[3H]L-655,708, a Novel Ligand Selective for the Benzodiazepine Site of GABAA Receptors which Contain the α5 Subunit
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Materials
[N-methyl-3H]Ro 15-1788 (75.3 Ci/mmol) and [7,9-3H]Ro 15-4513 (27.4 Ci/mmol) were from DuPont/New England Nuclear. Flunitrazepam was from Sigma; Ro 15-4513, Ro 15-1788 and Zolpidem were from Research Biologicals, Inc.
Synthesis of L-655,708 and [3H]L-655,708
L-655,708 (Table 1) was prepared from 5-methoxyanthranilic acid by following the previously described procedure of Watjen et al. (1990). For the preparation of [3H]L-655,708, starting from 5-benzyloxyanthranilic acid, the same procedure was used to prepare the benzyl derivative (
Selectivity of L-655,708
Competition of L-655,708 for benzodiazepine sites labelled by [3H]Ro 15-1788 or [3H]Ro 15-4513 in stably transfected cells revealed it to be selective for cell lines containing the α5 subunit, as shown in Table 2. L-655,708 was found to be 107-fold, 61-fold, 54-fold and 184-fold selective for the α5-containing receptors over receptors containing α1, α2, α3 and α6 subunits, respectively. Consequently, the compound was tritiated to investigate whether it could be used as a radioligand.
Binding of [3H]L-655,708 to rat hippocampal membranes
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DISCUSSION
This study demonstrates that L-655,708 binds to α5-containing receptors with more than 50-fold higher affinity than to any other GABAA-receptors. This is more selective than analogues of Ro 15-4513, which previously have been shown to have some selectivity (Hadingham et al., 1993; Gillard et al., 1994) and, as such, is the most α5-selective compound reported to date. [3H]L-655,708 is shown to bind rapidly and selectively to α5-containing receptors with high affinity, and so is the first
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