Abstract

Morphological profiling upon perturbation by compounds allows exploring bioactivity of small molecules in a broader cellular and functional context. Whereas phenotypic assays usually detect a particular phenotype, profiling approaches simultaneously monitor hundreds of parameters of a perturbed state and, thus, cover a wider bioactivity space. The Cell painting assay (CPA)(1) is a morphology-based profiling that employs high-content imaging and analysis of six stained cellular components and compartments to extract hundreds of morphological features. Morphological fingerprints are used to assess bioactivity and are compared with fingerprints of annotated compounds with known targets or activity. Profile similarity can lead to the generation of a target or mode-of-action hypothesis early on in the compound development process. We employed the Cell painting assay to assess the bioactivity of our in-house compound collection. Detected bioactivity allowed uncovering unanticipated activity for reference compounds or assigning a mode of action to thus far unexplored small molecules (2-5). Literature 1. Gustafsdottir SM, Ljosa V, Sokolnicki KL, Wilson JA, Walpita D, Kemp MM, et al. Multiplex Cytological Profiling Assay to Measure Diverse Cellular States. Plos One. 2013;8(12):https://doi.org/10.1371/journal.pone.0080999. 2. Zimmermann S, Akbarzadeh M, Otte F, Strohmann C, Sankar MG, Ziegler S, et al. A Scaffold-Diversity Synthesis of Biologically Intriguing Cyclic Sulfonamides. Chemistry. 2019;25(68):15498-503. 3. Schneidewind T, Brause A, Pahl A, Burhop A, Mejuch T, Sievers S, et al. Morphological Profiling Identifies a Common Mode of Action for Small Molecules with Different Targets. Chembiochem. 2020;21:3197-207. 4. Akbarzadeh M, Deipenwisch I, Schoelermann B, Pahl A, Sievers S, Ziegler S, et al. Morphological profiling by means of the Cell Painting assay enables identification of tubulin targeting compounds. Cell Chem Biol. 2021; 10.1016/j.chembiol.2021.12.009 5. Schneidewind T, Brause A, Scholermann B, Sievers S, Pahl A, Sankar MG, et al.