GNE-495

MAP4K4 (IC50 = 3.7 nM)
GNE-495 is a strong MAP4K4 inhibitor that works well to promote angiogenesis. GNE-495 exhibits minimum body stability, permeability, efficient ideal cellular equilibrium, and MAP4K4 adsorption [1].

GNE-495 是一种强效 MAP4K4 抑制剂，可以很好地促进血管生成。 GNE-495 表现出最低的身体稳定性、渗透性、有效的理想细胞平衡和 MAP4K4 吸附 [1]。

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GNE-495 对 MAP4K4 表现出强效的生化抑制活性，IC50 为 3.7 ± 1.4 nM。
在人脐静脉内皮细胞 (HUVEC) 迁移实验中，GNE-495 能够抑制细胞迁移，IC50 为 0.057 ± 0.004 µM，该实验用于评估抗血管生成活性。
该化合物保持了良好的激酶选择性，这与其结合 MAP4K4 的折叠 P 环构象有关，这种构象仅在少数激酶中存在。[1]

高剂量的 GNE-495（25 和 50 mg/kg）被腹腔注射给新生小鼠幼崽。在检查的每个物种中，GNE-495 都表现出良好的内部特性，包括低清除率、适度的终端半相衰减和适当的杠杆水平 (F=37–47%) [1]。

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向新生小鼠腹腔注射 GNE-495 可剂量依赖性地延迟视网膜血管向外生长，并诱导异常的视网膜血管形态（例如，血管内皮细胞出现长的膜突起）。
这些体内表型重现了在诱导性 Map4k4 基因敲除小鼠中观察到的视网膜血管缺陷，证实了该化合物的体内活性及其对 MAP4K4 的靶向作用。[1]

针对 MAP4K4 的生化 IC50 测定使用 Z'-LYTE 检测方法，数据代表至少三次独立实验的平均值。
对于该系列中的部分化合物，也使用了 Caliper LabChip 3000 (LC3K) 技术测定 IC50。[1]

GNE-495 的抗血管生成细胞活性通过 HUVEC 细胞迁移实验进行评估。
该实验测量对细胞迁移的抑制，细胞迁移是血管生成的关键过程。报告的 IC50 值是抑制 50% 细胞迁移所需的浓度。[1]

Rats, Mice and Pups
For the brain cassette study, three male Sprague-Dawley (SD) rats receive an intravenous (IV) bolus dose of six test substances (for example, GNE-495; 0.5 mg/kg). GNE-495 is injected intravenously in bolus doses of 1 mg/kg in female CD-1 mice for the mouse PK study. GNE-495 (5 mg/kg) is additionally injected intravenously (PO) into female CD-1 mice. The rat brain cassette PK is dosed at 2 mL/kg, and all other doses are administered at 5 mL/kg. Water and food are available to animals at all times, and they are not fasted before receiving a dose. Three blood samples (~60 μL) are taken from each mouse up to either 9 or 24 hours after the compound of interest is given. This is done using a serial sampling method, and it involves taking three blood samples from each mouse. After being mixed with K2EDTA and placed on ice or in a cold Kryorack immediately after collection, the blood is then centrifuged to separate the plasma. Blood samples are centrifuged for 10–15 minutes at 4°C while being spun at a speed of 1000-2000× g within an hour of being drawn. This process yields the plasma. The plasma samples are stored at -70 to -80°C until analysis. For neonate PK, 3-day-old CD1 pups are injected with 25 mg/kg and 50 mg/kg GNE-495. Blood samples are taken intraperitoneally at the times specified, retinas are taken one hour after the dose, snap frozen in liquid nitrogen, and kept at -80°C until analysis. The concentrations of plasma and retinal lysate are assessed using LC/MS/MS.

