Using luminescent ADP-Glo™ Max assay to detect activity modulators of transmembrane
ATPases and ABC transporters
Zegzouti Hicham, Alves Juliano, Vidugiris Gediminas and Goueli Said
2012
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
4. Comparison of detection limits of ADP and
phosphate detection assays
1. Abstract
Na+/K+ ATPase activity detection using luminescent ADP assay compared to colorimetric
and Fluorescent Pi detection assays
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.
ADP-Glo Max detects the activity of small amount of ATPase with high SB and high Signal to Noise
2. ADP-Glo™ is a positive detection Assay
for product formation
5. Screening inhibitors with ADP-Glo™ Max
generates meaningful data
Add ADP-Glo Reagent
6. Stimulation of MDRs ATPase activity by reference
compounds detected by ADP-Glo™ Max
ATPase assay using 5mM ATP and 10µg of membranes expressing different transporters
ATPase assay with MDR
expressing membranes
ADP-Glo Max Assay
ADP-Glo™ Max detects high and low activity MDRs with low amount of membrane
preparations (10µg)
7. Inhibition of basal and stimulated ATPase activity
of MDRs detected by ADP-Glo™ Max
Screening the LOPAC library for Na+/K+ ATPase inhibitors and compound interference
in the ADP-Glo™ Max Assay
MDR activators
Pgp: Verapamil
BCRP: Sulfasalazine
MRP1: NEM-GS
MRP2: Probenecid
MRP3: Benzbromarone
Add ADP-Glo Max Detection Reagent
 Step 1: Depletion of unconsumed ATP after the ATPase reaction.
 Step 2: ADP is converted into ATP that is detected via a luciferase/luciferin reaction.
ADP-Glo™ Max assay is adequate for screening for MDRs Inhibitors
 Luminescent signal is proportional to ADP produced and the ATPase activity.
3. ADP-Glo™ Max can be used with a broad
range of ATP concentrations
ADP conversion curves at different ADP/ATP concentrations
Na+/K+ ATPase inhibitors
8. General features of ADP-Glo™ Max assay
The known Na+/K+ ATPase inhibitors Ouabain and Dihydroouabain were successfully identified during the screen.
ADP-Glo™ generates low hit rate and less compound interference during HTS
Dose response of two clinically relevant drugs using ADP-Glo Max assay
Ouabain Titration
[ATP + ADP]
Percent ADP in an ATP + ADP Mixture
ADP-Glo Max
100
80
60
40
20
10
5
4
3
2
1
0
5mM
329
262
191
128
65
28
13
11
9
6
4
1
2.5mM
304
245
180
120
60
26
13
12
9
7
5
1
120
100
80
60
40
20
IC50 = 0.66µM
0
0.01
1
242
191
137
88
43
21
12
11
8
6
4
1
0.5mM
191
149
106
69
39
20
12
11
8
6
4
1
Linearity: ADP-Glo™ Max assay detects up to 5mM ADP in a linear fashion.
Sensitivity: ADP-Glo™ Max generates a high Signal to background (SB) ratios with high Z’ values
at even low % conversions.
0.04mU Na+ /K+ ATPase,
1.2mM ATP, 15min at 37C
120
100
Luminescent assay: Less Compound interference.
80
60
40
Homogenous, non radioactive and Antibody Free
20
IC50 = 1.7µM
0
100
Ouabain, nM
1mM
140
0.04mU Na+ /K+ ATPase,
1.2mM ATP, 15min at 37C
10000
1000000
 High dynamic range: High Signal to Background at low % ATP to ADP conversion allows use of
lower amount of enzyme or membranes
Broad range of ATP conc. (linear from µM to mM range) allows detection of activities requiring
high Km for ATP like transporters.
Digoxin Titration
% Enzyme Activity
Signal to Background ratios
% Enzyme Activity
140
 Universal: Any ATPase, e.g. Na+/K+ ATPase, ABC transporters (MDRs).
1
10
100
1000
10000
100000
Digoxin, nM
Na+/K+ ATPase is effectively inhibited by cardiac glycosides, which are of significant
therapeutic value in the treatment of congestive heart failure
 Robust Assay (Z’ higher than 0.7)
Convenient: For rapid high-throughput screening for Novel inhibitors of the Na+/K+ pump and
for both MDR transport substrates and inhibitors without the need for cell culture.
More info: [email protected]
www.promega.com