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Competitive Binding Assays Made Easy with a Native Marker and Mass Spectrometric Quantification.

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Binding Assays with Native Markers
Competitive Binding Assays Made Easy with a
Native Marker and Mass Spectrometric
Georg Hfner and Klaus Theodor Wanner*
Dedicated to Professor H. Nth
on the occasion of his 75th birthday
Competitive binding assays represent a fundamental method,
not only in drug discovery, but also in many other fields of the
life sciences.[1] These assays are used to characterize the
affinity of a ligand to a defined target, for example, a
pharmacological receptor. The principle is simple. A marker,
that is, a ligand which addresses the desired binding site (the
target) with high affinity and selectivity, is required. The
affinity of test compounds for the target can then be
determined from their ability to displace the marker from
the target. Owing to the extraordinarily high sensitivity
required for the quantification of the marker, the application
of this principle is generally restricted to radioactive-ligand
(radioligand) binding assays and fluorescence assays. However, this means that the marker has to be labeled with a
suitable radioisotope (e.g. 3H, 35S, or 125I)[2] or an appropriate
fluorophore.[3] Besides the additional synthetic efforts, further
limitations, such as safety precautions and expensive disposal
complicate the practical work with radioligands. On the other
hand, the introduction of a fluorophore to the marker often
requires re-optimization of the structure of the marker, in
addition background fluorescence in binding experiments can
interfere with the measurements.[4]
In the mean time, mass spectrometry (MS) has emerged as
an analytical technique which is sensitive enough to provide
quantification in binding assays.[5] Quite elegant approaches
based on affinity selection with mass spectrometric detection
have been described recently. However, this method is
demanding, because all test compounds showing affinity
have to be monitored.[6]
We have attempted to establish competitive binding
assays, similar to radioligand binding assays, in which only
the marker has to be detected and quantified by mass
spectrometry. In this kind of binding assay, which we termed
competitive MS-binding assay, a native marker is used instead
of the radioligand. When choosing a possible marker from the
[*] Prof. Dr. K. T. Wanner, Dr. G. H8fner
Department Pharmazie
Zentrum f;r Pharmaforschung
Ludwig-Maximilians-Universit=t M;nchen
Butenandtstrasse 7, 81377 M;nchen (Germany)
Fax: (+ 49) 89-2180-77247
[**] This work was supported by the Fonds der Chemischen Industrie
and the BMBF.
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. Int. Ed. 2003, 42, 5235 –5237
pool of known ligands the only factors that need to be
considered are that the ligand must have sufficient affinity
and selectivity for the target and a detection limit that is as
high as possible with respect to mass spectrometry.
Usually, in radioligand binding experiments the amount of
bound radioligand is determined. Performing MS-binding
experiments, it may be preferable to quantify the nonbound
marker to avoid the additional step of dissociating the
receptor–marker complex. From the analytical point of
view, it is favorable to choose a marker and a receptor
concentration near the Kd value. Thus it can be arranged that
the amount of bound and nonbound marker are in the same
order of magnitude. Then, in the competitive binding experiment (in which the bound marker is displaced by the test
compound) the amount of nonbound marker increases
considerably and the analytical signal is significantly
As a model to investigate the feasibility of binding assays
following this concept we selected the dopamine D1-receptor
which is a typical G-protein coupled receptor used in the field
of drug screening. A porcine striatal membrane fraction (P2)[7]
served as a natural source containing D1-receptors in relatively high concentrations. SCH 23390 (Figure 1) was chosen
Figure 1. Representative MRM chromatogram of SCH 23390 from
binding experiments after HPLC separation (Supersphere 60 RP
select B; solvent: CH3CN/0.1 % HCOOH in H2O 1/1). a) Without
(+)-butaclamol, b) 30 nm (+)-butaclamol, c) 10 mm (+)-butaclamol.
Nonbound SCH 23390 was monitored at a transition from m/z 288.1
(precursor ion) to m/z 91.2 (product ion).[9]
as a native marker. This compound shows high affinity (Kd =
0.53 nm) and selectivity for D1-receptors.[8] Furthermore
preliminary studies revealed that electrospray mass spectrometry (ESI-MS) in the multiple reaction monitoring
(MRM) mode using a triple quadrupole mass spectrometer
coupled to a HPLC (LC-ESI-MS-MS) is a rather reliable
technique for the quantification of SCH 23390 in sample
DOI: 10.1002/anie.200351806
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The affinity of seven known dopamine antagonists for D1receptors was determined according to the new procedure
(Table 1). The binding experiments were performed according to standard radioligand binding assays for dopamine
receptors.[8–10] In comparison to such standard radioligand
binding assays, the commonly used 50 mm Tris-buffer (containing various additional salts) was substituted with a 50 mm
ammonium formiate solution of pH 7.4, to avoid suppression
of the LC-ESI-MS-MS signal of SCH 23390 in matrix samples.
