Image‐Based Morphological Profiling Identifies a Lysosomotropic, Iron‐Sequestering Autophagy Inhibitor

Abstract

Chemical proteomics is widely applied in small‐molecule target identification. However, in general it does not identify non‐protein small‐molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image‐based morphological profiling in the cell painting assay. A compound‐induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

DMT1 Inhibitors Kill Cancer Stem Cells by Blocking Lysosomal Iron Translocation

“…It selectively kills CSC by sequestering lysosomal iron, leading to the production of reactive oxygen species (ROS) via Fenton chemistry, lysosomal membrane permeabilization and subsequent cell death reminiscent of ferroptosis . Thus, strategies to retain iron in lysosomes represent an attractive therapeutic modalities to target and eradicate CSC. A key protein in the regulation of iron homeostasis is the divalent metal transporter 1 (DMT1) located within the plasma membrane and membranes of late endosomes and lysosomes, which enables the translocation of ferrous iron (Fe 2+ ) to the cytosol following iron endocytosis .…”

Identification of Biologically Diverse Tetrahydronaphthalen‐2‐ols through the Synthesis and Phenotypic Profiling of Chemically Diverse, Estradiol‐Inspired Compounds

“…Seventeen out of twenty nine (59 %) compounds had an induction value of >5 % at either 10 or 30 μM, confirming the library had a high degree of bioactivity overall (Table 1). [23] It has previously been shown that the cell painting assay can predict certain modes‐of‐action with high confidence, including lysosomotropism, [10,11] mitochondrial respiratory activity, [16,24] and modulation of tubulin polymerization [25] . This is due to the availability of reference compounds that closely cluster in relation to their biological fingerprint in the CPA [26] .…”

“…More specifically, image‐based morphological profiling strategies such as the cell painting assay (CPA) have been developed to identify biological activity in a more general sense [7–9] . By comparing changes in cellular and organellar morphology upon compound treatment, one can not only determine whether a compound is bioactive, but also whether it is similar to known sets of reference compounds, facilitating mode‐of‐action and target identification [10,11] …”

Identification of Readily Available Pseudo-Natural Products

“…25 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. [26][27][28][29][30] 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.…”