ANA-12

TrkB (Kd = 10 nM)

体外活性：ANA-12 直接且选择性地与 TrkB 结合，并抑制 TrkB 下游过程，而不改变 TrkA 和 TrkC 功能。在 nnr5 PC12-TrkB 细胞中，ANA-12 在浓度低至 10 nM 时即可阻止脑源性神经营养因子 (BDNF) 诱导的神经突生长。在 DRG 神经元中，ANA-12 消除了 BDNF 对增加内向电流的影响。激酶测定：Maxisorp ELISA 96 孔板用碳酸盐缓冲液 (pH 9.6) 中的不同浓度的 Trk BECD -Fc、20 mg/ml BSA 或 1 mg/mL IgG-Fc（多克隆抗 TrkB）包被过夜。 4°C。将板用 0.5% BSA 的 PBS 溶液在室温下饱和 2 小时，并在 PBS-Tween 0.05% 中彻底清洗。然后将 Bodipy–ANA-12 在 0.5% PBS-BSA 中室温孵育 1 小时，然后添加 0.5% PBS-BSA 中的 BDNF 再孵育 1 小时。在 PBS-Tween 0.05% 中进行大量洗涤后，通过 520 ± 10 nm 处的荧光对结合的 bodipy-ANA-12 量进行定量。外推分析的可检测范围通过用 bodipy-ANA-12 包被 ELISA 板并在 520 ± 10 nm 处读取荧光来评估。细胞测定：分别添加 BDNF (1 nM)、NGF (2 nM) 和 NT-3 (10 nM) 后，在 nnr5 PC12–TrkB、–TrkA 和 –TrkC 细胞中评估分子对神经突生长的调节。通过显微镜确定每个计数视野中具有直径超过 2 个细胞的神经突的细胞数量（每孔 2 个视野，每个条件 3 个孔）。连续 3 天，每 24 小时进行一次盲计数。

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N-T19类似物的筛选揭示了一种有效的TrkB拮抗剂。[1]
KIRA-ELISA和神经突生长评估显示，只有N-T19能够维持其在神经元和神经元样系统中的作用。然而，尽管N-T19在抑制TrkB活性方面表现出很高的功效，但其相对较低的效价促使我们寻找能保持N-T19原有的高效但效价更高的类似物。为此，使用Bioinfo-DB数据库进行第二轮计算机筛选，以鉴定共享相同分子支架的N-T19类似物（图4A）。14个新分子被鉴定为接近类似物，并在第一组使用KIRA-ELISA检测的功能筛选中进行了测试。四种化合物的活性最高，但只有1种（ANA-12）在重组细胞中表现出亚微摩尔效力。ANA-12的深入药理表征证实了该分子的高效（完全抑制）和效力（亚微摩尔）（图4B）。与母体化合物NT-19一样，ANA-12在神经元中显示出2位点的作用模式，但令人惊讶的是，它也在重组细胞中发挥作用。两种细胞体系（分别为50 μM和50 nM）在低亲和力位点和高亲和力位点上的电位具有可比性。 值得注意的是，在测试的14个分子中，ANA-12的结构与母体先导化合物的结构最接近，两个分子的区别只是在ANA-12中多了一个苯片段（图4A）。

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ANA-12直接选择性结合TrkB。[1]
然后，我们通过将荧光标记的化合物（见方法）与嵌合的TrkBECD-Fc或BSA或IgG-Fc孵育作为非特异性结合的阴性对照，确定ANA-12是否直接与TrkB结合。如图5A所示，ANA-12以剂量依赖的方式特异性结合到TrkB的细胞外结构域，而不与BSA或IgG-Fc结合。与TrkBECD-Fc的饱和结合研究表明，ANA-12与TrkBECD-Fc结合的Kd值为12 μM（图5B），对应于之前在KIRA-ELISA中观察到的功能性低亲和力位点。检测到的高亲和位点位于荧光检测的非线性范围内，因此表现为小的压痕。线性化和外推分析表明，ANA-12也与高亲和力位点结合，Kd约为10 nM（数据未显示）。ANA-12与TrkB结合的2位点拟合模型显示，高亲和力位点占总结合位点的20%（数据未显示），这一数值与KIRA-ELISA检测中观察到的30%相似（图4B）。
为了进一步研究ANA-12的结合特性，我们将BDNF加入到ANA-12/TrkB复合物中（图5B）。这导致与TrkBECD-Fc结合的ANA-12的最大数量减少了60%，而曲线没有向右移动（Kd, 16 μM），表明这是一种非竞争机制。综上所述，这些数据表明高亲和力和低亲和力的结合位点共存于TrkB的细胞外结构域，BDNF和ANA-12并不竞争TrkB上的相同位点。
新化合物与TrkB-d5的计算对接表明，推测的结合模式与N-T19相似（图5C）： ANA-12的内酰胺部分与TrkB的His299和His300主链原子相互作用，而化合物的无环酰胺部分与TrkB特异性Gln347和Asp298侧链之间形成氢键。该配体的形状适合TrkB-d5 ADEB β-片，特别是通过7元环与可接近的二硫桥Cys302-Cys347之间的疏水接触，以及通过与一束组氨酸残基（His299, His300, His335）的芳香相互作用。

