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Size-Specific Modulation of a Multienzyme Glucosome Assembly during the Cell Cycle 细胞周期中多酶糖体组装的尺寸特异性调节
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-08-08 DOI: 10.1021/acsbiomedchemau.3c00037
Miji Jeon, Danielle L. Schmitt, Minjoung Kyoung and Songon An*, 

Enzymes in glucose metabolism have been subjected to numerous studies, revealing the importance of their biological roles during the cell cycle. However, due to the lack of viable experimental strategies for measuring enzymatic activities particularly in living human cells, it has been challenging to address whether their enzymatic activities and thus anticipated glucose flux are directly associated with cell cycle progression. It has remained largely elusive how human cells regulate glucose metabolism at a subcellular level to meet the metabolic demands during the cell cycle. Meanwhile, we have characterized that rate-determining enzymes in glucose metabolism are spatially organized into three different sizes of multienzyme metabolic assemblies, termed glucosomes, to regulate the glucose flux between energy metabolism and building block biosynthesis. In this work, we first determined using cell synchronization and flow cytometric techniques that enhanced green fluorescent protein-tagged phosphofructokinase is adequate as an intracellular biomarker to evaluate the state of glucose metabolism during the cell cycle. We then applied fluorescence single-cell imaging strategies and discovered that the percentage of Hs578T cells showing small-sized glucosomes is drastically changed during the cell cycle, whereas the percentage of cells with medium-sized glucosomes is significantly elevated only in the G1 phase, but the percentage of cells showing large-sized glucosomes is barely or minimally altered along the cell cycle. Should we consider our previous localization–function studies that showed assembly size-dependent metabolic roles of glucosomes, this work strongly suggests that glucosome sizes are modulated during the cell cycle to regulate glucose flux between glycolysis and building block biosynthesis. Therefore, we propose the size-specific modulation of glucosomes as a behind-the-scenes mechanism that may explain functional association of glucose metabolism with the cell cycle and, thereby, their metabolic significance in human cell biology.

葡萄糖代谢中的酶已经进行了大量的研究,揭示了它们在细胞周期中生物学作用的重要性。然而,由于缺乏测量酶活性的可行实验策略,特别是在活的人类细胞中,解决它们的酶活性以及由此预期的葡萄糖流量是否与细胞周期进展直接相关一直是一个挑战。人类细胞如何在亚细胞水平上调节葡萄糖代谢以满足细胞周期中的代谢需求,在很大程度上仍然难以捉摸。同时,我们已经表征了葡萄糖代谢中的速率决定酶在空间上被组织成三种不同大小的多酶代谢组装体,称为葡糖体,以调节能量代谢和构建块生物合成之间的葡萄糖流量。在这项工作中,我们首先使用细胞同步和流式细胞术技术确定,增强的绿色荧光蛋白标记的磷酸果糖激酶足以作为细胞内生物标志物来评估细胞周期中的葡萄糖代谢状态。然后,我们应用荧光单细胞成像策略,发现显示小尺寸葡糖体的Hs578T细胞的百分比在细胞周期中发生了显著变化,而具有中等尺寸葡糖团的细胞的百分比仅在G1期显著升高,但是显示出大尺寸葡糖体的细胞的百分比在细胞周期中几乎或最低限度地改变。如果我们考虑一下我们之前的定位-功能研究,这些研究表明葡萄糖体的组装大小依赖于代谢作用,这项工作强烈表明,葡萄糖体的大小在细胞周期中受到调节,以调节糖酵解和构建块生物合成之间的葡萄糖流量。因此,我们提出葡萄糖体的大小特异性调节是一种幕后机制,可以解释葡萄糖代谢与细胞周期的功能关联,从而解释其在人类细胞生物学中的代谢意义。
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引用次数: 0
Functional Diversity in Radiolabeled Nanoceramics and Related Biomaterials for the Multimodal Imaging of Tumors 放射标记纳米陶瓷和相关生物材料在肿瘤多模态成像中的功能多样性
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-08-08 DOI: 10.1021/acsbiomedchemau.3c00021
David G. Calatayud*, Marina Lledos, Federico Casarsa and Sofia I. Pascu*, 

