AF38469

Sortilin ( IC50 = 330 nM )

AF38469已进行进一步研究。应当注意，在3HNTS测定中，神经紧张素本身的结合IC50为360 nM，因此AF38469与这种sortilin底物基本相等。 在CEREP下运行的约70个靶标的标准选择性panel中，AF38469在10μM下没有显示出>50%的抑制或刺激。重要的是，AF38469对NTR1受体没有活性。此外，AF38469对已知结合酸性分子的选定靶点（δ-阿片类、GPR40、PPARδ、EP1、血管紧张素AT1、内皮素ETA和B、MMP-12）没有活性。因此，总体而言，AF38469的选择性分析表明其与sortilin具有高度特异性的相互作用[1]。

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成功获得了sortilin和AF38469的共晶体，并通过X射线晶体学确定了其结构，分辨率为2.78Å。图1显示了AF38469晶体结构与sortilin结合的神经紧张素C末端区域结构的叠加。AF38469与sortilin的相互作用与神经紧张素的C末端Leu残基相似。AF38469通过其羧酸与Arg292形成盐桥，CF3基团与Leu侧链的iPr占据相同的疏水结合口袋，酰胺N-H与Tyr318形成氢键供体相互作用。吡啶甲基与Phe317形成疏水π相互作用，类似于神经降压素Ile12的疏水相互作用。该结构还有助于使上述许多结构-活性关系合理化，例如间甲氨基酸5取代基的关键作用、5-Ph（10f）的无活性（由于尺寸排除）以及羧酸官能团的关键作用。 图2显示了AF38469与先前报道的小分子抑制剂AF40431的结合模式的叠加。AF38469和AF40431表现出许多常见的相互作用，包括神经降压素的盐桥和亮氨酸口袋结合特征，但有趣的是，前者的吡啶环和甲基取代基与后者的吡喃环和甲基取代基几乎一致。 预计sortilin-AF38469复合物的结构将用于通过经典的基于结构的药物设计进一步推动化学型的优化[1]。

AF38469 (1 mg/kg；iv) 在十二烷基苯磺酸钠中具有低循环容量（血容量为 0.7 L/kg）和低清除率（4.8 L/h/kg），半衰期（t1/2）为 1.2 h[1]。用AF38469治疗西方饮食喂养的小鼠可以降低血浆胆固醇和肝脏细胞因子的表达。它与西方饮食喂养的小鼠肝脏VLDL分泌减少和肝脏胆固醇7α水解酶表达升高有关，但不影响饮食诱导的肥胖或胰岛素抵抗[2]

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Sort1抑制剂降低了WD喂养小鼠的血浆胆固醇水平、肝脏VLDL分泌和肝脏促炎细胞因子[2]
最近，已经鉴定出一种口服生物可利用的Sort1抑制剂，AF38469，IC50值约为330 nM。为了确定AF38469对Sort1的药理学抑制是否可以改善脂质稳态和炎症，我们给补充了AF38469的WD小鼠喂食，以达到估计的4 mg/kg日剂量，这与已发表的药代动力学数据一致。AF38469治疗在8周内没有影响体重增加或肝脏重量（图7A，B）。治疗组和对照组的肝脏TG和胆固醇水平没有显著差异，尽管我们注意到AF38469治疗的小鼠肝脏TG下降了约30%（P=0.19）（图7C-E）。两组的血浆AST和ALT水平相似，表明AF38469治疗8周与肝毒性无关（图7F，G）。血脂参数分析显示，AF38469显著降低了30%的血浆胆固醇（图8A），但不影响血浆TG水平（图8B）。为了进一步了解AF38469的降胆固醇作用，我们首先测量了肝脏VLDL的分泌，因为之前的遗传学研究表明肝脏Sort1在调节肝脏VLDL分泌中起作用。有趣的是，AF38469治疗的小鼠显示出肝脏VLDL分泌显著减少（图8C）。胆汁胆固醇分泌和胆汁酸合成是影响循环胆固醇水平的两种主要胆固醇消除机制。AF38469治疗不影响胆囊总胆固醇含量（图8D）。胆汁酸代谢分析显示，AF38469治疗的小鼠肝脏胆汁酸水平显著降低（图8E），而胆囊胆汁酸、肠道胆汁酸和总胆汁酸池大小没有变化（图8E）。肝脏基因表达谱分析发现，在AF38469治疗的小鼠中，肝脏CYP7A1被显著诱导，而其他胆固醇代谢基因（ABCG5、ABCG8、HMGCR、LDLR）没有改变（图8F）。在AF38469治疗的小鼠中，趋化因子MCP1和促炎细胞因子IL-1β和IL-6的肝脏mRNA表达显著降低，但TNFα的表达没有降低（图8F）。较低的肝细胞因子可能为AF38469治疗的小鼠CYP7A1 mRNA表达较高提供了可能的解释。

