Info

2.32 nM

(G)-Methadone

50 mM

7.73 nM

Library 1

10 nM

2.76 nM

Library 1

1 mM

2.03 nM

Library 2

10 nM

5.02 nM

Library 2

1 mM

7.92 nM

Binding Assays

Fig. 7.12 Schematic flowchart of the competitive MS binding assay for «-opioid receptors including liberation of bound marker and test compounds, respectively. After incubation of the target («-opioid receptor) in the presence of the marker (morphine) and a compound library, the binding samples are centrifuged to separate bound from nonbound marker. Subsequently, the nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation (route 1).

Fig. 7.12 Schematic flowchart of the competitive MS binding assay for «-opioid receptors including liberation of bound marker and test compounds, respectively. After incubation of the target («-opioid receptor) in the presence of the marker (morphine) and a compound library, the binding samples are centrifuged to separate bound from nonbound marker. Subsequently, the nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation (route 1).

In the case that a signal reduction for the nonbound marker is caused by a compound library, the respective pellet remaining after centrifugation is resuspended in buffer containing a large excess of competitor [(+)-methadone] to liberate the bound marker and bound library components. After centrifugation, the marker and the hits liberated from the target are analyzed in the resulting supernatant by LC-ESI-MS/MS (route 2).

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 mM (G)-methadone) to liberate the unknown bound ligand (as well as the bound marker). Then the supernatants obtained by cen-trifugation were analyzed by LC-ESI-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 m-opioid receptor as the marker [65].

7.3.1.2.3 Competition Assays Taking the Depletion of Marker and Ligand into Account

Unlike radioligand binding assays that only allow quantitation of the radioligand itself, binding assays based on MS detection offer the opportunity to quantify any additional ligand. This allows another interesting application: competitive MS binding assays observing both the concentration of the marker and the test compound at the same time (via their nonbound portion). In competition curves, the concentration of the bound (and accordingly in this case the nonbound) marker is plotted against the concentration of the test compound. Of course, in the strict sense of the law of mass action, the free and not the nominal concentration of the test compound is relevant for the competition curve. Competition curves generated by radioligand binding assays however, are usually based on the nominal concentration of the test compound which can deviate substantially from its free concentration (due to specific as well as nonspecific binding). Therefore, quanti-tation of the nonbound test compound would enhance the accuracy of the affinity determined for the test compound by eliminating this source of error.

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

log (c naloxonefree / M)

Fig. 7.13 Representative binding curve obtained by nonlinear regression from a competitive MS binding assay for m-opioid receptors, in which naloxone competes with morphine as marker. The points describe nonbound morphine quantified by LC-ESI-MS/MS at concentrations of nonbound naloxone determined simultaneously by LC-ESI-MS/MS.

to the binding curve shown in Fig. 7.13 in which the concentration of the non-bound marker is plotted against the concentration of free naloxone.

Taking depletion of the marker [according to Eq. (7), Section 7.3.1) and the ligand (by analyzing the binding curve based on the concentration of free naloxone) into account, a Ki-value of naloxone for m-receptors of 1.6 nM was calculated (using a Kd value of 2.0 nM for morphine, according to Raynor et al. [66]). Without correction for free naloxone, a Ki value of 7.9 nM was obtained. Interestingly, the first but not the second value is in surprisingly good accord with the result of Raynor et al., who in a conventional radioligand binding assay with [3H]DAMGO as marker determined a Ki value of 1.4 nM for naloxone at human m-receptors [66].

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