Nanotechnology advances have the potential to assist toward the earlier detection of diseases, giving increased accuracy for diagnosis and helping to personalize treatments, especially in the case of noncommunicative diseases (NCDs) such as cancer. The main advantage of nanoparticles, the scaffolds underpinning nanomedicine, is their potential to present multifunctionality: synthetic nanoplatforms for nanomedicines can be tailored to support a range of biomedical imaging modalities of relevance for clinical practice, such as, for example, optical imaging, computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET). A single nanoparticle has the potential to incorporate myriads of contrast agent units or imaging tracers, encapsulate, and/or be conjugated to different combinations of imaging tags, thus providing the means for multimodality diagnostic methods. These arrangements have been shown to provide significant improvements to the signal-to-noise ratios that may be obtained by molecular imaging techniques, for example, in PET diagnostic imaging with nanomaterials versus the cases when molecular species are involved as radiotracers. We surveyed some of the main discoveries in the simultaneous incorporation of nanoparticulate materials and imaging agents within highly kinetically stable radio-nanomaterials as potential tracers with (pre)clinical potential. Diversity in function and new developments toward synthesis, radiolabeling, and microscopy investigations are explored, and preclinical applications in molecular imaging are highlighted. The emphasis is on the biocompatible materials at the forefront of the main preclinical developments, e.g., nanoceramics and liposome-based constructs, which have driven the evolution of diagnostic radio-nanomedicines over the past decade.

纳米技术的进步有可能有助于早期发现疾病,提高诊断的准确性,并有助于个性化治疗,尤其是在癌症等非传染性疾病的情况下。纳米颗粒是支撑纳米药物的支架,其主要优势是其具有多功能性的潜力:纳米药物的合成纳米平台可以定制为支持一系列与临床实践相关的生物医学成像模式,例如光学成像、计算机断层扫描(CT)、磁共振成像(MRI),单光子发射计算机断层扫描(SPECT)和正电子发射断层扫描(PET)。单个纳米颗粒有可能结合大量造影剂单元或成像示踪剂,封装和/或结合到成像标签的不同组合,从而为多模态诊断方法提供手段。与分子物种作为放射性示踪剂参与的情况相比,这些布置已被证明对可以通过分子成像技术获得的信噪比提供了显著的改进,例如,在使用纳米材料的PET诊断成像中。我们调查了在高度动力学稳定的放射性纳米材料中同时掺入纳米颗粒材料和成像剂作为具有(预)临床潜力的潜在示踪剂的一些主要发现。探索了功能的多样性和合成、放射性标记和显微镜研究的新进展,并强调了分子成像的临床前应用。重点是处于主要临床前发展前沿的生物相容性材料,例如纳米陶瓷和基于脂质体的构建体,它们在过去十年中推动了诊断性放射性纳米药物的发展。
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引用次数: 0
Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents 结核分枝杆菌吲哚-3-甘油磷酸合酶活性位点残基在抗结核药物潜在靶点中的作用研究
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-07-26 DOI: 10.1021/acsbiomedchemau.3c00029
David W. Konas, Sarah Cho, Oshane D. Thomas, Maryum M. Bhatti, Katherine Leon Hernandez, Cinthya Moran, Hedda Booter, Thomas Candela, Joseph Lacap, Paige McFadden, Savannah van den Berg, Alyssa M. Welter, Ashley Peralta, Cheryl A. Janson, Jaclyn Catalano and Nina M. Goodey*, 

Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

结核分枝杆菌耐药性正在出现,需要新的药物靶点。色氨酸的生物合成是结核分枝杆菌复制和毒力所必需的。吲哚-3-甘油磷酸合成酶(IGPS)催化结核分枝杆菌色氨酸生物合成的一个步骤,并被认为是一个潜在的抗感染靶点,但我们对这种酶的了解有限。为了帮助抑制剂的设计并获得这种酶的更深入的机制,需要了解活性位点氨基酸在配体结合和催化中的作用。在这项工作中,我们探索了保守的活性位点氨基酸Glu57、Lys59、Lys119、Glu168和Glu219的作用。每个到Ala的突变导致所有可检测活性的丧失。Glu57Gln、Lys59Arg、Lys119Arg、Glu168Gln和Glu219Asp突变导致大的活性损失,而Glu219Gln具有增强的活性。酶数据的分析得出以下主要结论:(A)Lys119可能是CdRP环闭合步骤中的催化酸。(B) Glu168稳定带电的反应中间体,也可以是催化碱。(C) Glu57、Glu219和Lys119形成紧密排列的三联体,其中Glu57和Glu219调节Lys119的pKa,从而调节整体活性。这增加了对分子间和分子内相互作用的理解,并证明了结核分枝杆菌IGPS活性位点的高度协调性,为未来研究这一潜在的新药靶点提供了新的机制信息和指导。
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引用次数: 0
Chemoenzymatic Synthesis of 3′-Deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP) 化学酶法合成3′-脱氧-3′,4′-二脱氢胞苷三磷酸(ddhCTP)
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-07-25 DOI: 10.1021/acsbiomedchemau.3c00014
James H. Lee, James M. Wood, Steven C. Almo, Gary B. Evans, Lawrence D. Harris and Tyler L. Grove*, 