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Sort1抑制剂不影响WD喂养小鼠的空腹血糖和糖耐量[2]
为了更全面地了解药理学Sort1抑制的代谢作用，我们接下来评估了AF38469对WD喂养小鼠胰岛素敏感性的影响。AF38469治疗不影响空腹血糖或空腹胰岛素浓度（图9A，B）。此外，AF38469治疗不影响空腹血浆游离脂肪酸浓度（图9C）或葡萄糖耐量试验中的葡萄糖耐量（图9D）。这些结果表明，AF38469不影响WD喂养小鼠的胰岛素敏感性。

如前所述（Andersen，2013，提交），用10摩尔过量的AF438469结晶1小时。将1μl sortilin-AF38469与1μl储层混合，并在24孔结晶板中在292 K下使用坐滴蒸汽扩散法与500μl储器平衡。在0.1 M HEPES Tris pH 7.3、0.4 M丙二酸钠、27%（w/v）PEG 3350和4.5%（v/v）甘油中获得了最佳的衍射晶体。晶体在两周内生长到200 x 100 x 50μm的尺寸。通过向储器中加入100μl 80%（v/v）PEG400对晶体进行脱水，并将其从母液中装入Litholoops（分子尺寸）中，用环路边缘轻轻接触滴孔侧面，将多余的母液浸入，并在液氮中快速冷却。在瑞士光源（SLS）的X06DA（PXIII）光束线上，使用PILATUS 2M-F探测器在100 K下收集了3600个振荡图像的完整单波长（λ=0.9Å）数据集，振荡角度为0.1o。衍射图像在XDS中处理，在SCALA中缩放。使用PHASER程序，使用基于sortilin与神经降压素复合物（PDB ID 3F6K）结构的搜索模型进行分子置换。在PHENIX中进行了刚体细化、配体坐标和约束的生成、省略图的计算和细化。使用Coot进行建模和分析。使用Molprobity对最终模型质量进行了分析。使用PyMol[1]进行叠加并制备结构图。[1]

Mice: Sort1 floxed mice on a C57BL/6N background were used. The targeting strategy is illustrated in Fig. 1A. The NeoR cassette was removed by crossing Sort1 floxed founders with the FLP deleter strain on a C57BL/6J background. Cre-mediated recombination results in the deletion of exon 2 and exon 3 and subsequent frameshift of the Sort1 gene. L-Sort1 KO mice were generated by crossing Sort1 floxed mice with the albumin-cre deleter strain on a C57BL/6J background. LysM-Sort1 KO mice were generated by crossing Sort1 floxed mice with the LysM-cre deleter strain on a C57BL/6NJ mixed background. Littermates without the cre transgene were used as WT controls. Mice were housed in micro-isolator cages with corn cob bedding under a normal light-dark cycle. WT C57BL/6J mice were purchased from Jackson Laboratory. The standard chow diet was PicoLab Rodent Diet 20 containing 13% fat calories and no added cholesterol. WD (TD.88137) contained 42% fat calories and 0.2% cholesterol. Male C57BL/6J mice were used for the AF38469 study. AF38469 was mixed with powdered WD and the estimated daily dose of ∼4 mg/kg was calculated based on daily food intake of ∼4 g per mouse. The control group was given powdered WD. Powdered WD was placed in a dish inside the cage and replaced every 2 days. Only male mice were used for this study. [2]

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Glucose tolerance test: After 7 weeks of WD feeding, control and AF38469-treated mice were fasted overnight and received a single intraperitoneal injection of glucose at 2 g/kg body weight. A drop of blood was collected from a tail nick at the indicated time, and glucose was measured with a OneTouch Ultra glucometer. These mice were continued on WD or WD supplemented with AF38469 for 1 week and euthanized for tissue collection.[2]

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Measurement of VLDL secretion Male C57BL/6J mice, at 12 weeks of age, were given powdered WD or powdered WD supplemented with AF38469 to provide an estimated daily dose of ∼4 mg/kg. After 2 weeks, mice were fasted for 6 h (9:00 AM to 3:00 PM). Tyloxapol was diluted in sterile PBS and administered to mice via tail vein injection as a single dose of 300 mg/kg. Blood was collected by tail nicking at 0 h (right before injection) and at 1.5 and 3 h post injection for TG measurement.[2]

Rat pharmacokinetics: [1]
AF38469 has a low volume of distribution (relative to blood volume of 0.7 l/kg) and low clearance (relative to liver blood flow of ca. 4.8 l/h/kg), and a half-life of 1.2 h. The initial exposure in plasma is high, due in part to the low volume of distribution. The oral bioavailability is 35%. Simple calculations based on Cmax, free fraction and the sortilin potency suggest that a free plasma concentration of AF38469 ca. 2 fold higher than its IC50 is obtained at Cmax from an oral 2 mg/kg dose.

