Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries
Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries
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DOI：
10.1073/pnas.0604348103
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发表时间：
2006-08-01
影响因子：
11.1
通讯作者：
Austin, Christopher P.
中科院分区：
文献类型：
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作者：
Inglese, James;Auld, Douglas S.;Austin, Christopher P.
通过高通量筛选（HITS）化合物来鉴定分子靶标调节剂是药物开发的支柱。 HITS 越来越多地用于识别基因、通路和细胞功能的化学探针，最终目标是全面描述化学结构和生物活性之间的关系。实现这一目标需要能够从初级筛选中有效生成药理学数据并可靠地分析与大型化学库相关的生物活性范围的方法。传统的 HTS 测试单一浓度的化合物，不适合这项任务，因为 HTS 经常出现假阳性和假阴性，并且需要大量的后续测试。我们开发了一种定量 HITS (qHTS) 范例，并使用丙酮酸激酶进行测试，可在单个实验中生成超过 60,000 种化合物的浓度-响应曲线。我们证明这种方法是精确的，不易受样品制备变化的影响，并且可以鉴定具有广泛活性的化合物。对浓度-反应曲线进行分类，以快速识别具有各种效力和功效的丙酮酸激酶激活剂和抑制剂，并直接从初级筛选中阐明结构-活性关系。 qHTS 与传统单浓度 HTS 的比较表明，单点筛查中假阴性的发生率很高。这项研究证明了 qHTS 准确分析大型化学库（> 10(5) 种化合物）中每种化合物的可行性。 qHTS 产生丰富的数据集，可以立即挖掘可靠的生物活性，从而为化学基因组学提供平台，并加速药物发现先导化合物的识别。
High-throughput screening (HITS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HITS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HITS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for > 60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional sing le-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (> 10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.