1 Drug Discovery Institute, University of Pittsburgh Medical School, Pittsburgh, PA, USA.
2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
SLAS Discov. 2019 Jul;24(6):669-681. doi: 10.1177/2472555219829740. Epub 2019 Feb 25.
Mcm2-7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its cellular importance, few small-molecule inhibitors of this complex are known. Based upon our genetic analysis of synthetic growth defects between alleles and a range of other alleles, we have developed a high-throughput screening (HTS) assay using a well-characterized mutant (containing the allele) to identify small molecules that replicate such synthetic growth defects. During assay development, we found that aphidicolin (inhibitor of DNA polymerase alpha) and XL413 (inhibitor of the DNA replication-dependent kinase CDC7) preferentially inhibited growth of the strain relative to the wild-type parental strain. However, as both strains demonstrated some degree of growth inhibition with these compounds, small and variable assay windows can result. To increase assay sensitivity and reproducibility, we developed a strategy combining the analysis of cell growth kinetics with linear discriminant analysis (LDA). We found that LDA greatly improved assay performance and captured a greater range of synthetic growth inhibition phenotypes, yielding a versatile analysis platform conforming to HTS requirements.
Mcm2-7 是真核复制解旋酶的分子马达，该复合物的调节是细胞 S 期调控的主要焦点。尽管该复合物具有重要的细胞意义，但已知的该复合物的小分子抑制剂很少。基于我们对合成生长缺陷等位基因和其他一系列等位基因之间的遗传分析，我们开发了一种使用经过充分表征的突变体（包含等位基因）的高通量筛选（HTS）测定法，以鉴定复制这种合成生长缺陷的小分子。在测定法的开发过程中，我们发现阿非迪可林（DNA 聚合酶α抑制剂）和 XL413（DNA 复制依赖性激酶 CDC7 的抑制剂）优先抑制突变体相对于野生型亲本菌株的生长。然而，由于这两种菌株对这些化合物都表现出一定程度的生长抑制，因此可能会出现较小且变化的测定窗口。为了提高测定法的灵敏度和重现性，我们开发了一种结合细胞生长动力学分析和线性判别分析（LDA）的策略。我们发现 LDA 大大提高了测定法的性能，并捕获了更大范围的合成生长抑制表型，从而产生了一种符合 HTS 要求的多功能分析平台。
