GABA_A receptor subtype selectivity and structure-activity relationships of flavonoids

A flavonoid, 6-hydroxyflavone, was previously reported to bind to the benzodiazepine (BZ) site on GABA_A receptors with moderate binding affinity. In the present study, we showed that 6-hydroxyflavone partially potentiated GABA-induced currents in native GABA_A receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6-hydroxyflavone displayed significant preference for α2- and α3-containing subtypes, which were thought to mediate anxiolytic effect, compared to α1- and α5-containing subtypes expressed in HEK 293T cells. In mice, 6-hydroxyflavone exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6-hydroxyflavone as a promising drug candidate for the treatment of anxiety-like disorders. In addition, GABA_A receptor structure-efficacy relationships for flavones were also studied. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogues, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2’-hydroxyflavone analyzed in the present study, as well as 6,2’-dihydroxyflavone reported earlier. Whole-cell patch clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABA_A receptors acting through benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. Moreover, 2’-hydroxyflavone was shown to be a neutralizing modulator, different in efficacy from its structural analogue, 6,2’-dihydroxyflavone, a negative modulator of GABA_A receptors. The fact that flavone analogues differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2’-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure-efficacy relationships for flavones acting at GABA_A receptors, and hence facilitation of flavone-based drug development