For unbiased assessment of compound bioactivity, we employed the cell painting assay (CPA). This multi-featured morphological assay enables the identification of a number of bioactivities and mechanisms of action (MoAs) without prior target hypotheses. It monitors bioactivity in a very broad sense and, has been proven to be a highly valuable method for comparison of bioactivity for PNPs with different fragment combinations, connectivities and stereoisomeric relationships. Briefly, treated and untreated U2OS cells are stained with 6 different stains that are specific for different compartments of the cell, and imaged in 5 different channels by microscope. The images are then processed with CellProfiler 31 and further analyzed using in-house written scripts during which the single-cell data is aggregated to compound level. The extracted morphological features are normalized to DMSO controls using a modified Z-score and the final generated profiles are investigated for their similarity to a set of pre-defined 13 clusters using a subprofile analysis. Details of the assay are described in the Supporting Information. The degree of bioactivity in the CPA is represented by the Induction value, i.e.