Clemons P A, Koehler A N, Wagner B K, Sprigings T G, Spring D R, King R W, Schreiber S L, Foley M A
Howard Hughes Medical Institutes at Harvard University, Cambridge, MA 02138, USA.
Chem Biol. 2001 Dec;8(12):1183-95. doi: 10.1016/s1074-5521(01)00086-2.
Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems.
We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides.
Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.
化学遗传学提供了一种利用小分子对蛋白质功能进行空间和时间控制来研究生物学的系统方法。作为互补方法，对结构多样且复杂的小分子进行表型和蛋白质组学筛选，不仅可能产生有趣的生物学功能单个探针，还能获得有关小分子集合及其成员与生物系统相互作用的全局信息。
我们报告了一种通用的高通量方法，可将高容量珠子转化为适用于表型和蛋白质组学分析的阵列储备溶液。来自定向多样性合成的聚苯乙烯珠子被单独排列到孔中。结合的化合物被裂解、洗脱并重新悬浮，以生成储备溶液的“母板”。我们技术平台开发的第二阶段包括优化的裂解和洗脱条件、一种新颖的珠子排列方法，以及将小分子储备溶液自动分配到“子板”中，以便直接用于化学遗传学分析。这种文库格式化策略实现了我们所谓的注释筛选，即文库的每个成员都用生物学分析数据进行注释。通过对一个由4320个成员组成的结构多样且复杂的二氢吡喃甲酰胺编码文库中的708个成员进行排列和筛选，验证了这一阶段。
我们的“一珠多储备溶液”文库格式化策略是旨在推进化学遗传学的技术平台的核心要素。注释筛选为生物学为化学提供信息提供了一种手段，这与传统（“研究者发起”）小分子筛选中化学为生物学提供信息的方式互为补充。
