SC79

Akt

合成化合物SC79抑制PHAKT-GFP质膜易位，但增强细胞质溶胶中Akt的磷酸化和活化。 SC79增强Akt三种亚型的磷酸化，并在多种细胞类型中提高Akt的活化。 SC79特异性增强Akt磷酸化和激活受体酪氨酸激酶和gpcr介导的信号传导。 SC79直接结合Akt并将其转化为更容易被上游激酶磷酸化的活性构象。 SC79减少神经元兴奋毒性并防止中风引起的神经元死亡。[1]

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在 HEK293、HeLa、HL60、NB4 和 HsSulton（B 细胞）细胞中，SC79 减少 PHAKTM-GFP 质膜易位并增加所有三种 Akt 亚型的磷酸化。 SC79 减轻神经元兴奋毒性并阻止中风引起的神经元死亡。[1] SC79 增加 MitoSox 阳性细胞产生超氧化物的能力，同时减少 BRAT1 敲低细胞的增殖。 [2]

在永久性局灶性脑缺血的小鼠模型中，SC79（0.04 mg/g，腹腔注射）可激活细胞质中的 Akt，并模拟 Akt 信号传导的主要细胞功能，从而增加神经元存活率。 [1]

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为了确定SC79是否可以导致Akt过度激活并防止完整生物体中谷氨酸介导的神经毒性，我们使用了缺血性卒中模型。研究人员对小鼠进行大脑中动脉闭塞（MCAO），引起闭塞区域大量细胞死亡。大多数中风的临床症状，如瘫痪、失语、视觉障碍和记忆丧失，都是由缺氧-葡萄糖剥夺（OGD）引起的神经元死亡引起的，这是血液流动不足的结果。脑卒中诱发的OGD在受影响的脑区诱导突触间隙谷氨酸的显著升高，进而导致大量兴奋性毒性引起的神经元细胞死亡（27,28）。Akt被认为是治疗中风引起的神经元死亡的潜在靶点（29-32）。与此一致的是，在小鼠中使用SC79进行i.p.预处理可以有效地预防中风诱导的Akt失活（图4 C和D）。因此，SC79可以保护皮质区和纹状体免受兴奋性毒性诱导的脑损伤。SC79的作用是强大的，单剂量SC79, 0.04 mg/g体重（相当于0.5 μM），在MCAO后24小时将新皮质病变大小减少35%，MCAO后1周减少40%以上（图4E）。当多次注入SC79时，观察到更为剧烈的效果（图4F）。

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大鼠围手术期给予SC79（一种选择性Akt激活剂，可透过细胞和血脑屏障）0.05 mg/kg × 3 i.p.或载药i.p.。MCAO 1小时，再灌注2小时后，测定14C-α-氨基异丁酸（14C- aib，分子量104 Da）的传递系数（Ki）和3h -葡聚糖体积（分子量7万Da）分布，测定血脑屏障破坏程度。同一时间点，采用四氮唑染色法测定梗死面积。在另一组大鼠中，给予更高剂量的SC79 （0.5 mg/kg × 3）以确定梗死面积。与未给药的MCAO/再灌注动物相比，SC79增加了缺血再灌注皮质（IR-C, +32%, p < 0.05）和对侧皮质（CC, +35%, p < 0.05）的Ki。SC79对右旋糖酐分布体积无显著影响。SC79治疗显著降低皮质梗死占总皮质面积的百分比（12.7±1.7% vs 6.9±0.9%,p < 0.001）。SC79剂量增加10倍对皮质梗死面积无显著影响。与我们的假设相反，我们的数据表明，尽管血脑屏障破坏增加，但SC79减少了缺血再灌注皮层的梗死面积。我们的数据表明Akt的激活对于治疗窗口内早期脑缺血再灌注神经元存活的重要性，并且神经保护的机制可能与SC79的血脑屏障作用无关。[3]

