Amplex red assay, a standardized in vitro protocol to quantify the efficacy of autotaxin inhibitors.

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 (IC(50)) 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).