Kinetic Assays for mGAT1

For the establishment of saturation and competitive MS binding assays described above, association and dissociation assays with mass spectrometric quantitation of the native marker had also been conducted.

In association assays, a constant NO 711 concentration (in the region of Kd) was incubated with the target. Using the previously described method, the binding experiments are terminated after different periods of time and quantified by LC-ESI-MS/MS. The binding curve derived from the data is shown in Fig. 7.20. It

Fig. 7.17 Structures of compounds tested in competitive MS binding assays quantifying bound marker.

is clearly visible that the binding of the marker to the target reaches steady state after approximately 30 min. The observed association rate constant kobs calculated from these experiments amounted to 0.19 G 0.01 min-1.

A further characteristic of ligand-target interaction is the dissociation rate constant. Dissociation experiments are not only an important criterion for the establishment of binding assays, they also can show the reversibility of the specific marker binding at the target [16], which is usually verified by dissociation experiments (see also Section 7.2.1). In the MS binding assays presented here, GABA was used as a competitor to initiate dissociation. The experiment was then termi-

Fig. 7.18 Representative binding curve for competitive MS-binding experiments quantifying bound marker. Compound (S)-3b was tested (see Fig. 7.17 for structure). Data points represent specific binding of the marker NO 711 (mean G SEM from triplicate values).

Table 7.5 Affinities (mean G SEM, n = 3) for GAT1 inhibitors at mGATl-membrane preparation obtained in competitive binding experiments using NO 711 for competitive MS binding quantifying bound marker and [3H2]NO 711 in competitive radioligand binding [80].

Test compound MS binding Radioligand

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