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研究人员在这里报道，Akt激活剂SC79保护肝细胞免受TNF-α-诱导的凋亡，并保护小鼠免受d-半乳糖(d-Gal)/脂多糖（LPS）诱导的TNF-α-介导的肝损伤和损伤。SC79不仅在TNF-α刺激下增强核因子-κB （NF-κB）促生存信号，而且增加细胞FLICE （FADD-like il -1β-转换酶）抑制蛋白L和S （FLIPL/S）的表达，从而抑制procaspase-8的活化。此外，预处理PI3K/Akt抑制剂LY294002可逆转sc79诱导的所有肝保护作用。这些结果强烈提示SC79对TNF-α-诱导的肝细胞凋亡具有保护作用，提示SC79可能是一种有希望改善肝损伤发展的治疗药物。SC79可保护肝细胞免受TNF-α介导的凋亡，并可保护小鼠免受Gal/ lps诱导的肝损伤和损伤。SC79对TNF-α的细胞保护作用是通过akt介导的NF-κB活化和FLIPL/S上调来实现的。[5]

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先前的研究表明，Akt的激活可能减轻蛛网膜下腔出血（SAH）后的早期脑损伤（EBI）。本研究旨在确定铁代谢是否参与SAH后Akt激活的有益作用。因此，我们使用了一个新的分子SC79来激活实验性SAH大鼠模型中的Akt。将大鼠随机分为sham、SAH、SAH +载药、SAH + SC79 4组。结果证实，SC79有效增强了SAH后颞叶氧化应激的防御，减轻了EBI。有趣的是，我们发现SC79磷酸化Akt可减少SAH后细胞表面转铁蛋白受体介导的铁摄取，并促进铁转运蛋白介导的铁转运。结果表明，SC79降低了脑组织中的铁含量。此外，受损的Fe-S簇生物发生得到恢复，含Fe-S簇酶活性的丧失得到恢复，表明受损的线粒体功能恢复到健康水平。这些发现表明，铁稳态的破坏可能导致EBI， Akt的激活可能调节铁代谢以减轻铁毒性，进一步保护SAH后的神经元免受EBI。［6］

Hela 细胞血清饥饿 1 小时，并用 IGF (100ng/mL) 或 SC79 (4 μg/mL) 处理 30 分钟。将蛋白酶抑制剂添加到裂解缓冲液中，其中含有 250 mM 蔗糖、20 mM HEPES、10 mM KCl、1.5 mM MgCl2、1 mM EDTA 和 1 mM EGTA。用 25G 针多次穿过细胞，然后将其置于冰上 20 分钟。此时，收集整个细胞裂解物。将细胞裂解物以 100,000 g 离心 30 分钟。通过收集上清液获得胞质级分。代表膜部分的沉淀在裂解缓冲液中洗涤。 SDS-PAGE 用于分离总细胞裂解物、胞质和膜部分。然后使用蛋白质印迹法检查磷酸 Akt (S473)、总 Akt、微管蛋白（胞质标记物）和 Orai1（膜标记物）的存在。

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Akt质膜易位抑制剂的筛选。为了验证我们基于细胞的高通量筛选方法，我们首先使用生物活性化合物文库（大约3000种化合物）进行了中试筛选。筛选是在哈佛医学院化学和细胞生物学研究所（ICCB）进行的。实验的每一步都在高吞吐量模式下进行（图S3）。根据我们的初步数据，我们将血清饥饿（0.1%血清）的细胞与化合物一起培养30分钟，然后用IGF诱导PHAkt-GFP膜易位。我们的目标是鉴定直接抑制PtdIns(3,4,5)P3/Akt信号通路的化合物。因此，我们选择使用较短的孵育时间来排除间接阻断GFP-PH膜易位的化合物（例如，通过影响转录或翻译）。从先导筛选中，我们鉴定出21种阳性击中化合物（图S4和表S1）。正如预期的那样，几种已知的PI3激酶抑制剂和非特异性抑制PI3激酶活性的化合物被鉴定为阳性命中化合物，验证了我们的高通量筛选策略和方法。然后我们使用几个合成化合物文库进行了高通量筛选。ICCB化合物来自多种来源，包括商业文库、面向多样性的有机合成文库、已知的生物活性化合物和由不同合成策略产生的历史化合物。当ICCB购买化合物文库时，他们选择了富含复杂杂环化合物和高分子量化合物（平均分子量为350-400道尔顿）的集合，因为这些类型的化合物更有可能在高通量筛选中提供有趣的结果。此外，他们还试图减少潜在“有害”化合物的数量，即那些含有可能使它们不稳定或有毒的基团的化合物。特别是，它们消除了不稳定的亚胺，含有游离羧基的化合物，以及含有可能螯合金属的构建块元素的化合物。表S2是用于当前筛选的文库列表，其中包括6万多种合成化合物。高通量筛选进行了两次，以尽量减少假阳性击中化合物的数量。从第一次筛选中，我们确定了大约446个阳性命中化合物，其中125个在第二次筛选中被确认（表S3和图S5）。我们后来发现，其中25种阳性化合物可以产生自荧光，它们对PH-Akt膜转运的影响实际上是背景荧光大大增强的结果（表S3） [1]

