Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis

Abstract

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a pro-survival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents, including acquired resistance. We aimed to identify small-molecule autophagy inhibitors using a HTS/HCA approach through a phenotypic, cell image-based assay, in order to screen multiple biological targets simultaneously and to screen compounds in a physiologically relevant environment. LC3 is a component of the autophagosome, which undergoes a cytoplasmic redistribution from diffuse to punctate dots during autophagy. We employed HeLa cells stably expressing EGFP-LC3 in a primary phenotypic screen. As a first step, a “Validation Library” of about 8,000 pre-selected compounds, about 25% of which had known biological activity and the others representing a range of chemical structures, was run in duplicate both to assess screening suitability and likely hit rate, and to give a valuable preview of possible active structures or biological targets. The primary screen of about 0.25 million compounds yielded around 10,500 positive compounds. These were tested in a suite of further cellular assays designed to eliminate unwanted positives, together with the application of chemi- and bioinformatics to pick out compounds with known biological activity. These processes enabled the selection of compounds that were the most promisingly active and specific. The screening “tree” identified, amongst others with as yet unidentified targets, chemical series active against autophagy-relevant biological targets ULK or Vsp34, validating the phenotypic screening methods selected. Finally, about 400 compounds were fully qualified after following this triage. The development of the assays, compound screening process and the compound triage is described.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

“…To identify small molecule autophagy inhibitors with new mechanisms of action, a high-content, imagebased phenotypic screen was employed that quantifies autophagosomal structures in MCF7 cells after autophagy induction ( Supplementary Table 1). [13][14][15] Briefly, autophagy was induced in MCF7 cells stably expressing EGFP-LC3 by amino acid starvation (starv. ; Earle's balanced salt solution, EBSS) or by rapamycin (rapa.)…”

Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8–Atg3 Protein–Protein Interaction

“…Targeting downstream events is attractive in circumventing this potential pitfall. Autophagy inhibitors that specifically prevent autophagosome expansion have been reported in humans and are being considered for anticancer drug therapy …”

Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34

“…Target‐agnostic cellular assays monitoring changes in phenotype followed by target identification and validation offer unbiased opportunities for the discovery of novel chemotypes and cellular targets linked to the biological process of interest by means of phenotypic changes . Herein, we report the discovery of a potent autophagy inhibitor by means of a phenotypic screening approach monitoring small‐molecule‐induced impairment of autophagy . The compound, named autophinib, targets the lipid kinase vacuolar protein sorting 34 (VPS34), which is a promising target for selective autophagy modulation …”

“…To identify novel autophagy inhibitors, we employed a previously described cellular assay . In brief, autophagy was induced in MCF7 cells stably expressing EGFP‐LC3 (enhanced green fluorescent protein tagged light chain 3; MCF7‐LC3) by amino acid starvation using Earle's balanced salt solution (EBSS), or by inhibition of mTOR using rapamycin.…”