传导途径，涉及到多种生理和病理过程。对其深入研究不仅有助于我们理解细胞如何响应外界信号并调控各种细胞功能，还可以为疾病的预防和治疗提供新的策略。随着研究的深入，未来可能会发现更多的关于这一信号通路的调控机制和治疗潜力。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↓

2024-10-12

·生物探索

Science | 解旋酶介导的连续突变系统：实现哺乳动物细胞上的原位定向进化

引言人类基因组中存在多达三十亿的碱基对，因此在基因组学的核心挑战和最终愿景，是描述该三十亿碱基对中，每一个碱基对于特定基因调控及下游蛋白质功能的影响。因此，如何系统且高通量地对目标基因组序列进行诱导突变，成为一项核心技术挑战。当研究者聚焦于单一基因组位点时，如何利用区域性的靶向诱变，模拟自然进化过程，揭示序列与表型之间的关系，领域内仍然缺乏较好的技术平台。 2024年10月11日，来自博德研究所（Broad Institute）和哈佛大学丹娜法博癌症研究中心（Dana-Farber Cancer Institute）的Fei Chen和Bradley E. Bernstein研究组在Science上在线发表题目为Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes的研究论文（哈佛大学博士研究生Xi Dawn Chen和博后研究员陈则宇为共同第一作者），通过融合解旋酶（Helicase）和脱氨酶（deaminase）形成解旋编辑器（Helicase Editor），并通过nCas9或dCas9将解旋酶编辑器引导至特定基因组靶向区域，成功实现了在人类细胞上原位基因组上的长程，位置饱和的散在诱导突变，创立了在哺乳动物细胞上的原位定向进化系统，并在多种人类细胞系上实现筛选抗药性单碱基突变，RNA剪切酶活位点和原位增强子中的功能性碱基等重要生物学过程。研究人员首先选择了PcrA M6解旋酶作为切入点，融合了可诱导C->U突变（DNA复制后体现为C->T或反链的G->A）的胞苷脱氨酶AID形成HE，并将靶向特定基因组位点的sgRNA，nCas9(D10A)和HE转染进入HEK293T细胞并突变72小时。研究人员发现只有细胞同时存在HE系统和nCas9-sgRNA系统时，目标靶向区域才会被显著突变。该突变方向以单侧为主，长度可达1kb以上，突变率平均在每个碱基0.5%左右。其中反链的G->A突变为主导突变类型，随着突变时间延长，每条单链上的突变也会随之累积。因此，研究人员将该系统命名为HACE（Helicase-assisted continuous editing）系统，即解旋酶介导的连续突变系统。接下来，研究人员建立了模块化的HACE体系，搭建了不同解旋酶（BLM,NS3h,PcrA, PcrA M6等），不同脱氨酶（AID, rAPOBEC, TadA, TadDE）及不同Cas9（nCas9 D10A, nCas9 H840A, dCas9）的功能性描述矩阵，系统性的描述了每个组合条件下的突变率，突变方向，G->A和C->T的偏好，同时也实现了A->G或T->C的突变，使得HACE系统可在不同使用场景下，根据目标区域的突变预期特点，来进行工具盒式的置换，实现不同目的的单碱基筛选。下一步，研究人员将HACE系统用于癌细胞的耐药性机制筛选中。研究人员将靶向MEK1外显子区域的HACE系统导入到A375细胞中，并使用Trametinib和Selumetinib两个MEK1抑制剂进行耐药性点突变筛选。在经历长达20天的耐药性筛选后，研究人员发现存活下来的A375细胞中，富集了G128D，G202E，E203K等多个点突变。利用单碱基编辑器和MAPK通路的活性报告系统验证发现，这些点突变可显著增强MAPK活性，并促进细胞在药物环境下生存和扩增。PDB结构分析进一步发现，以上突变位点均落在药物结合区域周围，阐释了MEK1突变的抗药机制。进一步的，研究人员将HACE系统应用于鉴定影响RNA剪切的体内酶活突变位点。研究人员将RNA剪切报告系统导入到HEK293FT细胞中，同时设计好靶向SF3B1的多个sgRNA，再将HACE系统整体导入含有报告系统的细胞中。通过报告系统中被异常剪切的mRNA（GFP报告）强度进行亚群分选，筛选出对于RNA剪切有重要影响的细胞亚群，并对于其靶向区域的基因型进行鉴定。研究人员发现并用单碱基编辑和先导编辑验证了多个可影响RNA剪切的SF3B1突变位点，并在PDB结构分析中确认了这些突变所属的蛋白区域是与RNA结合相关的部分。最后，研究人员设计了多条靶向CD69增强子的sgRNA，并利用HACE系统对于CD69基因的增强子进行突变筛选，并找到多个重要的转录因子结合区域，如ETS/IRF, GATA, RUNX等，揭示了这些转录因子家族可对CD69基因表达产生重要影响。进一步的，在探究RUNX结合区域是如何影响CD69表达的过程中，研究人员发现HACE所筛选的单碱基突变没有强制的人工连锁，而用于验证的单碱基编辑器，由于突变存在强制的人工连锁，则在破坏RUNX结合域的同时，又引入了GATA结合区域，导致单碱基编辑在分子库筛选中大概率无法识别该重要调控区域。因此，研究人员之后采用先导编辑，完善了对HACE筛选位点的系统性验证环节。模式图（Credit: Science）综上，HACE是可编程，可多靶向，可连续突变，低频，有方向性，靶区域内随机，位置饱和，罕见强制连锁的连续突变器，可实现哺乳动物原位基因组的定向进化。参考文献 http://doi.org.libproxy1.nus.edu.sg/10.1126/science.adn5876 责编|探索君排版|探索君文章来源|“BioArt” End 往期精选围观一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版）热文 Cell | 是什么决定了细胞的大小？热文 Nature | 2024年值得关注的七项技术热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望热文 CRISPR技术进化史 | 24年Cell综述

