ANA-12 is a potent and selective TrkB antagonist that exhibits anxiolytic and antidepressant activity in mice.

TrkB:10 nM(Kd)

In male Sprague-Dawley rats, ANA-12 (3 μg/dose) inhibits the medial nucleus of the solitary tract (mNTS) BDNF's effect on reducing food intake. In male C57BL/6 mice, ANA-12 (0.5 mg/kg, intraperitoneally) demonstrates antidepressant effects against lipopolysaccharide-induced depressive-like behavior. In wild-type male mice, ANA-12 reverses ethanol consumption and induces D3 receptor downregulation; however, it is ineffective in D3R -/- mice.

In nnr5 PC12-TrkB cells, ANA-12 inhibits neurite outgrowth induced by brain-derived neurotrophic factor (BDNF) at concentrations as low as 10 nM. It selectively binds directly to the TrkB receptor, suppressing the downstream TrkB pathway without altering the functions of TrkA and TrkC. Furthermore, ANA-12 nullifies the effect of BDNF on increasing the inward current in DRG neurons.

ANA-12 Binding assay: Maxisorp ELISA 96-well plates are coated with various concentrations of Trk BECD -Fc, 20 mg/ml BSA, or 1 mg/mL IgG-Fc (polyclonal anti-TrkB) in a carbonate buffer (pH 9.6) overnight at 4°C. Plates are saturated with 0.5% BSA in PBS for 2 hours at room temperature and extensively washed in PBS-Tween 0.05%. Bodipy–ANA-12 is then incubated in 0.5% PBS-BSA for 1 hour at room temperature before the addition of BDNF in 0.5% PBS-BSA for another hour. After extensive washes in PBS-Tween 0.05%, the amount of bodipy–ANA-12 bound is quantified by fluorescence at 520 ± 10 nm. Detectability range for extrapolation analysis is assessed by coating ELISA plates with bodipy–ANA-12 and reading fluorescence at 520 ± 10 nm.

Modulation of neurite outgrowth by molecules is assessed in nnr5 PC12–TrkB, –TrkA, and –TrkC cells after addition of BDNF (1 nM), NGF (2 nM), and NT-3 (10 nM), respectively. The number of cells bearing neurites longer than 2 cells in diameter in each counting field is microscopically determined (2 fields per well, 3 wells per condition). Counting is performed blind each 24 hours for 3 days. (Only for Reference)