Nezavist (DCUK-OEt) and aminoquinoline analogues interact with the etomidate site and display a complex mode of modulatory action of the GABAA receptor.

The aminoquinoline compound Nezavist (DCUK-OEt; ethyl 5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylate) is under preclinical and early clinical investigation for chronic pain, alcohol use disorder (AUD), and relapse prevention in nicotine dependence. It and its metabolite DCUKA (5,7-dichloro-4-(3,3-diphenylureido)quinoline-2-carboxylic acid) have been shown previously to be allosteric positive modulators of GABAA receptors. Here, we investigated the two new derivatives BCUKA (5,7-dichloro-4-(3,3-dibutylureido)quinoline-2-carboxylic acid) and BCUK-OEt (ethyl 5,7-dichloro-4-(3,3-dibutylureido)quinoline-2-carboxylate) and demonstrated that all four compounds enhance GABA-induced currents more strongly in β3-than in β1-containing receptors. Moreover, the presence of γ2 impacts on modulation with compound-specific differences. Previously observed α-isoform specificity does not extend to α6, which responds as α1. Mutational analysis using β3N265S showed that the etomidate binding site contributes to the positive allosteric effects, particularly for DCUK-OEt. The mutation had less impact when γ2 was present, suggesting involvement of additional binding sites. The presence of multiple allosteric sites is supported by radioligand binding studies where DCUKA and BCUKA displaced [3H]flunitrazepam (benzodiazepine site), whereas DCUK-OEt and BCUK-OEt enhanced its binding allosterically. Docking confirmed strong interactions at the etomidate site (transmembrane β+/α- interface), with the best ranked poses of DCUK-OEt forming stabilizing hydrogen bonds and salt bridges. DCUKA and BCUKA also fit well into the high-affinity benzodiazepine site, though likely act there as silent binders. This comprehensive study demonstrates that Nezavist (DCUK-OEt) and its derivatives are multi-site GABAA receptor modulators. Their actions depend on subunit composition and involve both known (extracellular and trans-membrane) and proposed novel allosteric sites. These findings support ongoing drug development for AUD and related conditions.