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ANA-12影响与TrkB相关的细胞功能，但不影响TrkA和TrkC。[1]
神经突生长被用来验证ANA-12对细胞过程的影响（图5、D和E）。我们观察到，在表达trkb的细胞中，浓度低至10 nM的ANA-12可以阻止bdnf诱导的神经突生长，证实了KIRA-ELISA检测中观察到的高效效（图5D）。在浓度高达10-100 μM时，ANA-12完全消除了BDNF的作用，因为即使在3天后也没有观察到单个神经突起或分支。为了评估该化合物对TrkB的选择性，我们使用了另外两种表达TrkA或TrkC的nnr5-PC12细胞系，它们的神经突生长分别依赖于NGF和NT-3（图5E）。在这些细胞系中，ANA-12对神经突的生长没有影响。浓度高达100 μM的ANA-12孵育3天后，对NGF-和nt -3依赖性的神经突长度和分支没有影响，证实了其对trkb相关信号的特异性。

在成年 C57BL6/129SveV F1s 小鼠中，ANA-12（0.5 mg/kg，腹腔注射）可降低大脑中的 TrKB 活性，减少焦虑和抑郁相关行为，而不影响神经元存活。在雄性 C57BL/6 小鼠中，ANA-12（0.5 mg/kg，腹腔注射）对脂多糖诱导的抑郁样行为显示出抗抑郁样作用。在雄性 Sprague-Dawley 大鼠中，ANA-12（3 μg/剂）可阻断内侧孤束核 (mNTS) BDNF 减少食物摄入的作用。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。在雄性 CocSired 大鼠中，ANA-12（0.5 mg/kg，腹腔注射）可逆转可卡因自我给药的减少。

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初级传入末端的NMDA受体可以通过增加神经递质释放来促进痛觉过敏。在大鼠和小鼠中，我们发现鞘内NMDA诱导神经激肽1受体（NK1R）内化（测量P物质释放）的能力需要事先注射BDNF。在原发性传入神经中选择性敲除NMDA受体可减少NMDA诱导的NK1R内化，从而证实了这些受体在突触前的位置。BDNF的作用是由原肌球蛋白相关激酶B （trkB）受体介导，而不是由p75神经营养因子受体（p75(NTR)）介导，因为它不是由proBDNF产生的，并且被trkB拮抗剂ANA-12抑制，而不被p75（NTR）抑制剂t1 - pep5抑制。这些作用可能是通过trkB受体的截断形式介导的，因为在背根神经节（DRG）神经元中几乎没有全长trkB的表达。Src家族激酶抑制剂阻断BDNF的作用，表明trkB受体通过Src家族激酶磷酸化促进这些NMDA受体的激活。培养DRG神经元的Western blot结果显示，BDNF增加了NMDA受体NR2B亚基的Tyr（1472）磷酸化，已知具有增强作用。膜片钳记录显示，BDNF而非proBDNF增加了培养的DRG神经元的NMDA受体电流。在神经性疼痛模型中，nmda诱导的NK1R内化也可以通过脂多糖激活背角小胶质细胞来实现。这些作用被BDNF清道夫、trkB受体拮抗剂和Src家族激酶抑制剂所减弱，这表明小胶质细胞释放的BDNF在神经性疼痛的初级传入事件中增强了NMDA受体。[1]