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通过延时荧光成像确认阳性命中。在这项研究中，筛选了6万多种化合物，很难滴定出每种化合物的最佳浓度。以5 mg/ml的浓度保存于DMSO中。在我们的筛选中，将100 nl的化合物原液转移到50µl的测定体积中，得到500道尔顿化合物的最终浓度为20µM。这是工商银行通常使用的浓度。我们筛选试验的一个潜在问题是，由于孵育时间相对较短，转移的化合物可能无法在每个孔中均匀扩散。因此，某些正打击化合物对PH-Akt膜易位的影响可能是局部浓度非常高的结果。为了选择最有效的化合物进行进一步的表征，我们使用活细胞在35毫米板上培养进行了剂量范围实验。第二次筛选后确定的125种阳性命中化合物（图S5）是从几家公司购买的。新鲜原液用DMSO （5 mg/ml）新鲜配制。将原液直接加入培养基中，得到3种不同的终浓度（4、8、16µg/ml）。为了延时活细胞成像，将HeLa-PH-EGFP细胞镀于35mm玻璃底皿中，培养24 - 48小时。将细胞在2ml Leibovitz L15培养基中血清饥饿1至2h，然后用1ml新鲜的含所需浓度的每种化合物的无血清Leibovitz 3l15培养基替换培养基。预孵育30 min后，加入IGF1 (5 ng/mL)，在40×油物镜下每5 ~ 10 min拍照。测定细胞膜与邻近细胞质的相对荧光强度。在16µg/ml或低于16µg/ml时，对PH-EGFP膜易位抑制大于75%的化合物被鉴定并指定为确认命中。55个已确认命中化合物的代表性实时图像和结构如图S6和图S7所示。[1]

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圆二色性（CD） [1]
远紫外CD （260-195 nm）在氮气净化的Jasco-810分光偏光计上于25°C下进行。数据采集在1 mm石英试管中，带宽为1 nm，响应时间为2 s，扫描速度为10 nm/min，扫描4次。纯n端6his标记的重组全长人Akt1购自Millipore （www.millipore.com/catalogue/item/14-279）。采用化学合成纯化的SC79。蛋白质和配体样品在50 mM Tris pH 7.5, 150 mM NaCl中制备。Akt1与SC79在37°C下孵育30分钟。数据集一式两份。二级结构百分比的测定采用程序K2D （http://www.embl.de/~andrade/k2d.html）和K2D2 （http://www.ogic.ca/projects/k2d2/）。

将 HsSultan 或 NB4 细胞 (2.5 × 105) 接种于 24 孔板中，加入 500 μL 不含酚红的 RPMI 培养基（补充有 10% FBS）。每种化合物 (8 µg/mL) 在孵育 24 小时后添加，然后培养过夜（16-20 小时）。每个孔接受 50 微升 MTT 溶液（PBS 中 5 mg/mL）。孵育2小时后，将500 L异丙醇和0.1 M HCl直接添加到每个孔中以溶解紫色甲臜晶体。在570 nm波长处，离心去除细胞碎片后测量吸光度。

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western blotting分析SC79诱导的Akt胞质磷酸化。[1]
将Hela细胞血清饥饿1小时，然后用IGF （100ng/ml）或SC79（4µg/ml）处理30分钟。细胞在含有250 mM蔗糖、20 mM HEPES、10 mM KCl、1.5 mM MgCl2、1 mM EDTA、1 mM EGTA并添加蛋白酶抑制剂的裂解缓冲液中裂解。25G针多次传代细胞，冰敷20分钟。此时取细胞总裂解液。细胞裂解液以100,000g离心30分钟。收集上清液作为细胞质组分。球团用裂解缓冲液洗涤，代表膜分数。用SDS-PAGE分离细胞总裂解液、胞浆和膜组分，用western blotting分析磷酸化Akt （S473）、总Akt、微管蛋白（胞浆标记物）和Orai1（膜标记物）。