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For the efficacy study in the neonatal retinal angiogenesis model, newborn mouse pups received intraperitoneal (IP) injections of GNE-495 daily from postnatal day 1 (P1) to P5 or P6.
Doses used were 25, 50, and 100 mg/kg. The compound was formulated in a solution containing 5% DMSO and 95% corn oil.
Retinas were harvested at P6 or P7, flat-mounted, and stained with Isolectin-B4 to visualize blood vessels for analysis of avascular area and vascular morphology. [1]

In mouse, following a 1 mg/kg IV dose, GNE-495 showed a clearance (CL) of 19 mL/min/kg, a terminal half-life (T1/2) of 1.5 h, a volume of distribution (V) of 1.6 L/kg, and an AUC extrapolated to infinity (AUCinf) of 5.3 hµM.
Following a 5 mg/kg oral dose in mouse, the maximum plasma concentration (Cmax) was 1.4 µM at 1.0 h (Tmax), with an oral bioavailability (F%) of 47%.
The unbound brain concentration measured at 1 h post-dose was very low (0.008 µM), indicating minimal CNS penetration.
In rat, CL was 7.5 mL/min/kg, T1/2 was 3.4 h, V was 1.2 L/kg, and oral F% was 40%.
In dog, CL was 8.9 mL/min/kg, T1/2 was 1.8 h, V was 1.1 L/kg, and oral F% was 37%.
The compound was highly plasma protein bound in mouse (94.4%) and rat (97.8%).
In vitro, it showed moderate MDCK cell permeability (Papp = 7.3 x 10^-6 cm/s) with an efflux ratio of 2.3.
It demonstrated low metabolic clearance in human and mouse liver microsomes (HLM CLint = 7 µL/min/mg; MLM CLint = 13 µL/min/mg). [1]

The predecessor compound 1 was poorly tolerated in multiday studies, which was hypothesized to be due to its significant CNS penetration (unbound brain concentration = 14.6 µM).
GNE-495 was specifically designed to minimize brain penetration (unbound brain concentration = 0.008 µM) to mitigate this potential toxicity risk. The exact toxicity profile of GNE-495 in repeated-dose studies is not detailed in this literature. [1]

[1]. Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis. ACS Med Chem Lett. 2015 Jun 29;6(8):913-8.

GNE-495 (compound 13) was developed from a structure-based design effort aimed at maintaining potent MAP4K4 inhibition while reducing central nervous system (CNS) penetration by increasing topological polar surface area (TPSA) and modifying other physicochemical properties.
The binding mode was confirmed by an X-ray co-crystal structure (PDB: 42KS), showing that the compound maintains key interactions with the hinge region and induces a folded P-loop conformation involving Tyr36.
It is described as a valuable tool compound for investigating the biological functions of MAP4K4 in various disease contexts, including angiogenesis. [1]

C22H20FN5O2

405.4249

405.16

C, 65.18; H, 4.97; F, 4.69; N, 17.27; O, 7.89

1449277-10-4

1449277-10-4

89730041

Yellow solid powder

1.5±0.1 g/cm3

734.5±60.0 °C at 760 mmHg

398.0±32.9 °C

0.0±2.4 mmHg at 25°C

1.712

2.54

101

2

6

4

30

668

0

FC1=C([H])C([H])=C([H])C(=C1[H])C1C([H])=C([H])C2=C(C(N([H])[H])=NC([H])=C2C(N([H])C2([H])C([H])([H])N(C2([H])[H])C(C2([H])C([H])([H])C2([H])[H])=O)=O)N=1

FYXCIBJXJYBWPX-UHFFFAOYSA-N

InChI=1S/C22H20FN5O2/c23-14-3-1-2-13(8-14)18-7-6-16-17(9-25-20(24)19(16)27-18)21(29)26-15-10-28(11-15)22(30)12-4-5-12/h1-3,6-9,12,15H,4-5,10-11H2,(H2,24,25)(H,26,29)

8-amino-N-[1-(cyclopropanecarbonyl)azetidin-3-yl]-2-(3-fluorophenyl)-1,7-naphthyridine-5-carboxamide

GNE 495; GNE495; GNE-495

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: ~2.2 mg/mL (~5.4 mM)

配方 1 中的溶解度: ≥ 0.22 mg/mL (0.54 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 2.2 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: ≥ 0.22 mg/mL (0.54 mM) (饱和度未知) in 10% DMSO + 90% (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。
例如，若需制备1 mL的工作液，可将 100 μL 2.2mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*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 3 中的溶解度: ≥ 0.22 mg/mL (0.54 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 2.2 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。