Luesch Hendrik, Abreu Pedro
Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2009;572:261-77. doi: 10.1007/978-1-60761-244-5_17.
In the last few years, genomic tools have been incorporated in natural product approaches to drug discovery, including understanding mechanisms of action which cannot be elucidated from phenotypic screens such as cell viability assays. The characterization of perturbed biological pathways and target identification are important for the evaluation of the compounds' potential as drug leads and for subsequent medicinal chemistry efforts; however, general procedures to tackle this task are lacking. The combination of high-throughput screening and genomic-scale assays has proven to be a powerful tool to aid in the identification of mechanisms and potentially of protein targets, not only in yeast but also mammalian cells. Arrayed libraries of cDNAs can be transfected into cancer cell lines in a high-throughput fashion to generate variants of spatially separated cancer cells with increased gene dosages for one particular cDNA. Cells overexpressing gene products that are directly targeted by a small molecule or that lie in the perturbed pathway may be less susceptible to the effects of the compound. This fact provides the basis for drug susceptibility screens employing cDNA libraries. The general procedures to optimize and execute those screens and subsequently validate putative screening hits are discussed in detail.
在过去几年中，基因组工具已被纳入天然产物药物发现方法中，包括了解细胞活力测定等表型筛选无法阐明的作用机制。对受干扰的生物途径进行表征和确定靶点，对于评估化合物作为药物先导物的潜力以及后续的药物化学研究工作很重要；然而，目前缺乏处理这项任务的通用程序。高通量筛选和基因组规模测定的结合已被证明是一种强大的工具，不仅有助于在酵母中，也有助于在哺乳动物细胞中识别作用机制和潜在的蛋白质靶点。可以将阵列式cDNA文库以高通量方式转染到癌细胞系中，以产生特定cDNA基因剂量增加的空间分离癌细胞变体。过表达被小分子直接靶向或处于受干扰途径中的基因产物的细胞，可能对该化合物的作用不太敏感。这一事实为利用cDNA文库进行药物敏感性筛选提供了基础。本文详细讨论了优化和执行这些筛选以及随后验证假定筛选命中物的通用程序。
