Library Screening and Competition Assays for -Opioid Receptors

2 Library Screening and Competition Assays for / -Opioid Receptors 

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. 

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 //-opioid receptor ligand. 

Accordingly, library 2 in contrast to library 1 must contain (at least) one ligand that, in concentrations of 1 pM and 10 nM, is capable of reducing the specific binding of morphine to the //-opioid receptor. Considering the composition of the two libraries, the conclusion that the component with an affinity for the //-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). 

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]. 

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