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.

Keywords: autophagy; cell painting; lysosome; proteomics; target identification.

Scheme 1

Synthesis of a cinchona alkaloid‐derived compound library. a) Molecular structures of previously identified autophagy inhibitor oxautin‐1, newly discovered inhibitor autoquin, and the four most abundant cinchona alkaloids. b) Synthesis of C2‐functionalised derivatives using the Borono–Minisci reaction. c) Synthesis of C3‐functionalised derivatives using selective C−H activation followed by Suzuki coupling. See Table 1 for details of the R‐groups investigated.

Figure 1

Validation of autoquin as an autophagy inhibitor. a) Effect of autoquin on MCF7 cells stably expressing EGFP‐LC3 upon autophagy induction by amino acid starvation using EBSS; n=3, representative images, scale bar=100 μm; CQ=chloroquine (50 μm). b) Quantification of the reduction in EGFP‐LC3 puncta by autoquin. Data points are mean ± SEM of three independent experiments. c) Effect of autoquin and analogues 17 a and 20 (see Scheme 2 for structures and data) on LC3‐II and p62 levels as assessed by western blot; n=3, representative images shown. d) MCF7 cells stably expressing mCherry‐EGFP‐LC3 treated with vehicle or 5 μm autoquin for 24 h, scale bars=10 μm. e) Autophagosomes (AP; yellow puncta) and autolysosomes (AL; red puncta) from (d) were quantified and data represented as percentage of cell area. Bar graphs show mean ± SD from three biologically independent experiments. Data points represent individual cells pooled from the three independent experiments (n≥23 cells per replicate). Significance was determined from biological replicates using a two‐tailed, unpaired t‐test. ns=not significant, **p=0.0064.

Scheme 2

SAR of the quinuclidine ring and pull‐down probe synthesis. a) Autophagy inhibitory activity of analogues with variations vicinal to the quinuclidine ring, variations highlighted in red. b) Synthesis and autophagy inhibitory activities of pull‐down probes for target identification experiments.

Figure 2

Autoquin is a lysosomotropic compound that acts as a functional inhibitor of acid sphingomyelinase. a) Cell painting profiles of autoquin and its most biosimilar compounds oxautin‐1, perphenazine, loperamide, and toremifene. See Figure S3 for representative images. b) Representative fluorescence microscopy images of MC7 cells treated with lysosomotropic compounds for 3 h and stained with Lysotracker DND‐99; scale bar=110 μm. c) Quantification of (b), n=3, data is mean ± SD. d) Product/substrate ratio of the acid sphingomyelinase (left) and acid ceramidase (right) reaction in intact cells. n=3, data is mean ± SD. e) Product/substrate ratio of the acid ceramidase reaction in cell lysates. n=3, data is mean ± SD. Statistical significance comparing treated samples to the DMSO control for (d) and (e) was assessed using the Student's t‐test. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Autoquin increases lysosomal mass and sequesters Fe²⁺ to the lysosomes in MCF7 cells, causing an increase in lysosomal reactive oxygen species. a) Fluorescence microscopy images showing the subcellular localisation of Fe²⁺ (red) and lysosomes (green) by means of RhoNox‐M and Lysotracker deep red (DR) fluorescence, respectively. b) Flow cytometry quantification of Lysotracker DR intensity from cells treated according to (a). c) Flow cytometry quantification of RhoNox‐M intensity from cells treated according to (a). d) Flow cytometry quantification of intracellular Fe²⁺ using an alternative turn‐on fluorescent probe RhoNox‐1. e) Fluorescence microscopy images showing the subcellular localization of ROS (red) by means of fluorogenic reaction with CellROX deep red in MCF7 cells treated with autoquin (5 μm) for 24 h, scale bar=10 μm. Colocalization with Lysotracker DND‐26 (green) assessed by Pearson correlation coefficient (R). ****p<0.0001, unpaired Student's t‐test.