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Amplex red assay, a standardized in vitro protocol to quantify the efficacy of autotaxin inhibitors
Elli-Anna Stylianaki1, Christiana Magkrioti1, Eleanna Kaffe1
1Insitute for Fundamental Biomedical Research, Biomedical Sciences Research Center "Alexander Fleming," Athens, Attica 16672, Greece.
Abstract:
Autotaxin (ATX), a secreted lysophospholipase D responsible for the extracellular production of the bioactive phospholipid lysophosphatidic acid (LPA), is a therapeutic target in idiopathic pulmonary fibrosis and pancreatic cancer, among other disorders, promoting the synthesis of novel ATX inhibitors. Here, we present a protocol for detecting and characterizing ATX inhibitors using a fluorometry-based microplate assay. We describe steps for a first screening of compounds, half-maximal inhibitory concentration (IC50) quantification of initial hits, screening for false positives, and identification of the hits' mode of inhibition. For complete details on the use and execution of this protocol, please refer to Stylianaki et al.1.
Insights
This study introduces a new assay to find and study autotaxin (ATX) inhibitors. This method helps develop new treatments for diseases like pulmonary fibrosis and pancreatic cancer.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Autotaxin (ATX) is a key enzyme in producing lysophosphatidic acid (LPA).
- ATX is a therapeutic target for idiopathic pulmonary fibrosis and pancreatic cancer.
- Novel ATX inhibitors are needed for treating these diseases.
Purpose of the Study:
- To present a protocol for detecting and characterizing ATX inhibitors.
- To enable efficient screening and validation of potential ATX-targeting compounds.
Main Methods:
- Fluorometry-based microplate assay.
- Compound screening and hit identification.
- IC50 quantification and false positive screening.
- Determination of inhibition mode.
Main Results:
- The protocol allows for the detection and characterization of ATX inhibitors.
- It facilitates initial screening, IC50 determination, and mode of inhibition analysis.
- This assay is suitable for identifying novel ATX inhibitors.
Conclusions:
- The described microplate assay is a valuable tool for ATX inhibitor discovery.
- This protocol aids in the development of new therapeutic strategies targeting ATX.
- It supports the advancement of treatments for ATX-related disorders.