3′-Deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin during the early stages of the innate immune response. ddhCTP has been shown to act as a chain terminator of flavivirus RNA-dependent RNA polymerases. To date, synthesis of ddhCTP requires complicated synthetic protocols or isolation of the enzyme viperin to catalyze the production of ddhCTP from CTP. Recombinant viperin approaches preclude the production of highly pure ddhCTP (free of contaminants such as CTP), whereas the chemical synthesis involves techniques or equipment not readily available to most laboratories. Herein, we describe the chemoenzymatic synthesis of ddhCTP, starting from commercially available ddhC. We utilize these methods to produce milligram quantities of ddhCTP, ddhCDP, and ddhCMP. Using purified semisynthetic ddhCTP and fully synthetic ddhCTP, we also show ddhCTP does not inhibit NAD+-dependent enzymes such as glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, or lactate dehydrogenase, contrary to a recent report.

3′-脱氧-3′,4′-二氢胞苷三磷酸(ddhCTP)是一种新型抗病毒分子,由viperin酶在先天免疫反应的早期阶段产生。ddhCTP已被证明是黄病毒RNA依赖性RNA聚合酶的链终止子。迄今为止,ddhCTP的合成需要复杂的合成方案或分离酶viperin来催化由CTP产生ddhCTP。重组viperin方法排除了高纯度ddhCTP(不含CTP等污染物)的生产,而化学合成涉及大多数实验室无法获得的技术或设备。在此,我们描述了ddhCTP的化学酶合成,从市售的ddhC开始。我们利用这些方法生产毫克量的ddhCTP、ddhCDP和ddhCMP。与最近的一份报告相反,使用纯化的半合成ddhCTP和全合成ddhCT,我们还表明ddhCTP不会抑制NAD+依赖性酶,如甘油醛3-磷酸脱氢酶、苹果酸脱氢酶或乳酸脱氢酶。
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引用次数: 0
Targeting SHP2 with an Active Site Inhibitor Blocks Signaling and Breast Cancer Cell Phenotypes 用活性位点抑制剂靶向SHP2阻断信号传导和乳腺癌细胞表型
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-07-14 DOI: 10.1021/acsbiomedchemau.3c00024
Dhanaji M. Lade,  and , Yehenew M. Agazie*, 

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an oncogenic protein for which targeted therapies are being sought. In line with this idea, we have previously reported the development of a specific active site inhibitor named CNBDA that showed effectivity in suppressing the transformation phenotypes of breast cancer cells. To improve efficacy, we introduced limited modifications to the parent compound and tested potency in vitro and under cell culture conditions. Of these modifications, removal of one of the butyric acid groups led to the production of a compound named CNBCA, which showed a 5.7-fold better potency against the SHP2 enzyme activity in vitro. In addition, CNBCA showed better selectivity to SHP2 than the control PTPs (SHP1 and PTP1B) as determined by the phosphatase assay. Furthermore, CNBCA binds and inhibits enzyme activity of full-length SHP2 in cellular contexts, downregulates SHP2 mediated signaling, and suppresses breast cancer cell phenotypes, including cell proliferation, colony formation, and mammosphere growth. These findings show that targeting SHP2 with CNBCA is effective against the cancerous properties of breast cancer cells.