The concentration of the D1-receptors was set in the range of
the Kd value (about 0.5–0.8 nm). As a further difference to
radioligand binding assays the nonbound marker was separated by centrifugation (at 50 000 g) instead of filtration.[9]
As an example of our method, the determination of the
affinity of the dopamine antagonist (+)-butaclamol as a test
compound is illustrated: D1-receptors together with 1.25 nm
SCH 23390 and (+)-butaclamol (0–10 mm) were incubated for
Figure 3. Binding curve obtained by nonlinear regression for binding
experiments in which (+)-butaclamol competes with SCH 23390 for
the binding sites. The data points correspond to nonbound
SCH 23390 quantified by LC-ESI-MS-MS and are mean values ( s)
from four binding experiments.[9]
curve of the concentration of nonbound
SCH 23390 in the supernatant solution as a
function of the test compound (+)-butaclamol is characteristic for this kind of assays
Marker SCH 23390
Marker [ H]SCH 23390
based on the quantification of the marker in
IC50 [nm]
Ki [nm]
Ki [nm]
the supernatant solution. From such binding
36 3
11 1
5.4 1.9
curves, the IC50 value, that is, the concenchlorpromazine
1700 40
620 10
300 40
tration of a test compound which reduces
620 170
220 60
110 2
the specific binding of the marker to 50 %,
13 000 700
4700 200
2500 500
5.9 1.0
1.9 0.3
2.7 0.6
can be deduced in analogy to conventional
> 10 000
> 10 000
> 10 000
binding assays. The mean of the IC50 values
1300 100
460 40
215 20
which were determined from three inde[a] Nonbound SCH 23390 was quantified by LC-ESI-MS-MS after centrifugation from binding experipendent experiments, was converted into a
ments performed in 50 mm ammonium formiate pH 7.4.[9] [b] IC50 and Ki values are expressed as
Ki value. This Ki value for (+)-butaclamol
arithmetic means standard error of the mean from three experiments. A Kd value of 0.72 nm obtained
correlates well with the Ki value determined
for [3H]SCH 23390 in ammonium formiate pH 7.4 was used for the calculation of the Ki values.[9]
in an analogously conducted conventional
[c] Bound [ H]SCH 23390 was quantified after the filtration of binding experiments performed in 50 mm
ammonium formiate pH 7.4.[9]
[3H]SCH 23390 (Table 1). The same applies
for the Ki values of the other six test
40 min and centrifuged afterwards. The amount of SCH 23390
compounds in this experiment. Further investigation is
in the supernatant solution after centrifugation was quantified
needed to explain the tendency of slightly enhanced Ki values.
without further sample preparation by ESI-LC-MS-MS with
Nevertheless, it is considerably more important that the rank
the aid of a calibration curve (Figure 1 and Figure 2). Figure 3
order of the Ki values obtained here is in agreement with the
shows a binding curve generated from the resulting data. The
rank order observed in radioligand binding assays. Consequently competitive MS-binding assays offer an attractive
alternative to the conventional procedures mentioned above.
Furthermore, with competitive MS-binding assays, it might be
possible to collect data from different binding sites in a
biological sample in one experiment. This could be achieved
by using different markers, which selectively address several
binding sites in the sample, in the same competitive binding
assay. Moreover, MS-binding assays could be modified in a
way that firstly, a library is screened for hits in a competitive
binding experiment and that in a second step, such bound hits
could be identified by mass spectrometry following their
liberation from their binding sites.
In conclusion we have described a new method to perform
competitive binding assays which is characterized by the mass
Figure 2. Calibration curve for SCH 23390. A matrix identical to the
spectrometric quantification of the marker. Among other
supernatant of the binding experiments was added to SCH 23390. The
benefits the main advantage that such competitive MSgiven data are mean values ( s) from four measurements.[9] F = peak
binding assays offer is that ligands for a binding site need
Table 1: IC50 and Ki values for dopamine antagonists at D1-receptors obtained by mass spectrometry and
by radioligand binding assays.
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2003, 42, 5235 –5237
not be labeled. The competitive MS binding assays at
dopamine D1-receptors using SCH 23390 as a native marker
resulted in affinity constants which were in good agreement
with the affinity constants determined in radioligand binding
assays. This simple procedure, its universal suitability, and the
advantage of using unmodified markers could help to
establish MS binding assays as a routine method in drug
Received: May 5, 2003 [Z51806]
Published Online: September 1, 2003
Keywords: analytical methods · binding assays · dopamine
receptors · drug screening · mass spectrometry
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Angew. Chem. Int. Ed. 2003, 42, 5235 –5237
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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