The ATPase assay is an in vitro membrane assay designed to indicate the nature of the interaction between the compound and the transporter. By measuring ATPase activity, both activation and inhibition of transporters can be investigated using membranes from baculovirus-infected insect cells or mammalian cell membranes containing high levels of human or rodent wild-type transporters.
ABC transporters mediate the transport of substrates against a concentration gradient using energy derived from ATP hydrolysis, which is proportional to the transporter activity and could easily be detected with a colorimetric method. To assess activation, ABC transporter-rich membranes are incubated with various (typically in 8) concentrations of the test article and the effect on basal ATPase activity is measured.
Compounds that stimulate ATPase are generally considered substrates for the transporter. To assess inhibition, a test article’ ability to modify the activity of a given ABC transporter stimulated with its prototypical substrates is examined. The activation and inhibition tests are complementary assays.
Stimulation detected in the activation assay indicate that the compound is a transported substrate of the transporter, while interactions detected in the inhibition test indicate interaction of the test compounds with the transporter, but do not give information on the nature (substrate or inhibitor) of the interaction. In some cases inhibitors or slowly transported compounds may inhibit the baseline transporter ATPase activity as well. Slowly transported substrates often do not stimulate the ATPase activity in a detectable extent; however the existing interaction can be identified in the inhibition assay.
Figure 1. Working principle of the ATPase assay
Figure 2. Vanadate sensitive ATPase activity of SB-ratMdr1b-Sf9-ATPase membranes
in the presence of different concentrations of test drugs
Figure 3. Activation of MDR1 ATPase activity and inhibition of verapamil stimulated
ATPase activity by digoxin in the MDR1-PREDEASY-ATPase assay