High-Content Drug Discovery Screening of Endocytosis Pathways

  • David A. Cardoso
  • Ngoc Chau
  • Phillip J. RobinsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)


Endocytosis is the dynamic internalization of cargo (receptors, hormones, viruses) for cellular signaling or processing. It involves multiple mechanisms, classified depending on critical proteins involved, speed, morphology of the derived intracellular vesicles, or substance trafficked. Pharmacological targeting of specific endocytosis pathways has a proven utility for diverse clinical applications from epilepsy to cancer. A multiplexable, high-content screening assay has been designed and implemented to assess various forms of endocytic trafficking and the associated impact of potential small molecule modulators. The applications of this assay include (1) drug discovery in the search for specific, cell-permeable endocytosis pathway inhibitors (and associated analogues from structure-activity relationship studies), (2) deciphering the mechanism of internalization for a novel ligand (using pathway-specific inhibitors), (3) assessment of the importance of specific proteins in the trafficking process (using CRISPR-Cas9 technology, siRNA treatment, or transfection), and (4) identifying whether endocytosis inhibition is an off-target for novel compounds designed for alternative purposes. We describe this method in detail and provide a range of troubleshooting options and alternatives to modify the protocol for lab-specific applications.

Key words

Endocytosis Pharmacology Therapeutics Drug discovery High-content screening 



clathrin-independent carriers/glycosylphosphatidylinositol-anchored protein-enriched endocytic compartments


clathrin-mediated endocytosis




Dulbecco’s Modified Eagle Medium


dimethyl sulfoxide


epidermal growth factor receptor


fetal bovine serum


fast endophilin-mediated endocytosis


green fluorescent protein


ImageXpress Micro


phosphate-buffered saline





We are grateful for financial support from the National Health and Medical Research Council Australia (GNT1069493, GNT1052494, GNT1047070, GNT 1105666, GNT1137064, and GNT1162515), Australian Research Council (DP180101781), Children’s Medical Research Institute (CMRI), and the University of Sydney and for equipment from the Australian Cancer Research Foundation, the Ramaciotti Foundation, and the Cancer Institute NSW. Endocytosis pathways (Fig. 1) were created with


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • David A. Cardoso
    • 1
  • Ngoc Chau
    • 1
  • Phillip J. Robinson
    • 1
    Email author
  1. 1.Cell Signalling Unit, Children’s Medical Research Institute, Faculty of Medicine and HealthThe University of SydneyWestmeadAustralia

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