Morphological Profiling Identifies a Common Mode of Action for Small Molecules with Different Targets

Unbiased morphological profiling of bioactivity, for example, in the cell painting assay (CPA), enables the identification of a small molecule's mode of action based on its similarity to the bioactivity of reference compounds, irrespective of the biological target or chemical similarity. This is particularly important for small molecules with nonprotein targets as these are rather difficult to identify with widely employed target‐identification methods. We employed morphological profiling using the CPA to identify compounds that are biosimilar to the iron chelator deferoxamine. Structurally different compounds with different annotated cellular targets provoked a shared physiological response, thereby defining a cluster based on their morphological fingerprints. This cluster is based on a shared mode of action and not on a shared target, that is, cell‐cycle modulation in the S or G2 phase. Hierarchical clustering of morphological fingerprints revealed subclusters that are based on the mechanism of action and could be used to predict target‐related bioactivity.