In Vitro Solubility 134 μg/ml, plasma free fraction 2% intrinsic clearance 0.4 l/h/kg
Oral (2 mg/kg) Cmax 12850 ng/ml
i.v. (1 mg/kg) Clb 0.03 L/h/Kg, t1/2 1.2 h, Vss 0.02 L/kg

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In principle a phthalimide could act as a pro-drug for an AF38469 type compound, with hydrolysis in vivo affording the corresponding phthalamic acid by an analogous process to that which lead to the liberation and identification of compound 2 itself (Scheme 1). Whilst an asymmetrically substituted phthalimide such as 1 would afford a mixture of regioisomers on hydrolysis (viz. 2 and 3), a symmetrical phthalimide would afford a single phthalamic acid (e.g. Scheme 4). To this end phthalimide 14 was investigated as a potential oral prodrug for phthalamic acid 10k. However, whilst oral administration of the phthalimide (14) did afford systemic exposure of phthalamic acid 10k, the free exposure of the acid was not an improvement on the exposure of AF38469 obtained via oral administration.

[1]. The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin. Bioorg Med Chem Lett. 2014 Jan 1;24(1):177-80.

[2]. Hepatocyte sortilin 1 knockout and treatment with a sortilin 1 inhibitor reduced plasma cholesterol in Western diet-fed mice. J Lipid Res. 2019 Mar;60(3):539-549.

Sortilin is a type I membrane receptor belonging to the vacuolar protein sorting 10 protein (VPS10P) family of sorting receptors. Sortilin is widely expressed in both the central nervous system and periphery. It mediates a number of important physiological functions via trafficking of, and signalling with, a variety of different protein partners. For example sortilin is involved in signalling via the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Indeed, in complex with the protein p75, sortilin has been reported to form the receptor for pro-neurotrophin-mediated apoptotic effects leading to degeneration and cell death in cellular and animal models.1 Sortilin has also been demonstrated to interact with apolipoprotein B100 in the Golgi and facilitate the export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol levels, a key contributor to atherosclerosis and ischemic heart disease. Recently, sortilin was also shown to function as a high affinity receptor for progranulin, and to mediate clearance of progranulin by binding followed by cellular uptake and distribution to lysosomes. [1]

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In summary we have identified a potent, selective and orally bioavailable inhibitor for the VPS10P family sorting receptor Sortilin. We hope and anticipate that AF38469 will serve as an important tool to further delineate the biology of Sortilin, and to facilitate evaluation of the therapeutic potential of this protein. [1]

C15H11F3N2O3

324.26

324.072

C, 55.56; H, 3.42; F, 17.58; N, 8.64; O, 14.80

1531634-31-7

1531634-31-7

72706115

Off-white to light yellow solid powder

3.432

79.29

2

7

3

23

456

0

O=C(NC1=CC=CC(C)=N1)C2=CC=C(C(F)(F)F)C=C2C(O)=O

JWCUSQCZMQIBMR-UHFFFAOYSA-N

InChI=1S/C15H11F3N2O3/c1-8-3-2-4-12(19-8)20-13(21)10-6-5-9(15(16,17)18)7-11(10)14(22)23/h2-7H,1H3,(H,22,23)(H,19,20,21)

2-[(6-methylpyridin-2-yl)carbamoyl]-5-(trifluoromethyl)benzoic acid

AF-38469; AF38469; 1531634-31-7; 2-[(6-Methylpyridin-2-Yl)carbamoyl]-5-(Trifluoromethyl)benzoic Acid; 2-((6-methylpyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid; CHEMBL3098745; MFCD28160694; 4n7e; SCHEMBL15903106; AF 38469

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)

DMSO: ~65 mg/mL (~200.5 mM)

配方 1 中的溶解度: 2.5 mg/mL (7.71 mM) (饱和度未知) 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), 悬浮液；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 2 中的溶解度: ≥ 2.08 mg/mL (6.41 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 20.8 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: 5%DMSO + Corn oil: 3.25mg/ml (10.02mM)

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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。