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MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑）细胞活力测定。[1]
将HsSultan或NB4细胞（2.5 × 105）在500 μL添加10%胎牛血清的无酚红RPMI培养基中接种于24孔板。孵育24小时后，加入每种化合物（8µg/ml），培养过夜（16-20 h）。每孔加入50微升MTT溶液（PBS中5 mg/ ml）。孵育2 h后，每孔中直接加入500 μL异丙醇和0.1 M盐酸，将紫色甲醛晶体溶解。离心清除细胞碎片后，在570nm波长处测定吸光度。

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神经元细胞培养与细胞毒性[1]
原代皮层或海马神经元的培养如前所述。为了诱导兴奋毒性，用Trisbuffered对照盐（CSS）溶液（120 mM NaCl, 5.4 mM KCl, 1.8 mMCaCl2, 25 mM Tris-HCl, pH 7.4和15 mM葡萄糖）对细胞进行预洗，并用含有50µM谷氨酸的CSS处理40分钟，然后在常规培养基中恢复4小时。在谷氨酸治疗前15 min和治疗期间给予SC79(4 μg/ml)。用计算机辅助细胞计数显微镜检查谷氨酸暴露4 h后的毒性。用Hoechst （0.5 ng/ml）和碘化丙啶（1 μg/ml）染色，分别测定总细胞和死亡细胞。孵育20 min后，在360 nm荧光显微镜下观察细胞。细胞死亡以死亡总细胞数之比确定，计数1000个细胞。

Permanent focal cerebral ischemia mouse model
0.04 mg/g
i.p.

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Permanent focal cerebral ischemia model [1]
The permanent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) essentially as described previously(11). Briefly, mice (C57 Black/6) weighing 17–25 g were anesthetized with 4% isoflurane/66% N2O/30% O2 and maintained with 1.5% isoflurane. Permanent focal ischemia was achieved as follows: a 2-mm hole was drilled at a site superior and lateral to the left foramen ovale to expose the left middle cerebral artery. The proximal portion of the left middle cerebral artery (MCA) was permanently occluded over a 1-mm segment distal to the origin of the lenticulostriate branches through the use of a bipolar coagulator. SC79 was injected intraperitoneally (0.04 mg/g mouse body weight) 5 min before permanent MCAO (Figure 4E). In another experiment, extra SC79 was injected (0.04 mg/g mouse body weight, once per hour for 6 hours) (Figure 4F).

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Akt activation in the brain assessed by immunohistochemistry [1]
The mouse brains were perfused from the apex of the heart with PBS and perfusion-fixed with 4% paraformaldehyde in PBS. They were then immersion-fixed overnight at 4°C in 4% paraformaldehyde with rocking and subsequently cryoprotected in 10% (2 hours), 15% (2 hours), 20% (2 hours), and 25% (overnight) sucrose in PBS at 4°C. The slices were then embedded in OCT compound and quickly frozen in isopentane. Coronal frozen sections (10-µm) were prepared on a cryostat and stored at -80 °C until use. The frozen sections were thawed, washed three times in PBS, permeabilized with 0.1% Triton X-100/PBS at room temperature for 5 min, and then blocked in 5% skim milk/3% BSA/PBS for 60 min. Total and phosphorylated Akt/PKB were detected using anti-Akt and anti-Phospho-Akt (Ser473) antibodies, respectively. The slides were incubated with primary antibodies (1:200) at 4 °C overnight, and with the secondary antibodies at room temperature for 2 h, and immunoreactivity visualized by the ABC method.

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Twenty-eight male Fischer 344 rats weighing 220–250 g were used. They were randomly divided into two groups, 14 rats in each group: (1) MCAO/reperfusion, (2) SC79 + MCAO/reperfusion. For the SC79 + MCAO/reperfusion groups, three doses of 0.05 mg/kg of SC79 dissolved in 5% DMSO in normal saline were administered i.p. 10 min before transient middle cerebral artery (MCA) occlusion, upon reperfusion, and one hour after reperfusion. For the MCAO/reperfusion group, at each injection time point, the same volume of vehicle was administered. In each group, 8 rats were used for determination of BBB permeability parameters and 6 rats were used to determine the size of infarcts. In an additional group of rats (SC79H + MCAO/reperfusion, n = 6), ten times the SC79 dose, 0.5 mg/kg × 3 was administered to determine the size of infarct and confirm the effects of higher dose SC79 on neuronal survival. All rats were ventilated through a tracheal tube with 2% isoflurane in an air-oxygen mixture for MCA occlusion. [3]

