Competition Assays for Dopamine D2 Receptors

The method was also applied to the D2 receptor. In this case however, an incubation medium with nonvolatile components frequently used in radioligand binding assays consisting of 50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2, 5 mM KCl and 1 mM EDTA was deliberately employed to demonstrate that the incubation medium in MS binding assays quantifying the nonbound marker is not restricted to volatile buffers. As in the D1 receptor binding assay, a crude membrane fraction of pig striatum was used as the source for the D2 receptors and with spiperone (Fig. 7.4) again the native form of a well established radioligand was chosen as a marker. Preliminary experiments showed, as expected, that the signal of the marker was substantially suppressed, when the nonbound marker was analyzed by LC-ESI-MS/MS directly out of the matrix of the binding sample. Therefore a solid phase extraction (SPE) method was employed to remove most of the interfering sample matrix and to enhance the spiperone signal, allowing the binding experiments to be successfully performed. After incubating spiperone in

Fig. 7.8 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine D2 receptors including matrix elimination. After incubation of the target (D2 receptor) in presence of the marker (spiperone) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified after SPE by LC-ESI-MS/MS.

Fig. 7.8 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine D2 receptors including matrix elimination. After incubation of the target (D2 receptor) in presence of the marker (spiperone) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified after SPE by LC-ESI-MS/MS.

a concentration of 1.25 nM with D2 receptors in a concentration of @400 pM in a total volume of 500 mL nonbound spiperone could be quantified reliably by LC-ESI-MS/MS after SPE of the supernatant obtained after centrifugation (Figs. 7.8, 7.9).

In this way, the affinities of (+)-butaclamol, chlorpromazine and (S)-sulpiride (Fig. 7.4) for the D2 receptor were characterized. The competition curves obtained [e.g. for (+)-butaclamol, Fig. 7.10] were as expected, but showed an unusually

Fig. 7.9 Nonbound spiperone in a competitive MS binding assay for dopamine D2 receptors monitored at a transition from 396.0 ! 123.0 m/z from binding samples without or with (+)-butaclamol. Intensity (I) is shown: (a) without (+)-butaclamol, (b) with 100 nM (+)-butaclamol, (c) with 10 mM (+)-butaclamol. (a-c) Representative

Fig. 7.9 Nonbound spiperone in a competitive MS binding assay for dopamine D2 receptors monitored at a transition from 396.0 ! 123.0 m/z from binding samples without or with (+)-butaclamol. Intensity (I) is shown: (a) without (+)-butaclamol, (b) with 100 nM (+)-butaclamol, (c) with 10 mM (+)-butaclamol. (a-c) Representative chromatograms after SPE on Oasis HLB cartridges followed by HPLC separation (RP8 column; solvent: CHsCN/0.1% HCOOH in H2O 30:70, 150 |iL min"1). All samples (supernatants) were spiked with haloperidol (0.875 nM, 376.0 ! 123.0 m/z) as internal standard.

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