Rap1A (IC50 = 3 μM, in vivo), Ha-Ras (IC50 > 20 μM, in vivo)[3]

RhoA 抑制剂（GGTI298 三氟乙酸酯）可显着降低 cAMP 激动剂增加的顶端 K+ 电导率[1]。当 GGTI298 三氟乙酸酯和 TRAIL 用于引起 DR5 依赖性细胞凋亡时，DR4 敲低会消除 NF-κB 激活，并使细胞更容易受到此过程的影响。三氟乙酸盐/TRAIL (GGTI298 ) 抑制 Akt 并增加 NF-κB。 DR5 敲低可阻止 GGTI298/TRAIL 产生的 IκBα 和 p-Akt 减少，表明 DR5 介导 GGTI298/TRAIL 诱导的这些分子的减少。另一方面，DR4 敲低使 GGTI298 /TRAIL 诱导的 p-Akt 降低更容易[2]。

体内小鼠回肠环实验表明，注射 TRAM-34、GGTI298 三氟乙酸酯或 H1152 与霍乱毒素联合注射可以剂量依赖性方式减少积液[1]。

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研究药物对骨巨细胞瘤（GCT）影响的主要障碍是缺乏动物模型。在这项研究中，我们创建了一种动物模型，其中GCT基质细胞存活并作为增殖肿瘤细胞发挥作用。使用GCT基质细胞的增殖细胞系来创建稳定的萤光素酶转导细胞系Luc-G33。对细胞系进行了表征，发现与野生型GCT基质细胞相比，细胞增殖率和单核细胞募集没有显著差异。我们将Luc-G33细胞皮下注射到裸鼠的背部或胫骨。使用IVIS 200生物发光成像（BLI）系统的实时活体成像评估活Luc-G33细胞的存在。肿瘤细胞最初在皮下肿瘤模型中增殖并在现场存活7周。我们还测试了唑来膦酸盐（ZOL）和香叶基转移酶-I抑制剂（GGTI-298）单独或其组合在Luc-G33移植裸鼠体内的抗肿瘤作用。400µg/kg的单独ZOL以及400µg/kg ZOL和1.16mg/kg GGTI-298的联合治疗降低了模型中的肿瘤细胞存活率。此外，ZOL、GGTI-298的抗肿瘤作用以及皮下肿瘤模型中的联合治疗也通过免疫组织化学（IHC）染色得到证实。总之，我们建立了一个GCT基质细胞的裸鼠模型，该模型可以无创、实时评估肿瘤的发展，并测试不同佐剂治疗GCT的体内效果。[4]

研究人员使用法尼烷基转移酶（FTase）和香叶基香叶基转移酶（GGTase）I的特异性抑制剂，以及洛伐他汀与香叶基天竺葵醇（GGOH）或法尼醇（FOH）的组合，研究蛋白质异戊二烯化在血小板衍生生长因子（PDGF）诱导的PDGF受体酪氨酸磷酸化中的作用。在完全抑制FTase依赖性加工的剂量下，用高度特异性FTase抑制剂FTI-277处理的NIH-3T3细胞对PDGF受体酪氨酸磷酸化或PDGF活化丝裂原活化蛋白激酶（MAPK）没有影响。相比之下，用GGTase I抑制剂GGTI-298处理这些细胞强烈抑制了受体酪氨酸磷酸化，与FTI-277联合处理没有额外效果。有趣的是，GGTI-298对PDGF激活MAPK的抑制作用只是部分的。此外，尽管抑制蛋白质香叶基香叶基化和蛋白质法尼基化的洛伐他汀阻断了PDGF受体酪氨酸磷酸化，但与GGOH而非FOH联合治疗逆转了洛伐他汀的阻断作用。此外，尽管观察到洛伐他汀可以阻断PDGF对MAPK的激活，但与GGOH而非FOH联合治疗可以恢复其激活。进一步的研究表明，受体酪氨酸磷酸化的抑制不是由于受体表达减少或GGTase II的抑制。因此，这些结果表明，PDGF受体酪氨酸磷酸化需要蛋白质香叶基香叶基化，但不需要蛋白质法尼基化，受体的酪氨酸磷酸化水平受GGTase I底物蛋白质的调节。[3]

细胞存活试验[2]
将细胞接种在96孔细胞培养板中，并在第二天用指定的试剂处理。如前所述，使用磺基罗丹明B测定法测定活细胞数。

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细胞凋亡检测[2]
根据制造商的说明，使用市售的Annexin V-PE凋亡检测试剂盒通过Annexin V染色评估细胞凋亡。通过蛋白质印迹法（如下所述）也检测到半胱天冬酶激活，作为凋亡的另一个指标。

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蛋白质印迹分析[2]
如前所述，制备全细胞蛋白裂解物并通过蛋白质印迹进行分析。

