The ATPase assay is an in vitro biochemical method used to assess whether a compound interacts with ABC transporters by measuring the rate of ATP hydrolysis. When an ABC transporter binds and transports a substrate, it hydrolyzes ATP; inhibitors reduce this rate. The assay is widely used in early-stage DMPK screening.
Principle of the ATPase Assay
Membrane vesicles containing the ABC transporter of interest are incubated with the test compound and ATP. The release of inorganic phosphate (Pi) from ATP hydrolysis is quantified colorimetrically (e.g., malachite green assay). Stimulation of ATPase activity above baseline indicates substrate interaction; inhibition of vanadate-sensitive ATPase activity indicates inhibitor behavior.
Anwendungen
The ATPase assay is commonly used for P-gp and BCRP screening in hit-to-lead and lead optimization phases. It offers higher throughput and lower cost compared to vesicle transport assays, making it suitable for screening compound libraries. Results guide prioritization of compounds for more definitive vesicle assay studies.
Limitations
Not all substrates stimulate ATPase activity proportionally, and some inhibitors are not detected reliably. The assay cannot definitively distinguish substrates from modulators and should be followed by a vesicle transport assay for regulatory submissions.
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