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Male, age-matched (6- to 8-week–old) C57BL/6 or BALB/c mice (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) weighing 16 to 18 g were used in the animal study. Mice were housed in humidity- and temperature-controlled rooms, with free access to food and water. Mice were pretreated with 10 mg/kg SC79 or dimethyl sulfoxide intraperitoneally at 0.5 hour before the i.p. administration of an agonistic anti-Fas Jo2 antibody at a lethal dose of 0.5 and 0.4 mg/kg for C57BL/6 and BALB/c mice, respectively, within 12 hours. Mouse IgG was used as a control for Jo2. After this lethal challenge, mice were monitored continuously for mortality. For analyses other than mortality, mice were sacrificed at various time points after Jo2 injection. Serum levels of alanine aminotransferase and aspartate aminotransferase were determined using a standard clinical automatic analyzer (model 7020; Hitachi, Kyoto, Japan). Immediately after the blood samples were obtained retro-orbitally, mice were sacrificed by cervical dislocation. The excised liver mass was sectioned, fixed overnight at 4°C in 10% formalin solution, dehydrated, paraffin embedded, cut at 3-mm thickness, and stained with hematoxylin and eosin for histologic examination. Liver tissues were extracted, immediately snap-frozen using liquid nitrogen, and stored at −80°C until analyzed. [4]

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Male, age-matched (6 to 8 wk old) C57BL/6 mice weighing 16–18 g were used in the study. Mice were housed in humidity- and temperature-controlled rooms, with free access to food and water. Ten mice from each group were pretreated intraperitoneally with 10 mg/kg SC79 or DMSO at 0.5 h before administration of 400 mg/kg ip of d-galactosamine (d-Gal) and 60 µg/kg of LPS for C57BL/6 mice. PBS was used as a control for d-Gal/LPS. Mice were euthanized at 12 h after Gal/LPS injection. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were determined using a standard clinical automatic analyzer. Immediately after taking the blood samples retroorbitally, mice were euthanized by cervical dislocation. The excised liver mass was sectioned, fixed overnight at 4°C in 10% formalin solution, dehydrated, paraffin-embedded, cut at 3-mm thickness, and stained with hematoxylin and eosin for histological examination. Liver tissues were extracted, immediately snap-frozen using liquid nitrogen, and stored at −80°C until analyzed. [5]

[1]. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10581-10586.

[2]. BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction. BMC Cancer. 2014 Jul 29;14:548.

[3]. Activation of Akt by SC79 decreased cerebral infarct in early cerebral ischemia-reperfusion despite increased BBB disruption. Neurosci Lett. 2018 Aug 10;681:78-82.

[4]. A Novel AKT Activator, SC79, Prevents Acute Hepatic Failure Induced by Fas-Mediated Apoptosis of Hepatocytes. Am J Pathol. 2018 May;188(5):1171-1182.

[5]. AKT activator SC79 protects hepatocytes from TNF-α-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury. Am J Physiol Gastrointest Liver Physiol. 2019 Mar 1;316(3):G387-G396.

[6]. Phosphorylation of Akt by SC79 Prevents Iron Accumulation and Ameliorates Early Brain Injury in a Model of Experimental Subarachnoid Hemorrhage. Molecules. 2016 Mar 10;21(3):325.

One caveat of using Akt activator as a drug for neurological disorders is that hyperactivation of Akt signaling may induce cancer. Nevertheless, induction of cancer by elevating PtdIns P3/Akt signaling is a progressive process and usually takes several months or even years. For example, in myeloid-specific PTEN knockout mice, we could not find any tumor until 3 months after the birth. When used as a suppressor of neuronal death caused by glutamate-excitotoxicity, Akt activator will only be given for several days, even several hours; thus it is unlikely that this type of treatment will lead to tumorigenesis. Interestingly, it was recently reported that activation of Akt1 decreases mammary epithelial cell migration, and Akt1 prevents an epithelial-to-mesenchymal transition that resembles events required for metastasis. Another report showed that in some acute myeloid leukemia (AML), activation of Akt surprisingly reduced leukemic cell growth by inhibiting FOXO, suggesting that Akt activator can even potentially be used to treat certain cancers. Akt is also a key enzyme involved in other processes such as cell migration, immune cell activation, embryonic development, hematopoetic and mesenchymal differentiation, and glucose homeostasis, thus SC79 may potentially be used to modulate cell function in other physiological and pathological situations such as wound healing, host defense, and blood glucose control in diabetes. For example, SC79 may have a potential benefit in regulating glucoregulatory responses and insulin sensitivity in type 1 and 2 diabetes. Phosphorylation and deactivation of GSK3b promotes glycogen synthesis resulting in decreased blood glucose. Akt-mediated GLUT4 translocation mediates glucose transport. GSK3b and FOXO also play a role in expression of genes in gluconeogenesis like G6Pase and PEPCK(4). In innate immunity, activating neutrophil functions by elevating PI3K/Akt pathway using PTEN inhibitor has been previously reported. SC79 may also offer similar effect by directly activating Akt. In addition, SC79 may also be effective in preventing myocardial infarction in heart attack, in which the acquired resistance to apoptosis is mediated at least in part by the sustained activation of Akt. Use of SC79 could exert a wide range of cardio-protective effects in myocardial ischemia/reperfusion-induced injury, myocardial hypertrophy, hypertension and vascular diseases by suppressing cell death and inducing angiogenesis by regulating eNOS.[1]