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LPS导致海马CA3和齿状回（DG）和前额叶皮质（PFC）的BDNF减少，而LPS增加伏隔核（NAc）的BDNF。地塞米松抑制实验显示lps处理小鼠下丘脑-垂体-肾上腺轴活动过度。腹腔注射7,8- dhf对lps诱导的抑郁样行为有抗抑郁作用，腹腔注射ANA-12可阻断其抗抑郁作用。令人惊讶的是，单独的ANA-12对lps诱导的抑郁样行为表现出抗抑郁样作用。此外，双侧向NAc输注ANA-12具有抗抑郁作用。此外，LPS导致CA3、DG和PFC的脊柱密度降低，而LPS增加了NAc的脊柱密度。有趣的是，7,8- dhf显著减弱了lps诱导的CA3、DG和PFC中p-TrkB和脊柱密度的降低，而ANA-12显著减弱了lps诱导的NAc中p-TrkB和脊柱密度的增加。 结论：lps诱导的炎症可能通过改变CA3、DG、PFC和NAc的BDNF和脊柱密度导致抑郁样行为，这可能参与了7,8- dhf和ANA-12的抗抑郁作用。[2]

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为了定位介导后脑室传递BDNF能量平衡效应的神经元，将脑室阈下剂量直接递送至内侧孤束核（mNTS）。mNTS BDNF可显著减少食物摄入，而这一作用被预先给予高选择性TrkB受体拮抗剂{[n2 -2-2-氧氮平-3-基氨基]羰基苯基苯并(b)噻吩-2-carboxamide (ANA-12)}阻断，表明TrkB受体激活介导了后脑BDNF对食物摄入的影响。由于BDNF和瘦素都与黑素皮质素信号相互作用以减少食物摄入，我们还研究了后脑瘦素的摄入抑制作用是否涉及后脑特异性BDNF/TrkB激活。后脑瘦素递送可显著增加后脑背迷走神经复合体内BDNF蛋白含量。为了评估BDNF/TrkB受体信号是否在瘦素信号的下游作用以控制能量平衡，瘦素和ANA-12被共同给予mNTS。TrkB受体拮抗剂可减弱瘦素的摄入抑制作用，提示mNTS TrkB受体激活有助于调解后脑瘦素的厌食作用。总的来说，这些结果表明，trkb介导的mNTS信号负调控食物摄入，部分地，瘦素给药到NTS的摄入抑制作用。[3]

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事实上，TrkB选择性拮抗剂ANA-12阻断BDNF通路逆转了慢性稳定的乙醇摄入，并强烈降低了D3R的纹状体表达。最后，我们对丁螺环酮进行了评估，丁螺环酮是一种被批准用于治疗焦虑症的药物，具有D3R拮抗剂活性（经分子模型分析证实），可有效抑制乙醇摄入。因此，通过D3R的DA信号对于乙醇相关的奖励和消耗是必不可少的，并且可能代表了断奶的新治疗靶点。[4]

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给药BDNF受体拮抗剂（TrkB受体拮抗剂ANA-12）逆转了雄性可卡因遗传大鼠的可卡因自我给药减少。此外，在自我服用可卡因的男性精子中，乙酰化组蛋白H3与Bdnf启动子的关联增加。综上所述，这些发现表明，父亲自愿摄入可卡因会导致种系的表观遗传重编程，对雄性后代的mPFC基因表达和对可卡因强化的抵抗力产生深远影响。[5]

将不同浓度的 Trk ^BECD -Fc、20 mg/ml BSA 或 1 mg/mL IgG-Fc（多克隆抗 TrkB）涂在 Maxisorp ELISA 96 孔板上，并在 4° 下放置过夜C，pH 为 9.6 的碳酸盐缓冲液。室温下在 0.5% BSA 的 PBS 溶液中溶解两小时后，将板在 0.05% PBS-Tween 中彻底清洗。在 0.5% PBS-BSA 中室温孵育一小时后，将 BDNF 添加到溶液中并再孵育一小时。使用 Bodipy-ANA-12 重复此过程。在 PBS-Tween 0.05% 中彻底洗涤后，通过 520 ± 10 nm 处的荧光测量结合的 bodipy-ANA-12 的量。为了确定外推分析的检测范围，将 ELISA 板涂有 bodipy-ANA-12，并测量 520 ± 10 nm 处的荧光。

