Competition Assays for Dopamine D1 Receptors

As described above conducting competitive MS binding assays is dependent on certain prerequisites. Crude membrane preparations from pig striatum could be shown to be a suitable source for D1 receptors in preliminary radioligand binding assays. Furthermore it turned out that the binding assays can be conducted in 50 mM ammonium formate instead of the Tris buffer generally used. A further decisive fact was that the D1 receptor density in the membrane preparation was high enough to reach a target concentration in the range of the Kd of SCH 23390 (Fig. 7.4) that was chosen as a marker.

Employing the D1 receptor selective compound SCH 23390 as marker, well known from radioligand binding assays (Kd for [3H]SCH 23390 = 0.53 nM) [60], had a two-fold advantage: first, the experimental conditions for the binding experiment could be adopted from respective radioligand binding assays, and secondly, it enabled a very simple validation of the results obtained from MS binding assays by means of radioligand binding assays. Finally, with the development of a LC-ESI-MS/MS method that allowed a reliable quantitation of SCH 23390 in concentrations below 100 pM, the requirements for using SCH 23390 as a marker in competitive MS binding assays were fulfilled.

The binding experiments were conducted with 1.25 nM SCH 23390 and D1 receptors in concentrations of roughly 0.5-0.8 nM in a total volume of 250 mL, applying different concentrations of the test compound. Separating the nonbound marker from the target preparation (containing the bound marker) was accomplished by centrifugation. The resulting supernatant was subsequently analyzed by LC-ESI-MS/MS without further purification (Fig. 7.5).

SCH 23390 SKF 83566 (-)-sulpiride / (+)-butaclamol
Flow Chart For Ans Receptors
Fig. 7.4 Structures of compounds used in competitive MS binding assays for dopamine Di and D2 receptors.
Binding Assays

Fig. 7.5 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine Di receptors. After incubation of the target (D1 receptor) in presence of the marker (SCH 23390) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation.

Fig. 7.5 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine Di receptors. After incubation of the target (D1 receptor) in presence of the marker (SCH 23390) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation.

Sch23390 Binding Assay

Fig. 7.6 Nonbound SCH 23390 in a competitive MS binding assay for dopamine Di receptors monitored at a transition from 288.1 ! 91.2 m/z from binding samples without or with (+)-butaclamol. Intensity (/) is shown: (a) without (+)-butaclamol, (b) with 30 nM (+)-butaclamol, (c) with 10 mM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column; solvent: CH3CN/0.1% HCOOH in H2O 1:1; 300 mL min"1).

Fig. 7.6 Nonbound SCH 23390 in a competitive MS binding assay for dopamine Di receptors monitored at a transition from 288.1 ! 91.2 m/z from binding samples without or with (+)-butaclamol. Intensity (/) is shown: (a) without (+)-butaclamol, (b) with 30 nM (+)-butaclamol, (c) with 10 mM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column; solvent: CH3CN/0.1% HCOOH in H2O 1:1; 300 mL min"1).

Figure 7.6 exemplifies the results for the dopamine antagonist (+)-butaclamol (Fig. 7.4).

In the absence of the test compound (total binding) the signal of the nonbound marker was low (Fig. 7.6a) and intensified with increasing concentrations of (+)-butaclamol (Fig. 7.6b). The signal obtained in the presence of 10 mM (+)-butaclamol (Fig. 7.6c) corresponds to the difference between the total marker concentration [Mtot] and nonspecific binding. Hence, the difference between the maximum signal (m [Mtot] - nonspecific binding) and the minimum signal (m [Mtot] - specific binding - nonspecific binding) represents specific SCH 23390 binding.

With the data obtained in this way competition curves could be generated for (+)-butaclamol (Fig. 7.7). A number of other dopamine antagonists were studied

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