Competition Assays for mGAT1

Additionally, MS binding assays comparable to competitive radioligand binding assays were conducted for the target-marker system mGAT1/NO 711. A wide variety of ligands with different lipophilicity and affinity to the target were tested (see Fig. 7.17). The experiments were conducted in a way that a constant concentration of both the target (10-20 mg protein per well, according to Bradford) and the marker (10 nM NO 711) were incubated with increasing concentrations of each test compound under conditions, as described for the saturation experiments. The bound native marker was then quantified by LC-MS/MS after the separation and liberation steps. From the binding curves obtained, the affinity constants of the test compounds (Ki) were calculated using the Cheng-Prusoff equation [see Eq. (3), Section 7.2.1], since marker-depletion was negligible (see Fig. 7.18 for a representative example for compound (S)-3b).

The results of the competitive MS binding assays quantifying the bound marker were again compared to conventional radioligand binding assays (based on [3H]NO 711) for the same test compounds. Table 7.5 shows the results of both the competitive MS binding studies and the competitive radioligand binding assays in comparison.

Figure 7.19 shows a direct correlation between pKi values derived from the MS binding assays described above and those from radioligand binding assays. The rise of the graph of 1.010 + 0.01604 shows (r2 > 0.99) that pKi values resulting from both binding assays are nearly identical.

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