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ANA-12 binding assay [1]
在4°C的碳酸盐缓冲液（pH 9.6）中，用不同浓度的TrkBECD-Fc（如图所示）、20 mg/ml BSA或1 mg/ml IgG-Fc（多克隆抗trkb）包被Maxisorp ELISA 96孔板，过夜。在室温下，用0.5%的BSA在PBS中饱和2小时，并用0.05%的PBS- tween广泛洗涤。将Bodipy-ANA-12在0.5% PBS-BSA中室温孵育1小时，再加入BDNF在0.5% PBS-BSA中孵育1小时，如图所示。在0.05% PBS-Tween中广泛洗涤后，在520±10 nm处荧光定量体脂- ana -12结合量。外推分析的检测范围通过在ELISA板上涂覆bodipy-ANA-12并在520±10 nm处读取荧光来评估。

在 nnr5 PC12-TrkB、-TrkA 和 -TrkC 细胞中，分别添加 BDNF (1 nM)、NGF (2 nM) 和 NT-3 (10 nM) 后评估分子对神经突生长的影响。显微镜对每个计数区域（每个条件三个孔，每个孔两个区域）中的细胞数量进行计数，其中神经突的直径超过两个细胞。三天内，每 24 小时在黑暗中进行一次计数。

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KIRA-ELISA [1]
如前所述，使用改良版KIRA-ELISA定量TrkB受体自磷酸化。将TetOn-rhTrkB细胞接种于96孔平板（每孔4 × 104个细胞），用1000 ng/ml强力霉素孵育过夜，诱导TrkB受体的表达。将皮质神经元接种于多聚氮薄包被的96孔平底培养板（每孔12 × 104个细胞），在37℃、5% CO2中培养7 - 8天。每次检测前验证TetOn-rhTrkB细胞的荧光水平。细胞用DMEM仔细清洗4次，然后用化合物处理20分钟，用BDNF刺激20分钟(重组细胞，4 nM；神经元，0.4 nM)，在含有0.5% BSA和25 mM HEPES（对照培养基）的DMEM中，在37°C， 5% CO2中。在冰上去除培养基停止实验，在室温下，通过添加增溶缓冲液（150 mM NaCl, 50 mM Hepes, 0.5% Triton X-100, 0.01%硫柳汞，2mm原钒酸钠，添加蛋白酶抑制剂混合物）使膜溶解1小时。将裂解液转移到预先包被抗gfp（1:5000）用于rhTrkB或抗TrkB （1 μg/ml）用于神经元TrkB的ELISA微滴板上。生物素化抗磷酸酪氨酸（0.5 μg/ml）和酶标链霉亲和素（1:4000）孵育后发现磷酸化。加入TMB后，用1 N盐酸酸化，在450 nm处读取吸光度。总TrkB也使用KIRA-ELISA定量，使用rhTrkB的单克隆抗gfp（1:3000）或神经元TrkB的单克隆抗TrkB（1:1000）沉淀受体，并使用rhTrkB的多克隆抗gfp（1:5000）或神经元TrkB的多克隆抗TrkB （1 μg/ml）检测受体。在细胞培养中，总TrkB信号在所有处理条件下都没有变化。在脑组织中，总TrkB的数量是可变的，用于对磷酸化TrkB获得的信号进行归一化。

In a vehicle of 17% dimethyl sulfoxide (DMSO) in phosphate-buffered saline, ketamine (ketamine hydrochloride, 10 mg/kg), 7,8-dihydroxyflavone (7,8-DHF; 10 mg/kg), and ANA-12, N2-(2-{[(2-oxoazepan-3-yl) amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide (0.5 mg/kg) are prepared on the day of injection. Ketamine (10 mg/kg), 7,8-DHF (10 mg/kg), and ANA-12 (0.5 mg/kg) are the doses that have been chosen. Mice receive intraperitoneal (i.p.) administration of all compounds.

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Administration of ANA-12 and in vivo KIRA-ELISA analysis [1]
F1 hybrids obtained by crossing C57BL/6 and 129SveV mice were used following the recommendation of the Banbury Conference on genetic background for studying mouse behavior. Adult C57BL6/129SveV F1s (3 months old) were randomly distributed into saline (1% DMSO dissolved in 0.9% NaCl solution) and ANA-12 (dissolved in saline solution) groups. A volume of 10 μl/g body weight was injected i.p. for saline and ANA-12 (0.5 mg/kg body weight) solutions. After 2 or 4 hours, mice were decapitated and brains were rapidly removed on ice. Striatum, cortex, and hippocampus were subsequently dissected when needed. Tissues were rapidly washed in ice-cold PBS, transferred into ice-cold KIRA-ELISA solubilization buffer, and left overnight at 4°C. Protein concentrations were determined, equal amounts of proteins were loaded, and KIRA-ELISA assays were performed as described above.