Mouse Ileal Loop Experiment[1]
The ileal loop experiment was performed in 6–8-week-old mice by a modified rabbit ileal loop assay originally described by De and Chatterje. Following gut sterilization, the animals were kept fasted for 24 h prior to surgery and fed only water ad libitum. Anesthesia was induced by a mixture of ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight). A laparotomy was performed, and the experimental loops of 5-cm length were constricted at the terminal ileum by tying with non-absorbable silk. The following fluids were instilled in each loop by means of a tuberculin syringe fitted with a disposable needle through the ligated end of the loop as the ligatured was tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (different concentrations in μm as indicated in Fig. 7), CT (1 μg) + H1152 (1 μm), and CT (1 μg) + GGTI298 (different concentrations in μm), a specific inhibitor of Rap1A. The intestine was returned to the peritoneum, and the mice were sutured and returned to their cages. After 6 h, these animals were sacrificed by cervical dislocation, and the loops were excised. The fluid from each loop was collected, and the ratio of the amount of fluid contained in the loop with respect to the length of the loop (fluid accumulation ratio in g/cm) was calculated as a reflection of the efficacy of various inhibitors.

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dissolved in DMSO, dilute in saline; 1.16 mg/kg; s.c. injection

Nude mice

[1]. The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem. 2013 Jul 12;288(28):20404-15.

[2]. Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis. Mol Cancer. 2010 Jan 29;9:23.

[3]. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem. 1996 Nov 1;271(44):27402-7.

[4]. A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone. Connect Tissue Res. 2015 Nov;56(6):493-503.

The apical membrane of intestinal epithelia expresses intermediate conductance K(+) channel (KCNN4), which provides the driving force for Cl(-) secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl(-) secretion via stimulation of Rap2-phospholipase Cε-[Ca(2+)]i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl(-) secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl(-) secretion and apical K(+) conductance (IK(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K(+) channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate IK(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated IK(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.[1]

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Background: Geranylgeranyltransferase I (GGTase I) has emerged as a cancer therapeutic target. Accordingly, small molecules that inhibit GGTase I have been developed and exhibit encouraging anticancer activity in preclinical studies. However, their underlying anticancer mechanisms remain unclear. Here we have demonstrated a novel mechanism by which GGTase I inhibition modulates apoptosis.
Results: The GGTase I inhibitor GGTI-298 induced apoptosis and augmented tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. GGTI-298 induced DR4 and DR5 expression and reduced c-FLIP levels. Enforced c-FLIP expression or DR5 knockdown attenuated apoptosis induced by GGTI-298 and TRAIL combination. Surprisingly, DR4 knockdown sensitized cancer cells to GGTI298/TRAIL-induced apoptosis. The combination of GGTI-298 and TRAIL was more effective than each single agent in decreasing the levels of IkappaBalpha and p-Akt, implying that GGTI298/TRAIL activates NF-kappaB and inhibits Akt. Interestingly, knockdown of DR5, but not DR4, prevented GGTI298/TRAIL-induced IkappaBalpha and p-Akt reduction, suggesting that DR5 mediates reduction of IkappaBalpha and p-Akt induced by GGTI298/TRAIL. In contrast, DR4 knockdown further facilitated GGTI298/TRAIL-induced p-Akt reduction.
Conclusions: Both DR5 induction and c-FLIP downregulation contribute to GGTI-298-mediated augmentation of TRAIL-induced apoptosis. Moreover, DR4 appears to play an opposite role to DR5 in regulation of GGTI/TRAIL-induced apoptotic signaling.[2]

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C27H33N3O3S.C2HF3O2

593.66

593.217

C, 58.67; H, 5.77; F, 9.60; N, 7.08; O, 13.47; S, 5.40

1217457-86-7

GGTI298;180977-44-0; GGTI298 Trifluoroacetate; 1217457-86-7; 205590-41-6 (HCl)

16078971

White to off-white solid powder

6.474

173.04

5

11

11

41

745

2

CC(C)C[C@@H](C(OC)=O)NC(C1=CC=C(NC[C@@H](N)CS)C=C1C2=C3C=CC=CC3=CC=C2)=O.O=C(O)C(F)(F)F

WALKWJPZELDSKT-UFABNHQSSA-N

InChI=1S/C27H33N3O3S.C2HF3O2/c1-17(2)13-25(27(32)33-3)30-26(31)23-12-11-20(29-15-19(28)16-34)14-24(23)22-10-6-8-18-7-4-5-9-21(18)22;3-2(4,5)1(6)7/h4-12,14,17,19,25,29,34H,13,15-16,28H2,1-3H3,(H,30,31);(H,6,7)/t19-,25+;/m1./s1

methyl (2S)-2-[[4-[[(2R)-2-amino-3-sulfanylpropyl]amino]-2-naphthalen-1-ylbenzoyl]amino]-4-methylpentanoate;2,2,2-trifluoroacetic acid

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: 2.5 mg/mL (4.21 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 25.0 mg/mL澄清DMSO储备液加入到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: 2.5 mg/mL (4.21 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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: ≥ 2.5 mg/mL (4.21 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 25.0 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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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。