Src同源磷酸酪氨酸磷酸酶2(SHP2)是一种正在寻求靶向治疗的致癌蛋白。根据这一想法,我们之前报道了一种名为CNBDA的特异性活性位点抑制剂的开发,该抑制剂在抑制癌症细胞的转化表型方面表现出有效性。为了提高效力,我们对母体化合物进行了有限的修饰,并在体外和细胞培养条件下测试了效力。在这些修饰中,去除一个丁酸基团导致产生了一种名为CNBCA的化合物,该化合物在体外对SHP2酶活性的效力提高了5.7倍。此外,通过磷酸酶测定测定,CNBCA对SHP2表现出比对照PTP(SHP1和PTP1B)更好的选择性。此外,CNBCA在细胞环境中结合并抑制全长SHP2的酶活性,下调SHP2介导的信号传导,并抑制乳腺癌症细胞表型,包括细胞增殖、集落形成和乳腺球生长。这些发现表明,用CNBCA靶向SHP2对乳腺癌症细胞的癌性是有效的。
{"title":"Targeting SHP2 with an Active Site Inhibitor Blocks Signaling and Breast Cancer Cell Phenotypes","authors":"Dhanaji M. Lade,&nbsp; and ,&nbsp;Yehenew M. Agazie*,&nbsp;","doi":"10.1021/acsbiomedchemau.3c00024","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.3c00024","url":null,"abstract":"<p >The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an oncogenic protein for which targeted therapies are being sought. In line with this idea, we have previously reported the development of a specific active site inhibitor named CNBDA that showed effectivity in suppressing the transformation phenotypes of breast cancer cells. To improve efficacy, we introduced limited modifications to the parent compound and tested potency <i>in vitro</i> and under cell culture conditions. Of these modifications, removal of one of the butyric acid groups led to the production of a compound named CNBCA, which showed a 5.7-fold better potency against the SHP2 enzyme activity <i>in vitro</i>. In addition, CNBCA showed better selectivity to SHP2 than the control PTPs (SHP1 and PTP1B) as determined by the phosphatase assay. Furthermore, CNBCA binds and inhibits enzyme activity of full-length SHP2 in cellular contexts, downregulates SHP2 mediated signaling, and suppresses breast cancer cell phenotypes, including cell proliferation, colony formation, and mammosphere growth. These findings show that targeting SHP2 with CNBCA is effective against the cancerous properties of breast cancer cells.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49768451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides 细胞色素P450与核糖体合成和翻译后修饰肽的生物合成有关
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-07-13 DOI: 10.1021/acsbiomedchemau.3c00026
Guannan Zhong*, 

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of exponentially increased natural products with characteristic chemical structures, topologies, and biosynthetic mechanisms as well as exceptional bioactivities including antibacteria, antitumors, and antiviruses. The biosynthesis of RiPP proceeds via a ribosomally assembled precursor peptide that undergoes varied post-translational modifications to generate a mature peptide. Cytochrome P450 (CYP or P450) monooxygenases are a superfamily of heme-containing enzymes that span a wide range of secondary metabolite biosynthetic pathways due to their broad substrate scopes and excellent catalytic versatility. In contrast to the enormous quantities of RiPPs and P450s, the P450 associated RiPP biosynthesis is comparatively limited, with most of their functions and timings remaining mysterious. Herein, this Review aims to provide an overview on the striking roles of P450s in RiPP biosyntheses uncovered to date and to illustrate their remarkable functions, mechanisms, as well as remaining challenges. This will shed light on novel P450 discovery and characterizations in RiPP biosyntheses.

核糖体合成和翻译后修饰肽(RiPP)是一类呈指数增长的天然产物,具有独特的化学结构、拓扑结构和生物合成机制,以及特殊的生物活性,包括抗菌、抗肿瘤和抗病毒。RiPP的生物合成通过核糖体组装的前体肽进行,该前体肽经历各种翻译后修饰以产生成熟肽。细胞色素P450(CYP或P450)单加氧酶是一个含血红素酶的超家族,由于其广泛的底物范围和优异的催化多功能性,它跨越了广泛的次级代谢产物生物合成途径。与大量的RiPP和P450形成对比的是,与P450相关的RiPP生物合成相对有限,它们的大部分功能和时间仍然神秘。在此,本综述旨在概述P450在迄今为止发现的RiPP生物合成中的显著作用,并说明其显著的功能、机制以及剩余的挑战。这将有助于阐明新的P450发现和RiPP生物合成中的特征。
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引用次数: 1
Hydrogen Sulfide Responsive Phototherapy Agents: Design Strategies and Biological Applications 硫化氢反应性光治疗剂:设计策略和生物学应用
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-06-23 DOI: 10.1021/acsbiomedchemau.3c00028
Musa Dirak, Sarp E. Turan and Safacan Kolemen*, 

