Fast dienophile reactions for in vivo click imaging

用于体内点击成像的快速亲二烯体反应

• 批准号: 7934959
• 负责人: RALPH WEISSLEDER, MD, PHD
• 金额: $ 39.68万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934959
• 关键词: Affinity; Alkynes; Antibodies; Avidity; Azides; Binding; Biocompatible; Biological; Biological Assay; Biological Models; Cancer Detection; Cancerous; Cell Survival; Cell surface; Cells; Chemicals; Chemistry; Clinical; Coculture Techniques; Complex; Data; Detection; Drug Kinetics; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Fluorescence; Fluorochrome; Freezing; Generations; Generic Drugs; Goals; Gold; Hour; Human; Image; In Vitro; Label; Lead; Life; Ligands; Magnetism; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Methods; Modeling; Molecular Target; Organ; Preclinical Testing; Preparation; Prodrugs; Reaction; Reagent; Reporter; Research; Signal Transduction; Source; Techniques; Technology; Temperature; Testing; Time; Tissue Microarray; Tissues; Toxic effect; Toxicity Tests; Validation; Work; base; cancer cell; catalyst; cell type; cost; cycloaddition; cytotoxicity; design; drug distribution; extracellular; in vivo; in vivo Model; molecular imaging; novel; public health relevance; receptor; research study; scale up; small molecule; therapeutic target

DESCRIPTION (provided by applicant): Biological and chemical amplification strategies are key to the design of successful molecular imaging agents. Several such strategies have been described including a) enzymatically activated prodrugs, b) covalent target binding, c) intracellular trapping, d) conformational changes upon target binding, e) pH induced fluorescence or magnetic changes f) increased avidity through multivalency, g) amplifying reporters, h) unnatural biorthogonal chemical reporters and i) pre-targeting. Some of these strategies have been extraordinarily robust but few possess intrinsic selectivity, are universally applicable for different classes of targets or are clinically translatable. One emerging chemical strategy for in vitro bioconjugation has been cycloaddition ("click chemistry"). Unfortunately, conventional reactions (e.g. between and azide and an alkyne) require elevated temperatures, a Cu(I) catalysts to be efficient or are simply too slow for in vivo use. We and others have discovered and tested a number of novel ring constrained reactants as more universal in vivo click reagents. In these reactions, a tetrazine replaces the azide functionality and readily reacts with constrained dienophile ligands. We have shown that the norbornene/ tetrazine click reaction can proceed orders of magnitude faster (in seconds as compared to hours-days with previous azide/alkyne reactions) and that reactions are very selective and specific. In preliminary data we have shown efficacy and compatibility of reactants with live cells. Importantly and extending this concept to transcyclooctenes, we have now shown that the technique works for intracellular targets as well as for extracellular targets. In parallel, proof-of-principle experiments shown that the technology allows in vivo clicking. The goal of this application is to further build on this cutting-edge technology and to develop generic amplifying in vivo click reactions for molecular imaging. In aim 1 we will perform more comprehensive cell based screens to identify lead compounds and conditions for dienophile/tetrazine " fast click reactions. In a second aim we will apply optimized compounds (antibody and/or small molecule conjugated dienophiles and tetrazine-fluorochromes) and conditions to biologically relevant in vivo models and ask a number of questions which may ultimately aid in the more widespread application of the technology: How efficient is in vivo click chemistry and what is the detection threshold in vivo? How does the strategy compare to current gold standards? Can the approach be used to measure EGFR target inhibition? Can the approach be used to quantitative drug distribution? Can the technology be used for multichannel imaging of several molecular targets? These experiments are a logical extension of our preliminary work and will likely result in broad, new imaging platforms. PUBLIC HEALTH RELEVANCE: The proposed research represents a new method for in vivo imaging of intra- and extracellular targets using biocompatible "click" reactions. The new method is very powerful as it harnesses very selective and specific chemistries and amplification strategies and has most recently been shown to work for intracellular targets as well.

描述(申请人提供)：生物和化学扩增策略是设计成功的分子显像剂的关键。已经描述了几种这样的策略，包括a)酶激活的前药，b)共价靶标结合，c)细胞内捕获，d)靶标结合时的构象变化，e)pH诱导的荧光或磁性变化f)通过多价态增加亲和力，g)扩增报告，h)非天然双正交化学报告和i)预靶向。这些策略中有一些非常稳健，但很少具有内在的选择性，对不同类别的靶点普遍适用，或者是临床可翻译的。一种新出现的体外生物偶联的化学策略是环加成(“点击化学”)。不幸的是，传统的反应(例如叠氮化物和炔烃之间的反应)需要更高的温度，铜(I)催化剂要么高效，要么太慢，无法在体内使用。我们和其他人已经发现并测试了一些新的环限制反应物，作为体内更通用的点击试剂。在这些反应中，四氮取代叠氮官能团，很容易与受约束的双烯亲核配体反应。我们已经证明，降冰片烯/四嗪点击反应可以更快地进行数量级(与以前的叠氮/炔反应相比，在几秒钟内进行)，并且反应具有非常高的选择性和特异性。在初步数据中，我们已经展示了反应物与活细胞的有效性和兼容性。重要的是，将这一概念扩展到跨环辛烯，我们现在已经表明，该技术不仅适用于细胞外靶标，也适用于细胞内靶标。同时，原理验证实验表明，该技术允许在体内点击。这项应用的目标是进一步建立在这一尖端技术的基础上，并开发用于分子成像的体内非专利放大点击反应。在目标1中，我们将进行更全面的基于细胞的筛选，以确定先导化合物和亲双烯/四嗪“快速点击反应”的条件。在第二个目标中，我们将把优化的化合物(抗体和/或小分子共轭双烯化合物和四嗪荧光素)和条件应用于体内相关的生物模型，并提出一些最终可能有助于该技术更广泛应用的问题：体内点击化学的效率如何，以及体内的检测阈值是多少？与当前的黄金标准相比，这一战略如何？该方法能用来测量EGFR靶点抑制吗？这种方法是否可以用于药品的定量分配？该技术能否用于多个分子靶标的多通道成像？这些实验是我们前期工作的合乎逻辑的延伸，并可能导致广泛的、新的成像平台。 与公共卫生相关：这项拟议的研究代表了一种新的方法，利用生物相容的“点击”反应对细胞内和细胞外的靶标进行体内成像。这种新的方法非常强大，因为它利用了非常选择性和特定的化学和扩增策略，最近被证明也适用于细胞内的靶标。