Fast dienophile reactions for in vivo click imaging

描述（由申请人提供）：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诱导的荧光或磁变化f)通过多价性增加亲和力，g)扩增报告，h)非自然双正交化学报告和i)预靶向。其中一些策略非常稳健，但很少具有内在选择性，普遍适用于不同类型的靶标或临床可翻译。一种新兴的体外生物偶联化学策略是环加成（“点击化学”）。不幸的是，传统的反应（例如叠氮化物和炔之间的反应）需要升高的温度，Cu(I)催化剂效率不高，或者对于体内使用来说太慢。我们和其他人已经发现并测试了一些新的环约束反应物作为更普遍的体内点击试剂。在这些反应中，四氮取代叠氮化物官能团，容易与受限的亲二烯配体反应。我们已经证明，降冰片烯/四嗪的点击反应可以进行数量级的快（在几秒内，而不是几个小时-天与以前的叠氮化物/炔的反应），反应是非常选择性和特异性的。在初步数据中，我们已经证明了反应物与活细胞的有效性和相容性。重要的是，将这一概念扩展到外环烯，我们现在已经证明该技术适用于细胞内靶点和细胞外靶点。与此同时，原理验证实验表明，该技术允许在体内点击。本应用程序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通用放大体内点击反应。在目标1中，我们将进行更全面的基于细胞的筛选，以确定二亲酚/四嗪“快速点击反应”的先导化合物和条件。在第二个目标中，我们将应用优化的化合物（抗体和/或小分子共轭二亲体和四氮-荧光染料）和条件，以生物相关的体内模型，并提出一些问题，这些问题可能最终有助于该技术的更广泛应用：体内点击化学的效率有多高，体内检测阈值是多少？与当前的金本位相比，该战略如何？该方法可用于测量EGFR靶抑制吗？该方法能否用于定量药物分配？该技术能否用于多个分子靶点的多通道成像？这些实验是我们初步工作的合理延伸，可能会产生广泛的新成像平台。

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.