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

TrkB:10 nM(Kd)

在雄性Sprague-Dawley大鼠中,ANA-12（3 μg/次）阻断内侧核孤束核（mNTS）BDNF对减少食物摄入的作用.在雄性C57BL/6小鼠中,ANA-12（0.5 mg/kg,腹膜内）显示对脂多糖诱导的抑郁样行为的抗抑郁作用.在雄性野生型小鼠中,ANA-12逆转乙醇摄入并诱导D3受体下调,但在D3R -/- 小鼠中无效.

在nnr5 PC12-TrkB细胞中,ANA-12在低至10 nM的浓度下阻止脑源性神经营养因子（BDNF）诱导的神经突生长。ANA-12可选择性直接与TrkB受体结合,抑制TrkB下游通路,而不改变TrkA和TrkC功能。ANA-12在DRG神经元中,消除了BDNF对增加内向电流的作用。

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)