Neutron encoded activity based probes

药物被设计用来调节一种与疾病有关的关键蛋白质的功能；然而，几乎所有小的 分子具有非靶点，可以减轻或完全终止其治疗效果。不幸的是， 没有通用的方法可以以公正的方式识别药物结合的所有蛋白质靶标。 例如，组蛋白脱乙酰酶抑制剂(HDACIs)在许多疾病中显示出良好的临床活性； 然而，它们通常具有低到中等的特异性，可能部分通过或受到以下因素的阻碍： 未描述的脱离目标的相互作用。我们提出了一项战略，将允许快速和深入的 早期候选药物的药理学图谱：1)识别具有特殊功能的蛋白质靶点 置信度，2)通常用氨基酸残基专一性来定位相互作用的精确位置，3)对 代谢改变，4)区分代谢物的药理学，5)量化 细胞环境之间的药理活性。使用此方法，我们将量化所有 每一种抑制物都与活细胞中的蛋白质发生了作用。一个中子编码的条形码被添加到基于活动的 点击捕获和释放期间的探针，允许我们以名义质量无关的方式盲目追踪药物 同时引入量化渠道。条形码在同位素罚款中显露出来 结构，但不影响较低分辨率下的灵敏度 碎裂光谱。中子条形码是通过在元素和结果之间移动中子来实现的 在小分子框架内嵌入的相对论核质量缺陷的规定模式中。使用 这种方法我们可以自信地从体内系统中检索药物-蛋白质反应产物，而不需要考虑 HDAC抑制剂的代谢变化，测量药理学特征并确定分子 作用机制。我们的中心假设是，基于中子编码活性的探针药理学 分析与创新的基于片段的发现相结合以生成选择性HDACI将使 产生具有高度特征性和不同选择性的HDACi探针库。这将通过以下方式实现 三个具体目标：在目标1中，我们将使用一组新颖的和系统多样化的SP3富含片段 以产生对HDAC的边缘区域进行采样的HDACI，从而导致高度选择性的相互作用。在《目标2》中，我们 将测量我们的新型HDACi的药理学特征以及它们对组蛋白乙酰化的影响。在《目标3》中， 我们将开发一种多路复用条形码系统，以实现对毒品目标的更高检测和分辨率 整个蛋白质组。这克服了早期药物开发普遍存在的许多挑战 尤其是阻碍了特定的HDAC抑制剂的开发的努力。尽管HDACI 将是这项建议的总体重点，我们的方法对于任何领域的药物开发都是通用的。

Drugs are designed to modulate the function of a critical protein involved in a disease; however, almost all small molecules have off-targets which can mitigate or altogether terminate their therapeutic efficacy. Unfortunately, there are no general methods that can identify all the protein targets that a drug binds to in an unbiased manner. For example, Histone Deacetylase Inhibitors (HDACIs) show promising clinical activity in many diseases; however, they are generally of low to moderate specificity and may act in part through, or be hindered by, uncharacterized off-target interactions. We propose a strategy that will allow for rapid and deep pharmacological profiling of early drug candidates that 1) identifies protein targets with extraordinary confidence, 2) localizes the precise site of interaction often with amino acid residue specificity, 3) is robust against metabolic alterations, 4) distinguishes the pharmacology of metabolites, 5) quantitates differences in pharmacological activity between cellular contexts. With this approach we will quantitate all interactions that each inhibitor has with proteins in a living cell. An neutron-encoded ‘bar code’ is added to activity-based probes during a click capture and release that allows us to blindly trace the drug in a nominal mass independent manner and simultaneously introduce quantitation channels. The barcodes are revealed in the isotopic fine structure by high resolution mass spectrometry yet do not compromise sensitivity at lower resolution fragmentation spectra. The neutron bar code is implemented by moving neutrons between elements and results in a prescribed pattern of relativistic nuclear mass defects embedded in the framework of a small molecule. With this method we can confidently retrieve drug-protein reaction products from in vivo systems regardless of metabolic alterations to the HDAC inhibitors, measure a pharmacological profile and determine molecular mechanism of action. Our central hypothesis is that neutron encoded activity based probe pharmacological profiling combined with innovative fragment-based discovery to generate selective HDACIs will enable the generation of a library of highly characterized and diversely selective HDACI probes. This will be realized through three specific aims: in aim 1 we will employ a novel and systematically diverse group of sp3-enriched fragments to generate HDACIs that sample the rim region of HDACs leading to highly selective interactions. In aim 2 we will measure the pharmacological profile of our novel HDACi’s and their effect on histone acetylation. In aim 3, we will develop a multiplexed barcoding system which to enable higher detection and resolution of drug targets over the entire proteome. This overcomes many challenges universal to early stage drug development efforts that have frustrated the development of specific HDAC inhibitors in particular. Although HDACIs will be the general focus of this proposal our method is general for drug development in any area.