Hydrogen sulfide (H2S) is one of the critical gasotransmitters, which play important roles in regular physiological processes, especially in vital signaling pathways. However, fluctuations in endogenous H2S concentration can be linked to serious health problems, such as neurodegenerative diseases, cancer, diabetes, inflammation, cardiovascular diseases, and hypertension. Thus, it has attracted a great deal of attention in therapeutic applications, specifically in the field of phototherapy. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two subclasses of phototherapy, which utilize either reactive oxygen species (ROS) or local temperature increase upon irradiation of a photosensitizer (PS) to realize the therapeutic action. Phototherapies offer unique advantages compared to conventional methods; thus, they are highly promising and popular. One of the design principles followed in new generation PSs is to build activity-based PSs, which stay inactive before getting activated by disease-associated stimuli. These activatable PSs dramatically improve the selectivity and efficacy of the therapy. In this review, we summarize small molecule and nanomaterial-based PDT and PTT agents that are activated selectively by H2S to initiate their cytotoxic effect. We incorporate single mode PDT and PTT agents along with synergistic and/or multimodal photosensitizers that can combine more than one therapeutic approach. Additionally, H2S-responsive theranostic agents, which offer therapy and imaging at the same time, are highlighted. Design approaches, working principles, and biological applications for each example are discussed in detail.

硫化氢(H2S)是一种重要的气体递质,在正常的生理过程中发挥着重要作用,尤其是在重要的信号通路中。然而,内源性H2S浓度的波动可能与严重的健康问题有关,如神经退行性疾病、癌症、糖尿病、炎症、心血管疾病和高血压。因此,它在治疗应用中,特别是在光疗领域引起了极大的关注。光动力疗法(PDT)和光热疗法(PTT)是光疗的两个亚类,它们利用活性氧(ROS)或光敏剂(PS)照射后的局部温度升高来实现治疗作用。与传统方法相比,光疗法具有独特的优势;因此,它们非常有前途和受欢迎。新一代PS遵循的设计原则之一是构建基于活动的PS,它在被疾病相关刺激激活之前保持不活动状态。这些可激活的PS显著提高了治疗的选择性和疗效。在这篇综述中,我们总结了基于小分子和纳米材料的PDT和PTT试剂,它们被H2S选择性激活以启动其细胞毒性作用。我们结合了单模PDT和PTT药物,以及可以结合多种治疗方法的协同和/或多模式光敏剂。此外,强调了同时提供治疗和成像的H2S反应性治疗剂。详细讨论了每个例子的设计方法、工作原理和生物学应用。
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引用次数: 0
Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3 m6A-RNA写入酶METTL3抑制剂的结构设计
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-06-14 DOI: 10.1021/acsbiomedchemau.3c00023
Rajiv Kumar Bedi, Danzhi Huang, Yaozong Li and Amedeo Caflisch*, 

Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, N6-methyladenosine (m6A). METTL3 is the catalytic subunit that binds the co-substrate S-adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The m6A modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay. There is increasing evidence for an oncogenic role of METTL3 in acute myeloid leukemia. Here, we use structural and dynamic details of the catalytic subunit METTL3 for developing small-molecule inhibitors that compete with SAM. Starting from a hit identified by high-throughput docking, protein crystallography and molecular dynamics simulations were employed to guide the optimization of inhibitory activity. The potency was successfully improved by 8000-fold as measured by a homogeneous time-resolved fluorescence assay. The optimized compound is selective against the off-targets RNA methyltransferases METTL1 and METTL16.

甲基转移酶样3(METTL3)和METTL14形成异二聚体复合物,催化最丰富的内部信使核糖核酸修饰N6-甲基腺苷(m6A)。METTL3是结合共底物S-腺苷甲硫氨酸(SAM)的催化亚基,而METTL14参与mRNA结合。m6A修饰提供了对基因表达的转录后水平控制,因为它几乎影响mRNA生命周期的所有阶段,包括剪接、核输出、翻译和衰变。越来越多的证据表明METTL3在急性髓系白血病中具有致癌作用。在这里,我们使用催化亚基METTL3的结构和动力学细节来开发与SAM竞争的小分子抑制剂。从高通量对接确定的命中开始,蛋白质晶体学和分子动力学模拟被用于指导抑制活性的优化。通过均匀时间分辨荧光测定法测量,效价成功地提高了8000倍。优化的化合物对脱靶RNA甲基转移酶METTL1和METTL16具有选择性。
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引用次数: 1
Chemical Tools to Image the Activity of PAR-Cleaving Proteases 化学工具成像par切割蛋白酶的活性
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-05-27 DOI: 10.1021/acsbiomedchemau.3c00019
Irene Y. Lee, Piyapa Tantisirivat and Laura E. Edgington-Mitchell*, 

