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Published on: February 27, 2019
A high-throughput assay pipeline for specific targeting of frizzled GPCRs in cancer
Holly V Shaw1, Alexey Koval1, Vladimir L Katanaev2
1Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.
Abstract:
Frizzleds (FZDs) are a family of GPCRs controlling key events in all branches of the developmental Wnt signaling pathway. In this capacity these receptors are mostly active prenatally and have only a limited set of functions in the human adult. Numerous cancer types and subtypes were shown to be dependent on aberrant Wnt signaling and FZDs in particular. Taken together with their GPCR properties, this makes them an attractive drug target for the development of highly specific and efficient targeted therapies against cancer. Despite that, there are few chemical or other agents described targeting FZDs, and an even smaller number bears any clinical relevance. This sparsity dictates the necessity for broader efforts in order to advance in the Wnt pathway-targeting drug discovery. The current work describes the concepts and methodology of an inexpensive high-throughput screening followed by the pipeline of secondary assays in order to identify anti-FZD agents, which will efficiently deactivate Wnt signaling in cancer cells. Specifically, we describe a process and criteria for the selection, generation and statistical validation of a stable cancer cell line based on the well-described luciferase readout (TopFlash) which is then converted into a disease-representative high-throughput-ready screening system. We also provide information on the follow-up test sequence and the post-screening criteria to select FZD-targeting compounds among the hits and to validate them as such. We expect that use of this pipeline will boost the research on clinically valuable Wnt-targeting anti-cancer compounds.
Insights
Researchers developed a new, cost-effective high-throughput screening method to identify agents targeting Frizzleds (FZD) receptors. This approach aims to advance the discovery of novel Wnt pathway-targeting cancer therapies.
Area of Science:
- Molecular Biology
- Oncology
- Drug Discovery
Background:
- Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) crucial for Wnt signaling during development.
- Aberrant Wnt signaling and FZDs are implicated in numerous cancers, making FZDs attractive therapeutic targets.
- Limited availability of clinically relevant FZD-targeting agents necessitates new drug discovery efforts.
Purpose of the Study:
- To describe a methodology for inexpensive high-throughput screening (HTS) to identify anti-FZD agents.
- To develop a stable cancer cell line for Wnt signaling inhibition screening.
- To establish criteria for selecting and validating FZD-targeting compounds for cancer therapy.
Main Methods:
- Development and validation of a stable cancer cell line utilizing a luciferase readout (TopFlash).
- Implementation of a high-throughput screening pipeline for identifying potential FZD inhibitors.
- Establishment of secondary assays and post-screening criteria for hit validation and FZD targeting confirmation.
Main Results:
- A robust and statistically validated screening system was generated for Wnt signaling assessment.
- The pipeline facilitates the identification of compounds that can deactivate Wnt signaling in cancer cells.
- Criteria for selecting and validating FZD-targeting compounds were defined.
Conclusions:
- The described HTS pipeline offers an inexpensive and efficient approach to discover novel anti-cancer agents targeting Frizzleds.
- This methodology is expected to accelerate the development of clinically valuable Wnt pathway-targeting anti-cancer compounds.
- The study provides a framework for advancing drug discovery in Wnt-dependent cancers.
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