Printing small molecules as microarrays and detecting protein-ligand interactions en masse
Printing small molecules as microarrays and detecting protein-ligand interactions en masse
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DOI：
10.1021/ja991083q
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发表时间：
1999-09-01
影响因子：
15
通讯作者：
Schreiber, SL
中科院分区：
文献类型：
--
作者：
MacBeath, G;Koehler, AN;Schreiber, SL
识别任何感兴趣蛋白质的小分子配体的能力对于阐明蛋白质功能和开发新型药物都具有深远的影响。随着分池合成策略的引入和适当标记技术的开发，2 名化学家现在能够制备大量固定在聚合合成珠上的天然产物类化合物。 3 这些文库为发现新的蛋白质配体提供了丰富的分子来源。有了这样的文库，筛选这些化合物的有效方法就变得势在必行。已使用的一种方法是珠上结合测定。 4 将适当标记的目标蛋白与文库混合，随后通过显色或荧光联检测来鉴定展示同源配体的珠子。尽管这种方法很实用，但它受到可以有效筛选的蛋白质数量较少的限制。原则上，珠子可以去除一种蛋白质并用另一种蛋白质重新探测；然而，这个串行过程很慢并且仅限于几次迭代。为了识别细胞、组织或生物体中每种蛋白质的特定小分子配体，需要进行高通量测定，使每种化合物能够以并行方式针对许多不同的蛋白质进行筛选。为了解决这个问题，我们开发了一种称为小分子印刷 (SMP) 的技术。首先，将合成珠以每孔一颗珠的密度分配到聚丙烯微量滴定板中。然后，附着的化合物从珠子中释放出来，并溶解在少量合适的溶剂中。由于每个珠子上存在微量化合物，因此绝对需要后续测定的极度小型化。受 cDNA 微阵列技术的启发，我们使用高精度机器人5从每个孔中拾取少量溶解的化合物，并重复将大约 1 nL 的溶液输送到一系列化学衍生玻璃显微镜载玻片上的指定位置（每次打印运行约 150 张载玻片）。这导致载玻片上形成化合物的微小斑点（直径 200-250 μm）。每种化合物都包含一个共同的官能团，可介导与载玻片表面的共价连接。通过这种方式，化合物被排列并随后以极高的空间密度（> 1000 个点/cm2）固定在载玻片上。然后可以用不同的标记蛋白探测每张载玻片，并可以通过荧光连接测定来检测结合事件。虽然连接小分子与载玻片的连接体的存在减少了每种化合物可用的结合模式的数量
The ability to identify small molecule ligands for any protein of interest has far-reaching implications, both for the elucidation of protein function and for the development of novel pharmaceuticals. With the introduction of the split-pool strategy for synthesis1 and the development of appropriate tagging technologies, 2 chemists are now able to prepare large collections of natural product-like compounds immobilized on polymeric synthesis beads. 3 These libraries provide a rich source of molecules for the discovery of new protein ligands. With such libraries in hand, the availability of efficient methods for screening these compounds becomes imperative. One method that has been used is the on-bead binding assay. 4 An appropriately tagged protein of interest is mixed with the library and beads displaying cognate ligands are subsequently identified by a chromagenic or fluorescence-linked assay. Despite the utility of this approach, it is limited by the small number of proteins that can be screened efficiently. In principle, the beads can be stripped of one protein and re-probed with another; however, this serial process is slow and limited to only a few iterations. To identify a specific small molecule ligand for every protein in a cell, tissue, or organism, high-throughput assays that enable each compound to be screened against many different proteins in a parallel fashion are required.To address this issue, we have developed a technique that we refer to as small molecule printing (SMP). First, synthesis beads are distributed into polypropylene microtiter plates at a density of one bead per well. The attached compounds are then released from their beads and dissolved in a small volume of a suitable solvent. Due to the minute quantities of compound present on each bead, extreme miniaturization of the subsequent assay is an absolute requirement. Taking our cue from cDNA microarray technology, we use a high-precision robot5 to pick up a small volume of dissolved compound from each well and repetitively deliver approximately 1 nL of solution to defined locations on a series of chemically derivatized glass microscope slides (∼ 150 slides per print run). This results in the formation of microscopic spots of compound on the slides (200-250 μm in diameter). Each compound contains a common functional group that mediates covalent attachment to the slide surface. In this way, compounds are arrayed and subsequently immobilized on glass slides at extremely high spatial densities (> 1000 spots per cm2). Each slide can then be probed with a different tagged protein and binding events can be detected by a fluorescence-linked assay. While the presence of a linker connecting the small molecule to the slide reduces the number of binding modes available to each compound