TP003 is a non-selective benzodiazepine site agonist that induces anxiolysis via α2GABAA receptors

Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABA_A receptors (α1, α2, α3, and α5GABA_ARs), are highly effective against anxiety disorders, but also cause severe side effects greatly limiting their clinical application. Both, preclinical studies in genetically engineered mice, and preclinical and clinical trials with subtype-selective compounds indicate that undesired effects can in principle be avoided by targeting specific GABA_AR subtypes. While there is general consensus that activity at α1GABA_ARs should be avoided, controversy exists as to whether α2 or α3GABA_ARs need to be targeted for anxiolysis. While previous experiments in GABA_AR point-mutated mice demonstrated a critical role of α2GABA_ARs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABA_ARs. As most α1GABA_AR-sparing BDZ site agonists discriminate little between α2 and α3GABA_ARs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABA_AR modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABA_AR triple point-mutated mice, in which only either α1, α2, or α3GABA_ARs were left BDZ sensitive. These experiments revealed that TP003 behaves as a partial, rather non-selective BDZ site agonist in vitro that acts in vivo through α1, α2, and α3GABA_ARs (α5GABA_AR-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABA_ARs, but not of α3GABA_ARs. TP003 should therefore not be considered an α3GABA_AR selective agent. Previously published studies using TP003 should be interpreted with caution.