Opioid binding assays

Not many nonpeptide agonists are yet available, but such compounds have been described — for example, in the angiotensin, CCK, and opioid receptor systems. In fact, for a few receptors, such as the somatostatin, ghrelin, and complement C5A receptors, basically all compounds found by screening using binding assays are agonists. In contrast, for the majority of receptors for which... 

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

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

Fig. 7.12 Schematic flowchart of the competitive MS binding assay for //-opioid receptors inciuding iiberation of bound marker and test compounds, respectiveiy. After incubation of the target (/t-opioid receptor) in the presence of the marker (morphine) and a compound iibrary, the binding sampies are centrifuged to separate bound from nonbound marker. Subsequentiy, the nonbound marker in the resuiting supernatant is quantified by LC-ESI-MS/MS without further sampie preparation (route 1).

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... 

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

TABLE 1 Activity of Delta-Selective Ligands in Opioid Receptor Binding Assays and Delta GTP-y[35S] Assays... 

Several nonpeptidic 6-opioid agonists have been synthesized, although none are currently available clinically. These include BW373U86 which is —10 and 20 times more selective for 6-opioid receptor over the p and kappa opioid receptor, respectively, in receptor-binding assays [18]. BW 373U86 also demonstrated high potency (ED50 of 0.2 0.06 nM) in the MVD assay, and its... 

Although the analytical control of each pool was impossible due to the large number of individuals, using a tea bag introduced especially for this purpose, a purity check for the reaction was performed on each pool after each step, and this was assumed as a quality control for the whole library. The library was tested in p- and k-opioid receptor binding assays and in a cr-receptor binding assay. Some preliminary activities for one of the pools in... 

Rodgers, G. et al. 2003. Development of displacement binding and GTPy scintillation proximity assays for the identification of antagonists of the micro-opioid receptor. Assay Drug Dev. Technol. 1, 627-636. 

A series of cyclic conformationally constrained peptides related to somatostatin were designed, synthesized and tested for opioid receptor interaction by Hruby and his collaborators. Compounds (17)-(22) were found to be pure opioid antagonists (GPI) with high affinity (IC50 = 1.2 to 4.3 nM) and exceptional selectivity for p over S opioid receptors (Table 3.1) and with minimal or no somatostatin-like activity (ligand binding assays)[63-65]. 

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