Hallis Tina M, Kopp Andrew L, Gibson Jasmin, Lebakken Connie S, Hancock Michael, Van Den Heuvel-Kramer Kaitlyn, Turek-Etienne Tammy
Invitrogen Discovery Sciences, Madison,Wisconsin 53719, USA.
J Biomol Screen. 2007 Aug;12(5):635-44. doi: 10.1177/1087057107301499. Epub 2007 May 21.
A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator. To demonstrate the improvement in hit confirmation, the multiplexed assay was used to perform a small-library screen (7728 compounds) for serotonin 5HT1A receptor antagonists. Hits identified from analysis of the beta-lactamase reporter data alone were compared to those hits determined when the reporter and cytotoxicity data generated from the multiplexed assay were combined. Confirmation rates were determined from compound follow-up using dose-response analysis of the potential antagonist hits identified by the initial screen. In this representative screen, the multiplexed assay approach yielded a 19% reduction in the number of compounds flagged for follow-up, with a 37% decrease in the number of false hits, demonstrating that multiplexing a beta-lactamase reporter assay with a cytotoxicity readout is a highly effective strategy for reducing false hit rates in cell-based compound screening assays.
细胞分析用于药物发现存在一个固有问题，即它们对细胞毒性化合物敏感，这可能导致某些化合物筛选出现假阳性结果。为了减少在报告基因分析后再进行单独细胞毒性分析的需求，作者开发了一种多重分析方法，该方法将β-内酰胺酶报告基因的读数与均相细胞毒性指示剂读数相结合。文中讨论了多重分析形式开发的重要方面，包括结果表明在β-内酰胺酶报告基因分析中针对核因子κB和SIE途径拮抗剂的40种选定化合物的IC(50)值不受细胞毒性指示剂添加的影响。为了证明命中确认方面的改进，使用多重分析方法对血清素5HT1A受体拮抗剂进行了小型文库筛选（7728种化合物）。将仅从β-内酰胺酶报告基因数据分析中确定的命中结果与结合多重分析产生的报告基因和细胞毒性数据时确定的命中结果进行比较。通过对初始筛选确定的潜在拮抗剂命中结果进行剂量反应分析来进行化合物跟进，从而确定确认率。在这个代表性筛选中，多重分析方法使标记进行跟进的化合物数量减少了19%，假阳性命中数量减少了37%，这表明将β-内酰胺酶报告基因分析与细胞毒性读数相结合是一种在基于细胞的化合物筛选分析中降低假阳性率的高效策略。
