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Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis
J V Peppard1, C Rugg1, M Smicker1
1Lead Generation and Candidate Realization, R&D, Bridgewater, NJ 07059, U.S.A.
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.
Insights
Researchers screened 250,000 compounds to find small-molecule autophagy inhibitors for cancer therapy. They identified promising compounds targeting ULK and Vsp34, validating their high-content screening approach.
Area of Science:
- Oncology
- Cell Biology
- Drug Discovery
Background:
- Autophagy is a crucial cellular process implicated in cancer development and progression.
- It can act as both a tumor suppressor and a pro-survival mechanism, aiding cancer cells in resisting metabolic stress and chemotherapy.
- Understanding and modulating autophagy is a key strategy for developing novel cancer therapies.
Purpose of the Study:
- To identify novel small-molecule autophagy inhibitors using a high-throughput screening (HTS) and high-content analysis (HCA) approach.
- To validate the efficacy of phenotypic, cell image-based assays for simultaneous screening of multiple biological targets in a physiologically relevant context.
- To discover compounds that can overcome chemoresistance by targeting the pro-survival functions of autophagy.
Main Methods:
- Utilized HeLa cells stably expressing EGFP-LC3 for a primary phenotypic screen, monitoring autophagosome formation.
- Conducted a large-scale screen of approximately 250,000 compounds using HTS/HCA.
- Employed a multi-step triage process involving secondary cellular assays, cheminformatics, and bioinformatics to filter and select promising compounds.
Main Results:
- The primary screen identified approximately 10,500 initial positive compounds.
- A rigorous triage process successfully narrowed down the candidates to about 400 fully qualified compounds.
- The screening identified chemical series targeting autophagy-related kinases ULK and Vsp34, validating the phenotypic screening strategy.
Conclusions:
- The developed HTS/HCA phenotypic screening approach is effective for identifying small-molecule autophagy inhibitors.
- The study successfully identified specific compounds with activity against key autophagy regulators, providing potential leads for cancer drug development.
- This work validates the utility of image-based screening for discovering modulators of complex cellular pathways like autophagy in cancer research.