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Analysis of ANA-12 stability and bioavailability in mouse brains [1]
Analysis of stability in mouse serum and quantification in mouse brain were performed by TechMedILL facilities (École Supérieure de Biotechnologie de Strasbourg, Illkirch, France). Stability was assessed by incubating ANA-12 in mouse serum for 15, 30, 45, and 60 minutes at 37°C. Mixtures were homogenized and proteins precipitated with acetonitrile before being analyzed by liquid chromatography–mass spectrometry (Agilent LC-MS ESI qTOF connected to a C18-1 × 10 × 1.9 column). Bioavailability in mouse brain was assessed by injecting mice i.p. with ANA-12 (0.5 mg/kg) as described above. After 30 minutes, 1, 2, 4, and 6 hours (3 mice/time point), brains were removed and mashed in NaCl 0.9% before treatment with acetonitrile. The mixture was cleared by ultracentrifugation, and the compound was extracted using a series of dehydrations and resuspensions in acetonitrile/water (1/1 v/v) before being subjected to LC-MS for detection and analysis. Reference samples were prepared by adding known amounts of compound to blank mixtures (brains from saline-treated mice). Concentrations of ANA-12 (ng/g brain) were derived from the reference samples by calculating the area under the detection peak.

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In vivo cell death analysis [1]
The effects of subchronic treatments of ANA-12 on cell death in mouse brains were assessed using the TUNEL assay. Mice were randomly distributed into 4 groups and were i.p. injected with saline (containing 1% DMSO) or 0.5, 1.0, or 2.0 mg/kg of ANA-12 once a day for 1 week. Mice were sacrificed 24 hours after the last injection and perfused transcardially with PBS followed by 4% paraformaldehyde. Brains were postfixed overnight in 4% paraformaldehyde, and 50 μm–thick coronal sections were obtained using a vibratome. Free-floating sections were extensively washed in PBS and permeabilized in PBS containing 0.5% Triton X-100 for 90 minutes at room temperature. Sections were then rinsed and mounted on slides. Labeling of 3′ OH-DNA strand breaks was performed using the DeadEnd Fluorometric TUNEL system according to the manufacturer’s instruction. The reaction was terminated by rinsing the slides in SSC 2× and washing extensively with PBS. Sections were mounted with VECTASHIELD plus DAPI, and fluorescein was visualized at 520 nm using fluorescence microscopy. Positive controls were obtained by pretreating sections from saline-treated animals with DNAse (10 U/ml) for 90 minutes at 37°C in PBS containing 0.5% Triton X-100. Negative controls (absence of fluorescent spots) were obtained by processing the sections as described above without the terminal transferase.

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Behavioral testing of ANA-12 [1]
All behavioral testing was started at the same time of the day (1:00 pm) in quiet and separated rooms under bright ambient light conditions (800–900 lux, except for the open field, 300–400 lux). Behavioral tasks were performed 4 hours after injection of 0.5 mg/kg of ANA-12 using the same procedure as described above. To eliminate odor cues, all testing apparatus was thoroughly cleaned after each animal using the disinfectant Roccal.

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Effects of ANA-12 on anxiety-related behaviors [1]
Anxiety-related behaviors were tested using the open field test, elevated plus maze, and novelty-suppressed feeding paradigm.

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Drug Administration [2]
ANA-12, N2-(2-{[(2-oxoazepan-3-yl) amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide (0.5mg/kg, i.p.), was dissolved in 1% dimethylsulfoxide in physiological saline. The doses of 7,8-DHF and ANA-12 were also selected as previously reported (Ren et al., 2013 2014; Cazorla et al., 2011).

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Surgery and Bilateral Injection of ANA-12 into NAc [2]
Mice were anesthetized with pentobarbital (5mg/kg), and placed in a stereotaxic frame. Microinjection needles were placed bilaterally into the NAc shell (+1.7 AP, ±0.75 ML, -3.6 DV) (Paxinos and Watson, 1998). Twenty-four hours after surgery, LPS (0.5mg/kg) or saline (10ml/kg) was injected i.p. Twenty-three hours after injection of LPS (or saline), ANA-12 (0.1 nmol/L, 0.1 μL/min for 5min) or vehicle was injected bilaterally. Behavioral evaluation was performed 4 and 6 hours after the final infusion (Figure 3B).