Protease-activated receptors (PARs) comprise a family of four G protein-coupled receptors (GPCRs) that have broad functions in health and disease. Unlike most GPCRs, PARs are uniquely activated by proteolytic cleavage of their extracellular N termini. To fully understand PAR activation and function in vivo, it is critical to also study the proteases that activate them. As proteases are heavily regulated at the post-translational level, measures of total protease abundance have limited utility. Measures of protease activity are instead required to inform their function. This review will introduce several classes of chemical probes that have been developed to measure the activation of PAR-cleaving proteases. Their strengths, weaknesses, and applications will be discussed, especially as applied to image protease activity at the whole organism, tissue, and cellular level.

蛋白酶激活受体(PAR)包括一个由四个G蛋白偶联受体(GPCR)组成的家族,在健康和疾病中具有广泛的功能。与大多数GPCR不同,PAR通过蛋白水解裂解其细胞外N末端而被独特激活。为了充分了解标准杆数在体内的激活和功能,研究激活它们的蛋白酶也是至关重要的。由于蛋白酶在翻译后水平上受到严重调节,因此总蛋白酶丰度的测量作用有限。相反,需要蛋白酶活性的测量来告知它们的功能。这篇综述将介绍几类化学探针,这些探针已被开发用于测量PAR-离去蛋白酶的激活。将讨论它们的优点、缺点和应用,特别是应用于整个生物体、组织和细胞水平的图像蛋白酶活性。
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引用次数: 0
Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage 多重蛋白成像通过太平洋:光活性免疫荧光与迭代切割
Q3 Pharmacology, Toxicology and Pharmaceutics Pub Date : 2023-04-28 DOI: 10.1021/acsbiomedchemau.3c00018
Fei Ji, Moises Hur, Sungwon Hur, Siwen Wang, Priyanka Sarkar, Shiqun Shao, Desiree Aispuro, Xu Cong, Yanhao Hu, Zhonghan Li* and Min Xue*, 

Multiplex protein imaging technologies enable deep phenotyping and provide rich spatial information about biological samples. Existing methods have shown great success but also harbored trade-offs between various pros and cons, underscoring the persisting necessity to expand the imaging toolkits. Here we present PACIFIC: photoactive immunofluorescence with iterative cleavage, a new modality of multiplex protein imaging methods. PACIFIC achieves iterative multiplexing by implementing photocleavable fluorophores for antibody labeling with one-step spin-column purification. PACIFIC requires no specialized instrument, no DNA encoding, or chemical treatments. We demonstrate that PACIFIC can resolve cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells. To further highlight how PACIFIC assists discovery, we integrate PACIFIC with live-cell tracking and identify phosphor-p70S6K as a critical driver that governs U87 cell mobility. Considering the cost, flexibility, and compatibility, we foresee that PACIFIC can confer deep phenotyping capabilities to anyone with access to traditional immunofluorescence platforms.

多重蛋白质成像技术能够进行深入的表型分析,并提供有关生物样本的丰富空间信息。现有的方法取得了巨大的成功,但也在各种利弊之间进行了权衡,强调了扩展成像工具包的持续必要性。在这里,我们介绍了太平洋:迭代切割的光活性免疫荧光,一种新的多重蛋白质成像方法。PACIFIC通过一步旋转柱纯化实现抗体标记的可光裂解荧光团,实现迭代复用。PACIFIC不需要专门的仪器,不需要DNA编码或化学处理。我们证明PACIFIC可以解决福尔马林固定石蜡包埋(FFPE)样品和固定细胞中的细胞异质性。为了进一步强调PACIFIC如何帮助发现,我们将PACIFIC与活细胞跟踪相结合,并将磷酸-p70S6K确定为控制U87细胞迁移的关键驱动因素。考虑到成本、灵活性和兼容性,我们预计PACIFIC可以为任何使用传统免疫荧光平台的人提供深入的表型能力。
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引用次数: 0
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ACS Bio & Med Chem Au
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