In phenotypic compound discovery, conclusive identification of cellular targets and mode of action are often impaired by off-target binding. In particular, microtubules are frequently targeted in cellular assays. However, in vitro tubulin binding assays do not correctly reflect the cellular context, and conclusive high-throughput phenotypic assays monitoring tubulin binding are scarce, such that tubulin binding is rarely identified. We report that morphological profiling using the Cell Painting assay (CPA) can efficiently detect tubulin modulators in compound collections with a high throughput, including annotated reference compounds and unannotated compound classes with unrelated chemotypes and scaffolds. Small-molecule tubulin binders share similar CPA fingerprints, which enables prediction and experimental validation of microtubule-binding activity. Our findings suggest that CPA or a related morphological profiling approach will be an invaluable addition to small-molecule discovery programs in chemical biology and medicinal chemistry, enabling early identification of one of the most frequently observed off-target activities.

在表型化合物的发现中，细胞靶标和作用方式的最终鉴定通常会因脱靶结合而受损。特别是，微管在细胞测定中经常被靶向。然而，体外微管蛋白结合分析不能正确反映细胞环境，监测微管蛋白结合的结论性高通量表型分析很少，因此很少发现微管蛋白结合。我们报告说，使用细胞绘画测定 (CPA) 的形态学分析可以有效地检测具有高通量的化合物集合中的微管蛋白调节剂，包括带注释的参考化合物和具有不相关化学型和支架的未注释化合物类。小分子微管蛋白结合剂具有相似的 CPA 指纹，从而能够预测和实验验证微管结合活性。我们的研究结果表明，CPA 或相关的形态学分析方法将成为化学生物学和药物化学中小分子发现计划的宝贵补充，