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Experiment 4: BDNF/TrkB receptor signaling within the mNTS. [3]
All rats (n = 9) received two unilateral NTS intraparenchyma injections that were ∼20 min apart, and food intake was determined at 1, 3, 6, and 24 h after the second injection. Conditions were counterbalanced and were as follows: control condition (100 nl of DMSO followed by 100 nl of aCSF), ANA-12 condition (1 or 3 μg of ANA-12 followed by aCSF), BDNF condition (DMSO followed by 0.2 μg of BDNF), and the combination condition (either 1 or 3 μg of ANA-12 followed by 0.2 μg of BDNF). Body weight measurements were made immediately before and 24 h after the injection of the drugs.

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Experiment 6: hindbrain TrkB receptor signaling and leptin. [3]
All rats (n = 9) received two unilateral NTS parenchyma injections that were ∼20 min apart, and food intake was measured at 1, 3, 6, and 24 h after the second injection. The four counterbalanced conditions were as follows: control condition (100 nl of DMSO followed by 100 nl of sodium bicarbonate), ANA-12 condition (3 μg of ANA-12 followed by sodium bicarbonate), leptin condition (DMSO followed by 0.2 μg of leptin), and the combination condition (3 μg of ANA-12 followed by 0.2 μg of leptin). Body weight measurements were made immediately before and 24 h after the injection of drugs.

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Drugs and Treatments [4]
Ethanol, U99194A maleate, SB277011A hydrochloride, buspirone hydrochloride, 8-OH-DPAT and ANA-12 were dissolved in saline and intraperitoneally (i.p.) injected (in a volume of 10 ml/kg), except ANA-12 that was dissolved in 10% dimethyl sulfoxide. U99194A was used at 10 mg/kg (Harrison and Nobrega, 2009), SB277011A was used at 10 mg/kg (Song et al, 2012), buspirone was used in the range 0.1–10 mg/kg (Martin et al, 1992), 8-OH-DPAT was used at 1 mg/kg (Martin et al, 1992), and ANA-12 was used at 0.5 mg/kg (Cazorla et al, 2011).
In the two-bottle choice paradigm, after 30 days of voluntary alcohol-drinking procedure, D3R−/− and WT were randomly allocated to the eight experimental groups (n=6/10 per group): WT/vehicle, WT/U99194A, WT/SB277011A, WT/buspirone, D3R−/−/vehicle, D3R−/−/U99194A, D3R−/−/SB277011A, and D3R−/−/buspirone. Animals were i.p. injected once a day, for 14 consecutive days. On day 14, animals were sacrificed 1 h after the last administration and brain tissues were taken. In another set of experiments, after 30 days of voluntary alcohol-drinking procedure, mice were randomly allocated to five experimental groups (n=5/7 per group): WT naïve, WT/vehicle, WT/ANA-12, D3R−/−/vehicle, and D3R−/−/ANA-12. Animals were i.p. injected once a day, for 4 consecutive days with the selective Trkb antagonist ANA-12 at 0.5 mg/kg (Cazorla et al, 2011; Vassoler et al, 2013). On day 4, animals were sacrificed 1 h after the last administration and brain tissues were taken.

Systemic administration of ANA-12 inhibits TrkB in the brain.[1]
The purpose of this study was to develop a small molecule that can inhibit TrkB in the adult mammalian brain after systemic administration. We first tested whether ANA-12 was stable and not degraded into breakdown products in mouse serum before reaching the brain. To this end, ANA-12 was incubated in mouse serum for 15, 30, 45, and 60 minutes at 37°C. Liquid chromatography–mass spectrometry (LC-MS) analysis of the mixture did not reveal the presence of breakdown products and no degradation of ANA-12 over time was observed (Figure 6A).

We then determined whether ANA-12 crosses the blood-brain barrier and reaches the brain after systemic administration. For this purpose, ANA-12 (0.5 mg/kg) was injected i.p. into adult mice. Animals were sacrificed 0.5, 1, 2, 4, or 6 hours later and brains were processed for quantification of ANA-12 by LC-MS. Figure 6B shows that active concentrations of ANA-12 could be detected in the brain as early as 30 minutes (~400 nM) and up to 6 hours after the i.p. injection (~10 nM).

