Unbiased profiling approaches are powerful tools for small-molecule target or mode-of-action deconvolution as they generate a holistic view of the bioactivity space. This is particularly important for non-protein targets that are difficult to identify with commonly applied target identification methods. Thereby, unbiased profiling can enable identification of novel bioactivity even for annotated compounds. We report the identification of a large bioactivity cluster comprised of numerous well-characterized drugs with different primary targets using a combination of the morphological Cell Painting Assay and proteome profiling. Cluster members alter cholesterol homeostasis and localization due to their physicochemical properties that lead to protonation and accumulation in lysosomes, an increase in lysosomal pH, and a disturbed cholesterol homeostasis. The identified cluster enables identification of modulators of cholesterol homeostasis and links regulation of genes or proteins involved in cholesterol synthesis or trafficking to physicochemical properties rather than to nominal targets.

无偏分析方法是小分子目标或作用模式反卷积的强大工具，因为它们生成了生物活性空间的整体视图。这对于用常用的目标识别方法难以识别的非蛋白质目标尤其重要。因此，无偏见的分析可以识别新的生物活性，即使是注释的化合物。我们报告了一个大型生物活性簇的鉴定，该簇由许多具有不同主要目标的良好表征的药物组成，使用形态学细胞绘画测定和蛋白质组分析的组合。簇成员改变胆固醇稳态和定位，因为它们的物理化学特性导致溶酶体中的质子化和积累，溶酶体 pH 值的增加，和紊乱的胆固醇稳态。已识别的簇能够识别胆固醇稳态的调节剂，并将参与胆固醇合成或运输的基因或蛋白质的调节与物理化学特性联系起来，而不是与标称目标联系起来。