Abstract

ATPases are involved in multiple aspects of cellular
function and are attractive targets for novel cancer therapeutics. Key to
designing new drugs targeting ATPase activity is the measurement of the
inhibition of ATP turnover. ADP Glo is a common endpoint assay used in high
throughput screens. Although well suited to this role a continuous assay would
enable the efficient collection of more data at a greater number of time points
for mechanism of inhibition studies. With this in mind we compared available ATPase
detection technologies that could be read continuously. Although several
options gave a robust assay many were prone to interference with a number of
the tool compounds investigated. Full assay development was carried out with
the phosphate sensor kinetic ATPase assay as this showed no issues with
interference from the tool compounds. We successfully used this assay to
rapidly determine the mechanism of inhibition of two representative compounds
during the hit validation stage of a project, demonstrating this method can be
used for future studies.