Zegzouti Hicham, Alves Juliano, Vidugiris Gediminas and Goueli Said
Promega Corporation 2800 Woods Hollow Road Madison WI 53711 USA
Transmembrane ATPases play an important role in either importing the many metabolites necessary for cell metabolism or exporting the toxins, wastes, and drugs that can interfere with cellular processes. An important example of import protein is the sodium-potassium pump (or Na+/K+ATPase), which maintains the ionic concentration balance that is required for cell viability. Due to its activity modulation by cardiac glycosides, Na+/K+ ATPase emerged as a drug target for heart related conditions.
Another example of transporters important in drug discovery is the family of ATP-binding cassette (ABC) transporters that are associated with multi-drug resistance (MDR). To ensure drug candidates will have good bioavailability and distribution and less toxicity, it is important to discern drug interaction with these transporters early in drug discovery. These drug efflux pumps are characterized by their ATPase activity that is both coupled to the transporting event and is stimulated by transported drugs. Therefore, discovering drugs that modulate all these transporters ATPase activity or identifying how a drug affects it can lead to new therapeutic agents, or better understanding of the interaction, respectively.
Because these transporters usually have a low ATP turnover rate and/or high ATP requirements, a sensitive assay is needed with high throughput for early drug screening efforts. The ADP-Glo™ Max luminescent ADP detection assay provides a universal, homogeneous, and high-throughput method for measuring ATPase activity by quantifying the amount of ADP produced during the reaction. The assay is sensitive enough to detect low amounts of ADP in the presence of high amounts of ATP in a broad range of ATP concentrations (up to 5mM). This is of distinct advantage for drug transporters which typically have a high Km for ATP. The luminescent signal generated is proportional to the ADP concentration produced and it correlates to the extent of ATPase activity. We demonstrate the application of this assay for the identification of inhibitors of Na+/K+ ATPase activity and the analysis of substrates and inhibitors of the ABC transporters P-gp, BCRP, MRP1, MRP2, and MRP3. The ADP-Glo™ Max Assay can be used with virtually all ATPases and because of its exquisite sensitivity, only a small amount of transporter membranes is required to achieve a high signal to background ratio. The ADP-Glo™ Max luminescent detection scheme also minimizes interferences from reaction components that interfere with other detection schemes. This highly sensitive readily automatable assay can easily replace the low throughput, significantly less sensitive colorimetric or fluorescent ATPase assays that are based on inorganic phosphate detection. A comparison of the ADP-Glo™ Max assay to the latter showed that the luminescent assay has a lower limit of detection allowing both a savings in enzyme usage and better assay performance in attempts to identify relevant chemical probes.