Small-molecule screening made simple for a difficult target with a signaling nucleic acid aptamer that reports on deaminase activity
Small-molecule screening made simple for a difficult target with a signaling nucleic acid aptamer that reports on deaminase activity
复制标题
DOI：
10.1002/anie.200601695
复制
发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Brown, Eric A.
中科院分区：
文献类型：
--
作者：
Elowe, Nadine H.;Nutiu, Razvan;Brown, Eric A.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew。化学。Int。编辑。2006,45,5648-5652适用于化学基因组方法的酶分析发展的适用性和潜力。适配体是来自随机文库的单链DNA和RNA分子，能够结合不同的生物靶标，如小分子代谢物、肽、激素或蛋白质，具有高亲和力和特异性。适配体有多种用途，从研究到诊断和治疗应用。[2,3]核酸适体在化合物筛选中首次被证明有希望成为一种可置换配体，在此过程中，一组萘磺酸被测试了它们与血小板衍生生长因子中32p标记的适体竞争的能力核酸适体的一个有趣特性是它们在与靶结合时发生构象变化这一特性正逐渐被用于信号探针的设计，这些探针可以报告适体与其靶标的相互作用，因此，信号适体在分析开发和小分子筛选中具有真正的潜力。[6,7]最近，我们报道了一项概念验证研究，该研究利用一种对腺苷比AMP亲和力更高的信号DNA适配体实时监测碱性磷酸酶的酶活性。该实验采用了一种“结构转换”适配体，腺苷结合通过释放猝灭基团诱导高荧光状态。该试验可在96孔板中筛选，并且对已知的碱性磷酸酶抑制剂敏感。因此，信号核酸系统已经开始显示出作为报告系统的真正希望，它可以相对容易地针对各种检测进行定制。在本文报道的工作中，我们在ADA的自动高通量筛选（HTS）中对超过44000种化合物的小分子文库进行了严格的测试。ADA是嘌呤代谢的关键酶，可催化腺苷/脱氧腺苷不可逆脱胺为肌苷/脱氧肌苷。ADA是一种普遍存在的酶，以其在某些类型的严重联合免疫缺陷疾病中的作用而闻名该酶具有胞质和细胞外两种形式，其中后者与CD26相关，CD26在t细胞活化中被强烈上调越来越多的人认识到细胞外腺苷在减轻免疫和炎症方面的作用，因此ADA抑制剂可能具有真正的临床应用潜力尽管对这一靶点的兴趣日益浓厚，但ADA还没有一种简单、均质、敏感的HTS检测方法。常用的方法是通过监测265 nm的吸光度来测量氨生成b[12]或腺苷浓度的变化前者不容易自动化，后者的短波长检测容易受到筛选化合物本征吸光度的干扰。还描述了一种长波长耦合分析系统，但由于需要三种偶联酶，它本身就很麻烦。因此，迄今为止所描述的检测方法都不能被认为是小分子筛选的理想方法。为了开发ADA的筛选试验，我们采用了一种对腺苷具有高亲和力而对肌苷几乎没有亲和力的荧光信号适配体（图1a）。[15,16]该系统中的荧光信号来源于DNA适配体的5位末端的荧光素基团，并由两种不同结构状态（腺苷结合形式和自由形式）的比例决定。
5648 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2006, 45, 5648–5652 applicability and potential for the development of enzymatic assays suitable for a chemical genomic approaches. Aptamers are single-stranded DNA and RNA molecules derived from random libraries that are capable of binding to diverse biological targets, such as small-molecule metabolites, peptides, hormones, or proteins, with high affinity and specificity. Aptamers have found diverse utility ranging from research [1] to diagnostic and therapeutic applications.[2, 3] Nucleic acid aptamers were first shown to have promise as a displaceable ligand in compound screening in which a panel of a dozen naphthalensulfonic acids were tested for their capacity to compete a 32P-labeled aptamer from plateletderived growth factor.[4] An interesting property of nucleic acid aptamers is that they are known to undergo conformational changes upon target binding.[5] This characteristic is emerging as exploitable for the design of signaling probes that report on the interaction of aptamers with their targets and thus, signaling aptamers have genuine potential in assay development and small-molecule screening.[6, 7] Recently, we reported a proof-of-concept study in which the enzyme activity of alkaline phosphatase was monitored in real time by using a signaling DNA aptamer with a higher affinity for adenosine than for AMP.[8] The assay employed a “structureswitching” aptamer in which adenosine binding induced a state of high fluorescence through release of a quenching group. The assay was amenable to screening in a 96-well plate and was sensitive to known inhibitors of alkaline phosphatase. Thus, signaling nucleic acid systems have begun to show real promise as reporter systems that can be tailored with relative ease to a variety of assays. In the work reported herein, we have rigorously tested this potential in an automated, highthroughput screen (HTS) of ADA against a small-molecule library of more than 44000 compounds. ADA is a key enzyme in purine metabolism, catalyzing the irreversible deamination of adenosine/deoxyadenosine to inosine/deoxyinosine. ADA is a ubiquitous enzyme best known for its role in certain types of severe combined immunodeficiency diseases.[9] The enzyme has both cytosolic and extracelluar forms in which the latter is associated with CD26, which is strongly upregulated in T-cell activation.[10] Increasingly, extracellular adenosine is recognized to have a role in attenuating immunity and inflammation, and thus inhibitors of ADA may have real potential for clinical applications.[11] Despite growing interest in this target, ADA does not have a simple, homogenous, and sensitive assay amenable to HTS. Common methods measure ammonia produced [12] or the change in adenosine concentration by monitoring absorbance at 265 nm.[13] The former is not easily automated and the short-wavelength detection of the latter is prone to interference from the intrinsic absorbance of screening compounds. A long-wavelength coupled assay system has also been described,[14] but it is inherently cumbersome owing to the need for three coupling enzymes. Thus, none of the assays described to date could be considered ideal for small-molecule screening. To develop a screening assay for ADA, we employed a fluorescence-signaling aptamer with high affinity for adenosine and virtually no affinity for inosine (Figure 1 A).[15, 16] The fluorescence signal in this system is derived from a fluorescein group present at the 5о-end of the DNA aptamer and determined by the ratio of two different structural states, the adenosine-bound and free forms …