Oh Ho

morphine naloxone

amitryptiline

(+)-bicuculline

Fig. 7.11 Structures of compounds used in competitive MS binding assays for m-opioid receptors.

amitryptiline

(+)-bicuculline

Fig. 7.11 Structures of compounds used in competitive MS binding assays for m-opioid receptors.

Two artificial compound libraries were chosen as compound mixtures, of which library 1 was composed only of ''dummy'' ligands (acetanilide, amitryptiline, benzoic acid, (+)-bicuculline, 4-chloraniline, 2,3-dichloraniline, methylbenzoate, phenol, tramadol; see Fig. 7.11), whereas library 2 contained, in addition to these compounds, naloxone, a known m-opioid receptor ligand.

The assay was conducted at 10 nM morphine (Fig. 7.11) which had been chosen as native marker and a m-opioid receptor concentration of 5.5 nM (in 50 mM Tris-HCl, 5 mM MgCl2). After incubation and centrifugation to separate the non-bound marker, the resulting supernatants were directly analyzed by LC-ESI-MS/ MS. Control experiments without competitor (representing total binding) revealed 2.32 nM nonbound morphine whereas 7.73 nM nonbound morphine were found in the presence of 50 mM (G)-methadone (representing [Mtot] -nonspecific binding, Fig. 7.11, Table 7.3). Experiments employing the libraries at final concentrations of 1 mM and 10 nM per compound led in the case of library 1 to a concentration of nonbound morphine in the range of the control experiment without competitor. In the binding samples containing library 2, however, the concentration of nonbound morphine was significantly higher than in the control experiment without competitor (Table 7.3).

Accordingly, library 2 in contrast to library 1 must contain (at least) one ligand that, in concentrations of 1 mM and 10 nM, is capable of reducing the specific binding of morphine to the m-opioid receptor. Considering the composition of the two libraries, the conclusion that the component with an affinity for the m-opioid receptor must be naloxone is naturally trivial, but this issue could also be addressed by further examining the relevant binding samples (Fig. 7.12).

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

Conditions Morphine

Compound Concentration Nonbound

Control

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