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Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.[1]

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Background: BRAT1 (BRCA1-associated ATM activator 1) interacts with both BRCA1, ATM and DNA-PKcs, and has been implicated in DNA damage responses. However, based on our previous results, it has been shown that BRAT1 may be involved in cell growth and apoptosis, besides DNA damage responses, implying that there are undiscovered functions for BRAT1. Methods: Using RNA interference against human BRAT1, we generated stable BRAT1 knockdown cancer cell lines of U2OS, Hela, and MDA-MA-231. We tested cell growth properties and in vitro/in vivo tumorigenic potentials of BRAT1 knockdown cells compared to control cells. To test if loss of BRAT1 induces metabolic abnormalities, we examined the rate of glycolysis, ATP production, and PDH activity in both BRAT1 knockdown and control cells. The role of BRAT1 in growth signaling was determined by the activation of Akt/Erk, and SC79, Akt activator was used for validation. Results: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo. Cell migration was also remarkably lowered when BRAT1 was depleted. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown HeLa cells. Furthermore, both basal and induced activity of Akt and Erk kinases were suppressed in these cells, implicating abnormality in signaling cascades for cellular growth. Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration.[2]

C17H17CLN2O5

364.78

364.082

C, 55.98; H, 4.70; Cl, 9.72; N, 7.68; O, 21.93

305834-79-1

2810830

White to off-white solid powder

1.3±0.1 g/cm3

524.8±50.0 °C at 760 mmHg

271.2±30.1 °C

0.0±1.4 mmHg at 25°C

1.564

3.23

112

1

7

7

25

611

0

ClC1C([H])=C([H])C2=C(C=1[H])C([H])(C(C(=O)OC([H])([H])C([H])([H])[H])=C(N([H])[H])O2)C([H])(C#N)C(=O)OC([H])([H])C([H])([H])[H]

DXVKFBGVVRSOLI-UHFFFAOYSA-N

InChI=1S/C17H17ClN2O5/c1-3-23-16(21)11(8-19)13-10-7-9(18)5-6-12(10)25-15(20)14(13)17(22)24-4-2/h5-7,11,13H,3-4,20H2,1-2H3

ethyl 2-amino-6-chloro-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate

SC79; SC-79; 305834-79-1; SC79; ethyl 2-amino-6-chloro-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; SC 79; SC-79; 4H-1-Benzopyran-4-acetic acid, 2-amino-6-chloro-alpha-cyano-3-(ethoxycarbonyl)-, ethyl ester; 2-amino-6-chloro-alpha-cyano-3-(ethoxycarbonyl)-4h-1-benzopyran-4-acetic acid ethyl ester; MFCD02681303; SC 79

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 该产品在溶液状态不稳定，请现配现用。

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

DMSO: 72 mg/mL (~197.4 mM)
Water: <1 mg/mL
Ethanol: 72 mg/mL (~197.4 mM)

配方 1 中的溶解度: 5 mg/mL (13.71 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: ≥ 2.5 mg/mL (6.85 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 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 3 中的溶解度: ≥ 2.5 mg/mL (6.85 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 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 4 中的溶解度: ≥ 2.5 mg/mL (6.85 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900 μL 玉米油中，混合均匀。

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配方 5 中的溶解度: 2% DMSO+corn oil: 5mg/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。