EHT 1864 2HCl

Rac1 (Kd = 40 nM); Rac1b (Kd = 50 nM); Rac2 (Kd = 60 nM); Rac3 (Kd = 250 nM)

体外活性：EHT 1864 选择性抑制 Rac 诱导的片状伪足形成，并特异性逆转 Rac1 和 Tiam1 组成型激活突变体诱导的细胞转化。 EHT 1864 强烈损害稳定表达 H-Ras(61L) 蛋白的 NIH 3T3 细胞中致癌 Ras 诱导的细胞增殖。 EHT 1864 还通过抑制 APP 的 γ-分泌酶依赖性裂解来降低细胞外和细胞内 Aβ 肽的水平。在培养的海马锥体神经元中，EHT 1864 通过抑制 Rac1，挽救了 Rich2 敲低诱导的表型。激酶测定：对于抑制剂：GTPase 结合分析，将小份 GTPase 溶液（含有 1 μM 抑制剂）滴定到比色皿中的 1 μM 抑制剂溶液中。每次添加后 30 秒，在 λex = 360 nm、λem = 440 nm 处监测荧光各向异性的变化。所有数据分析和曲线拟合均使用 Microsoft Excel 和 Mac OS X 的 QuantumSofts ProFit 进行。 细胞测定：将稳定表达致癌 Ras 的 NIH 3T3 细胞接种于 96 孔板中。细胞在完全生长培养基中培养长达 4 天，可以单独使用，也可以添加 5 μM EHT 1864。然后使用 MTT 转化为甲臜产物来评估细胞生长。简而言之，将 MTT 试剂（用 PBS 稀释的 5 mg/ml 溶液）以 0.5 mg/ml 的终浓度添加到孔中，并将细胞在 37°C 下进一步孵育 4 小时。然后除去培养基，并通过添加 100 μl/孔 Me2SO 终止反应。使用酶标仪在 570 nm 处读取吸光度。

EHT 186440 mg/kg ip）显着降低豚鼠大脑中的 Abeta 40 和 Abeta 42 水平。

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EHT 1864阻止体内Aβ40和Aβ42的产生[2]
在豚鼠中测试了EHT 1864的影响，以确定在过表达野生型和人类突变APP的细胞系中观察到的Aβ40和Aβ42的减少是否可以在体内复制。我们使用正常的野生型白化豚鼠作为模型，因为豚鼠是生理APP加工和aβ产生的既定模型。此外，它们的Aβ40和Aβ42肽与人类Aβ相同，可以很容易地通过BIOSOURCE夹心ELISA检测到。
在大鼠身上进行的初步实验表明，口服EHT 1864后显示出良好的耐受性、脑外显率和无遗传毒性（Ames试验）。我们在豚鼠中选择了一种直接的给药方式，通过每天腹腔注射两种浓度（10和40mg/kg）在15天内给药EHT 1864。我们使用基于胍的提取方案来确保Triton可溶性和Triton不溶性aβ组分的回收。在对照组动物中，回收的Aβ40浓度为1220 pg/mg蛋白质EHT 1864（40mg/kg/天）将脑Aβ40降低了37%，p<0.05（通过威尔科克森检验）（图8A）。对于Aβ42，尽管测量值存在很大差异，可能是由于肽的量较少，但相同剂量的化合物EHT 1864（40mg/kg/天）导致Aβ42水平下降了23.6%。在10mg/kg的剂量下，EHT 1864也导致大脑中aβ40和aβ42的量略有减少（分别为12.8%和6%）。

为了进行抑制剂：GTPase 结合分析，将含有 1 μM 抑制剂的小份 GTPase 溶液滴定到含有 1 μM 抑制剂的比色皿中。每次添加后 30 秒在 λex = 360 nm 和 λem = 440 nm 处测量荧光各向异性。 Microsoft Excel 和 QuantumSoft 的 Mac OS X ProFit 用于所有数据分析和曲线拟合。

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HeLa细胞中NotchΔE转染和Notch-1切割试验[2]
用表达载体pSC2+ΔE3MV-6MT瞬时转染10cm平板中的HeLa细胞，该载体过表达截短的Notch-1，缺乏大部分Notch细胞外结构域，并具有C末端Myc标签（NotchΔE）。这种截短形式的Notch是γ-分泌酶的底物。转染后1天，将培养物与指定浓度的EHT 1864或γ-分泌酶抑制剂DAPT预孵育18小时，然后处理CelLytic-M裂解物，使用1:1000的抗Myc抗体通过蛋白质印迹检测Notch细胞内结构域（NICD）。

在 96 孔板中，表达致癌 Ras 的 NIH 3T3 细胞稳定铺板。细胞在完全生长培养基中生长，可以单独生长，也可以添加 5 μM EHT 1864，最多生长 4 天。下一步是将 MTT 转化为甲臜产品来测量细胞生长。总之，将 MTT 试剂（来自用 PBS 稀释的 5 mg/ml 溶液）以 0.5 mg/ml 的终浓度添加到孔中后，将细胞在 37°C 下再孵育 4 小时。之后，取出培养基，加入100μl/孔的Me₂SO终止反应。使用酶标仪，在 570 nm 处测量吸光度。

Male Hartley albino guinea pigs
40 mg/kg daily
i.p.

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In Vivo Delivery of Inhibitors—EHT 1864 or vehicle (physiological saline) were injected in male Hartley albino guinea pigs, weighing 250–270 g at delivery and obtained from Charles River Laboratories, once a day for 15 consecutive days by the intraperitoneal route. 1 h after the final administration, the guinea pigs were killed; brains were immediately extracted and immersed in an oxygenated (95% O2, 5% CO2) physiological saline bath placed on ice (1–2 °C); and superficial vessels were removed. The whole brains were dissected to provide left and right cortices, which were weighed, snap-frozen in liquid nitrogen, and stored at –80 °C, separately. The maximum time between sacrifice and snap freezing was less than 15 min.[2]

[1]. J Biol Chem . 2007 Dec 7;282(49):35666-78.

[2]. J Biol Chem . 2005 Nov 11;280(45):37516-25.

[3]. J Biol Chem . 2014 Jan 31;289(5):2600-9.

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.[1]

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beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.[2]

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Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.[3]

C25H29CL2F3N2O4S

581.47

580.117

C, 51.64; H, 5.03; Cl, 12.19; F, 9.80; N, 4.82; O, 11.01; S, 5.51

754240-09-0

9938202

White to off-white solid powder

6.922

90.1

2

10

10

37

770

0

Cl[H].Cl[H].S(C1C([H])=C([H])N=C2C([H])=C(C(F)(F)F)C([H])=C([H])C=12)C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])OC1=C([H])OC(=C([H])C1=O)C([H])([H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

LSECOAJFCKFQJG-UHFFFAOYSA-N

InChI=1S/C25H27F3N2O4S.2ClH/c26-25(27,28)18-4-5-20-21(14-18)29-7-6-24(20)35-13-3-1-2-10-33-23-17-34-19(15-22(23)31)16-30-8-11-32-12-9-30;;/h4-7,14-15,17H,1-3,8-13,16H2;2*1H

2-(morpholin-4-ylmethyl)-5-[5-[7-(trifluoromethyl)quinolin-4-yl]sulfanylpentoxy]pyran-4-one;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: ≥ 2.08 mg/mL (3.58 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 20.8 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: ≥ 2.08 mg/mL (3.58 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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: ≥ 2.08 mg/mL (3.58 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 4 中的溶解度: 25 mg/mL (42.99 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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配方 5 中的溶解度: 25 mg/mL (42.99 mM) in Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).
*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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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。