We then determined whether ANA-12 inhibits TrkB in the adult brain and determined the magnitude of TrkB inhibition in the brain 2 and 4 hours after the injection of 0.5 mg/kg of ANA-12. This time course was chosen based on what we previously observed with cyclotraxin-B, for which a minimum of 3 hours was required for TrkB deactivation in the brain. KIRA-ELISA quantification of phospho-TrkB revealed that 0.5 mg/kg of ANA-12 partially inhibited the total endogenous TrkB activity in the whole brain (8% at 2 hours, 25% at 4 hours; Figure 6C).

Although TrkB is widely distributed in the brain, it is possible that ANA-12 does not inhibit the receptor uniformly in different brain areas. This could lead to an apparent partial inhibition of TrkB in the whole brain with some structures being fully inhibited while others are not or are very little affected. We therefore determined the amplitude of inhibition between different brain areas. Again, 0.5 mg/kg of ANA-12 was injected into adult mice, and different brain structures (striatum, cortex, and hippocampus) were collected after 2 or 4 hours (Figure 6D). KIRA-ELISA analysis showed that TrkB inhibition was more effective in the striatum than in hippocampus and cortex 2 hours after injection. At 4 hours, inhibition was comparable among all the structures that were analyzed (25%–30%), yet TrkB in the striatum appeared to be slightly more inhibited than in the hippocampus and cortex. Together, these observations suggest that very low doses of ANA-12 are sufficient to partially inhibit TrkB activity homogenously throughout the brain after 4 hours.

ANA-12 does not affect neuron survival. [1]
Since inhibition of the BDNF/TrkB signaling can induce neuronal death in the central nervous system, we verified whether ANA-12 can be chronically administrated without toxic effects for the brain. For that purpose, adult mice received a daily injection of different doses of ANA-12 (0.5, 1.0, and 2.0 mg/kg) or saline solution for a week. After the last injection, mice were sacrificed and brains were processed for apoptosis detection using fluorescent TUNEL staining (Figure 9). Little or no TUNEL-positive cells could be detected in the brains of mice injected with saline or 0.5 or 1.0 mg/kg of ANA-12. It is noteworthy that examination of the whole brain revealed that the apoptotic cells were present only in the dentate gyrus of the hippocampus. However, a small increase in the number of TUNEL-positive cells was observed in the dentate gyrus of mice that received 2.0 mg/kg of ANA-12. No staining was detected in any other areas of investigation (cortex, striatum, CA1-3 areas of the hippocampus, hypothalamus, thalamus, substantia nigra, ventral tegmental area, pallidum, and raphe nucleus).

[1]. J Clin Invest. 2011 May 2; 121(5): 1846–1857.

[2]. Eur J Neurosci . 2014 May;39(9):1439-54.

[3]. Int J Neuropsychopharmacol . 2014 Oct 31;18(4):pyu077.

[4]. Am J Physiol Endocrinol Metab . 2012 May 1;302(10):E1252-60.

[5]. Neuropsychopharmacology . 2014 Jul;39(8):2017-28.

[6]. Nat Neurosci . 2013 Jan;16(1):42-7.

ANA-12 is a secondary carboxamide that is anthranilic acid in which the carboxy group has undergone condensation with the primary amino group of alpha-amino-epsilon-caprolactam, while the aryl-amino group has undergone condensation with the carboxy group of 1-benzothiophene-2-carboxylic acid. It is a selective, non-competitive antagonist of tropomyosin receptor kinase B (TrkB, also known as tyrosine receptor kinase B). It has a role as a tropomyosin-related kinase B receptor antagonist, an antidepressant and an anxiolytic drug. It is a secondary carboxamide, a member of caprolactams and a member of 1-benzothiophenes. It is functionally related to a 2-aminohexano-6-lactam and an anthranilic acid.

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Finally, other compounds sharing the 3-(acylamino)-ε-caprolactam scaffold of N-T19 and ANA-12 have been described as procognitive agents in mice, although the precise mechanism of action is not described. Given the central role of the BDNF/TrkB coupling in cognition and memory, it would be interesting to evaluate the effect of these compounds on TrkB receptors and to compare them with ANA-12 and N-T19. To the best of our knowledge, ANA-12 is the first nonpeptide antagonist of TrkB receptor that elicits strong and specific effects in vivo. This proteolytically stable small molecule constitutes a valuable pharmacological tool to enable us to better investigate the role of BDNF/TrkB signaling in pathophysiological situations and will serve as a lead compound for the design of potent orally bioavailable TrkB modulators. [1]

