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.

No abstract

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh‐pathway modulator Pipinib by means of cell‐based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4‐kinase IIIβ (PI4KB) and suppresses GLI‐mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl‐4‐phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

Cell-based screening is ap owerfula pproach to identify novel chemical modulators and biological components of relevant biological processes.The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis,a nd its deregulation playsacrucial role in carcinogenesis.T herefore,t he identification of new pathway members and regulators is of significant interest. By means of ac ell-based assaym onitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling.T arget identification and validation revealed that Pyrcoumin is ac ompetitive inhibitor of dCTP pyrophosphatase 1(dCTPP1). We demonstrate ayet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors.T hese findings indicate that dCTPP1 playsarole in regulation of Wnt/bcatenin signaling most likely through ad irect interaction with USP7.

No abstract