基因疗法临床研究

2024-09-12

·drugs

Ingredient in Hair Dye Led to Woman's Vision Loss

THURSDAY, Sept. 12, 2024 -- An ingredient in the hair dye a French woman used caused her to develop a vision-robbing retinopathy , researchers report. When she switched to a dye without these ingredients, called aromatic amines, her vision troubles resolved, according to a team led by Dr. Nicolas Chirpaz , an ophthalmologist at Edouard Herriot Hospital, in Lyon. Such cases could be rare, the researchers said, but spreading awareness of the danger "may allow prompt consideration to remove exposure to such hair dye" so eyes aren't permanently damaged, Chirpaz and colleagues said. They published their case report Sept. 12 in the journal JAMA Ophthalmology . As the French team noted, this isn't the first time hair dye has been linked to retinopathy: Three prior cases were reported in 2022 among "middle-aged women following exposure to hair dyes containing aromatic amines." In the latest case, a 61-year-old woman with no prior history of vision trouble came to doctors with progressively blurry vision in both eyes "a few days after dyeing her hair with hair dye containing aromatic amines," Chirpaz' group said. The aromatic amine in the dye used in this case was para-phenylenediamine. Upon examination, the woman was found to have multiple retinal detachments that resembled the damage that can occur in retinopathies that are tied to certain enzymes found in the eye, called MEK1 or MEK2. The woman's eyes also displayed an unhealthy "thickening of the neurosensory retina," the French team found. Tests were conducted to rule out a host of possible causes, including infections and even cancer. In the end, retinal damage caused by the ingredient in hair dye was "diagnosed based on the temporal association between symptoms and hair dye exposure," the team said. The woman soon switched her brand of hair dye, and her vision returned to 20/20 within a month. "Four years later, the patient reported using aromatic amines–free hair dye and has not experienced any recurrence," the study authors said. Chirpaz' team believes that aromatic amine chemicals such as para-phenylenediamine "disrupt" a neurochemical pathway that's essential to the health of what are known as retinal pigment epithelial cells. Cases of "retinopathy associated with the use of hair dye aromatic amines [RAHDAA]" remain rare, but doctors should be on the alert to the possibility when people show up with any retinopathy that doesn't have an easy explanation, the French team said. Whatever your topic of interest, subscribe to our newsletters to get the best of Drugs.com in your inbox.

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