MS Binding Assays

Radioligand binding assays are a relatively simple but at the same time a very important and efficient tool to study target-ligand interactions. Since they are very videly used in drug discovery and have been described extensively, this section only discusses their fundamental aspects and those aspects that are important for the description of the MS binding assays below . More detailed information can be found in the relevant literature [6, 7, 16-21]. 

Fig. 7.4 Structures of compounds used in competitive MS binding assays for dopamine Di and D2 receptors.

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 (Di 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. 7.6 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 pM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column solvent CH3CN/0.1% HCOOH in H2O 1 1 300 pL min ).

Fig. 7.7 Representative binding curve obtained by nonlinear regression from a competitive MS binding assay for dopamine Di receptors, in which (+)-butaclamol competes with SCH 23390 as marker. The points describe nonbound SCH 23390 quantified by LC-ESI-MS/MS. Data reflect means (+s) from binding samples, each performed in quadruplicate.

Table 7.1 Affinities (mean + SEM, n = 3) for dopamine antagonists at D] receptors obtained by MS-binding assays and radioligand binding assays, respectively [61],...

The reliability of the SPE-LC-ESI-MS/MS quantitation method of the nonbound marker spiperone was verified in identical binding assays employing [ H] spiperone as marker and (S)-sulpiride as test compound, again quantifying the nonbound marker but this time by scintillation counting. Both the run of the competition curve and the Ki value determined for (S)-sulpiride were in good accordance with the results from the MS binding assays (Table 7.2). 

MS binding assays are also useful for library screening with subsequent hit identification. The concept is simple. First, a library is searched for active compounds in a competitive binding assay. If the result is positive (which is indicated by an increase of the marker signal), the target bound hit is liberated and identified. 

Table 7.3 Nonbound morphine in MS binding assays at /i-opioid receptors [65],...

To this end, the pellets remaining from the competitive MS binding assay were, after several washing steps, resuspended in binding buffer and incubated with a great excess of competitor (50 pM (+)-methadone) to liberate the unknown bound ligand (as well as the bound marker). Then the supernatants obtained by centrifugation were analyzed by LC-ESl-MS/MS. In addition to morphine as the marker, naloxone was identified as the hit that had been searched for. Thereby, the relative concentrations of marker (2.93 nM) and hit (2.30 nM) pointed to the fact that the hit had a similar affinity to the //-opioid receptor as the marker [65]. 

This strategy was realized in a competitive MS binding assay examining the affinity of naloxone for //-opioid receptors (with morphine as marker under the conditions described above for library screening). The respective experiments led... 

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