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成功设计分子成像剂的关键。已经描述了几种这样的策略，包括a）酶促活化的前药，B）共价靶结合，c）细胞内捕获，d）靶结合后的构象变化，e）pH诱导的荧光或磁性变化，f）通过多价增加亲合力，g）扩增报道分子，h）非天然双正交化学报道分子和i）预靶向。这些策略中的一些非常强大，但很少具有内在的选择性，普遍适用于不同类别的目标或临床翻译。用于体外生物缀合的一种新兴化学策略是环加成（“点击化学”）。不幸的是，常规反应（例如叠氮化物和炔之间的反应）需要升高的温度、Cu（I）催化剂才有效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许多新的环约束反应物作为更通用的体内点击试剂。在这些反应中，四嗪取代叠氮化物官能团并容易与受限的亲二烯体配体反应。我们已经表明，异戊烯/四嗪点击反应可以更快地进行几个数量级（与先前的叠氮化物/炔反应的小时-天相比，在几秒钟内），并且反应是非常选择性和特异性的。在初步数据中，我们已经显示了反应物与活细胞的有效性和相容性。重要的是，将这一概念扩展到反式环辛烯，我们现在已经表明，该技术适用于细胞内靶点以及细胞外靶点。同时，原理验证实验表明，该技术允许在体内点击。本申请的目标是进一步建立在这种尖端技术的基础上，并开发用于分子成像的通用扩增体内点击反应。在目标1中，我们将进行更全面的基于细胞的筛选，以鉴定亲二烯体/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/或小分子缀合的亲二烯体和四嗪荧光染料）和条件，以生物相关的体内模型，并提出一些问题，这可能最终有助于更广泛的应用的技术：如何有效的是在体内点击化学和什么是在体内的检测阈值？该战略与当前的黄金标准相比如何？该方法能否用于测量EGFR靶点抑制？该方法能否用于定量药物配送？该技术能否用于多个分子靶点的多通道成像？这些实验是我们前期工作的逻辑延伸，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.