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In conclusion, our study shows that LPS-induced inflammation caused depression-like behavior, as well as alterations in BDNF protein and spine density within the hippocampus, PFC, and NAc. Furthermore, antidepressant effects were shown on LPS-induced depressive behavior by normalizing altered dendritic spines in the hippocampus and PFC with TrkB agonist 7,8-DHF and in the NAc with antagonist ANA-12. Therefore, abnormal BDNF-TrkB signaling in the hippocampus, PFC, and NAc may play a role in inflammation-induced depression. Finally, in MDD, TrkB agonists and TrkB antagonists could act as potential therapeutic drugs for patients with lower BDNF levels in the hippocampus and PFC, and those with higher levels of BDNF in the NAc. [2]

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Mesolimbic dopamine (DA) controls drug- and alcohol-seeking behavior, but the role of specific DA receptor subtypes is unclear. We tested the hypothesis that D3R gene deletion or the D3R pharmacological blockade inhibits ethanol preference in mice. D3R-deficient mice (D3R(-/-)) and their wild-type (WT) littermates, treated or not with the D3R antagonists SB277011A and U99194A, were tested in a long-term free choice ethanol-drinking (two-bottle choice) and in a binge-like ethanol-drinking paradigm (drinking in the dark, DID). The selectivity of the D3R antagonists was further assessed by molecular modeling. Ethanol intake was negligible in D3R(-/-) and robust in WT both in the two-bottle choice and DID paradigms. Treatment with D3R antagonists inhibited ethanol intake in WT but was ineffective in D3R(-/-) mice. Ethanol intake increased the expression of RACK1 and brain-derived neurotrophic factor (BDNF) in both WT and D3R(-/-); in WT there was also a robust overexpression of D3R. Thus, increased expression of D3R associated with activation of RACK1/BDNF seems to operate as a reinforcing mechanism in voluntary ethanol intake. Indeed, blockade of the BDNF pathway by the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and strongly decreased the striatal expression of D3R. Finally, we evaluated buspirone, an approved drug for anxiety disorders endowed with D3R antagonist activity (confirmed by molecular modeling analysis), that resulted effective in inhibiting ethanol intake. Thus, DA signaling via D3R is essential for ethanol-related reward and consumption and may represent a novel therapeutic target for weaning. [4]

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We delineated a heritable phenotype resulting from the self-administration of cocaine in rats. We observed delayed acquisition and reduced maintenance of cocaine self-administration in male, but not female, offspring of sires that self-administered cocaine. Brain-derived neurotrophic factor (Bdnf) mRNA and BDNF protein were increased in the medial prefrontal cortex (mPFC), and there was an increased association of acetylated histone H3 with Bdnf promoters in only the male offspring of cocaine-experienced sires. Administration of a BDNF receptor antagonist (the TrkB receptor antagonist ANA-12) reversed the diminished cocaine self-administration in male cocaine-sired rats. In addition, the association of acetylated histone H3 with Bdnf promoters was increased in the sperm of sires that self-administered cocaine. Collectively, these findings indicate that voluntary paternal ingestion of cocaine results in epigenetic reprogramming of the germline, having profound effects on mPFC gene expression and resistance to cocaine reinforcement in male offspring. [5]

C22H21N3O3S

407.49

407.13

C, 64.85; H, 5.19; N, 10.31; O, 11.78; S, 7.87

219766-25-3

2799722

White to off-white solid powder

4.786

126.01

3

4

4

29

628

0

S1C2=C([H])C([H])=C([H])C([H])=C2C([H])=C1C(N([H])C1=C([H])C([H])=C([H])C([H])=C1C(N([H])C1([H])C(N([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O)=O)=O

TUSCYCAIGRVBMD-UHFFFAOYSA-N

InChI=1S/C22H21N3O3S/c26-20(25-17-10-5-6-12-23-21(17)27)15-8-2-3-9-16(15)24-22(28)19-13-14-7-1-4-11-18(14)29-19/h1-4,7-9,11,13,17H,5-6,10,12H2,(H,23,27)(H,24,28)(H,25,26)

N-[2-[(2-oxoazepan-3-yl)carbamoyl]phenyl]-1-benzothiophene-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: 1.43 mg/mL (3.51 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 14.3 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.43 mg/mL (3.51 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 14.3 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 1 mg/mL (2.45 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 0.45 mg/mL (1.10 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 0.45 mg/mL (1.10 mM) (饱和度未知) in 5% DMSO + 95% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 6 中的溶解度: 2% DMSO+30% PEG 300+2% Tween 80+ddH2O: 2mg/mL .

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。