Kobe0065   中文名称: N-(3-氯-4-甲基苯基)-2-[2,6-二硝基-4-(三氟甲基)苯基]-肼硫代甲酰胺

Ras-Raf interaction; H-Ras·GTP (Ki = 46±13 μM)

Kobe0065 家族的化合物与 Ras·GTP 结合，并对激活 Ras 癌基因的癌细胞显示出抗增殖作用。这些化合物在细胞水平上的更大效力可能是由于它们对不同 Ras 家族小 GTP 酶具有相当广泛的结合特异性，如它们有效抑制 Ras·GTP 与其多种效应子（如 Raf、PI3K、和 RalGDS，以及称为 Sos 的调节器/效应器。在瞬时表达 H-RasG12V 的 NIH3T3 细胞中，下游激酶 MEK 和 ERK 的磷酸化可被 20 μM Kobe0065 和 Koho2602 有效抑制[2]。

口服Kobe0065和Kobe2602已被证明对含有K-ras(G12V)基因的人结肠癌SW480细胞异种移植物具有抗癌活性[1]。

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甘草次酸（GA）和Kobe0065 (Kobe)抑制小鼠异种移植瘤模型的肿瘤生长[3]
利用裸鼠A549细胞的异种移植来评估GA的抗肿瘤活性。与GA处理的小鼠相比，模型组的肿瘤大小显着增加。高剂量GA抑制肿瘤生长50%-60%，与Kobe0065 (Kobe)处理的动物相似。为了进行组织学分析，肿瘤组织切片用血红素和伊红（H&E）染色。与模型组比较，高剂量GA和神户组肿瘤细胞核坏死、固缩程度明显改善。肿瘤组织的免疫组化（IHC）显示，在GA治疗的反应中，ERK1/2磷酸化显著降低。此外，我们检测了GA-和Kobe0065 (Kobe)-处理组肿瘤组织中phospho-c-RAF、phospho-B-RAF和phospho-ERK1/2的表达。GA高剂量组c-RAF Ser259位点、B-RAF Ser729位点和Thr401位点、ERK1/2位点磷酸化水平降低。

生化分析[1]
H-Ras（残基1-166）和GST-c-Raf-1-Rasbinding domain (RBD；残基50-131)在大肠杆菌中产生并按照前面描述的方法纯化。在体外结合抑制实验中，将预载[γ35S] gtp - γ s的H-Ras（1-166）与GST-c-Raf-1-RBD（50-131）在25°C下孵育30分钟，并通过谷胱甘肽-sepharose树脂降低放射性来定量结合的H-Ras的数量。化合物的Ki值计算如图S1所示。在体内实验中，NIH 3T3细胞转染pEF-BOS-HA-H-RasG12V或pEF-BOS-HAK-RasG12V，在10% (vol/vol) FBS中培养18 h，然后在2% (vol/vol) FBS中培养1 h。细胞在50 mM Tris·HCl （pH 7.4）、150 mM NaCl、1% Nonidet P-40、10% （vol/vol）甘油、1 mM EDTA、1 mM DTT、磷酸酶抑制剂混合物中裂解，和蛋白酶抑制剂混合后，用抗h - ras抗体（C-20）和抗craft -1抗体（C-12）检测c-Raf-1共免疫沉淀，用抗pmek1 /2 （p217/p221）和抗erk1 /2 （p202/204）抗体检测磷酸化的MEK和ERK，用抗pakt抗体（Ser473）检测磷酸化的Akt，用抗RalA抗体固定在谷胱甘肽-sepharose树脂上用GST-Sec5（1-99）拉低的RalA·GTP。用抗ha抗体检测ha标记的H-RasG12V·GTP。体外检测重组c-Raf-1的激酶活性，采用Raf-1激酶检测试剂盒。体外GDP - gtp交换实验通过在25°C下培养600 nM GST-H-Ras（1 - 166）·GDP固定在谷胱甘肽-sepharose树脂上，11 μM [γ - 35s] gtp - γ -s (1,500 cpm/pmol)，纯化的6×His-tagged小鼠Son of sevenless (mSos)1(563-1,049)（每个180 nM），野生型或W729E突变体(5)在缓冲液B [50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT和20 mM咪唑]中进行。用液体闪烁计数法定量了树脂经剧烈洗涤后残留的放射性。在反应混合物中加入不同浓度的化合物，观察其抑制效果。

菌落形成分析[1]
将细胞（103 ~ 104）接种于2 mL含10% (vol/vol) FBS、0.33% SeaPlaque琼脂糖和其中一种化合物的DMEM中，并覆盖在底部琼脂上，该琼脂由4 mL含10% (vol/vol) FBS、0.6% SeaPlaque琼脂糖的DMEM和相同浓度的化合物组成，并在六孔培养板中培养。37℃孵育14 ~ 21 d后，在解剖显微镜下计数直径>200 μm的菌落数。

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细胞增殖试验[1]
细胞（2 × 103）接种于96孔板中，在含有2% (vol/vol) FBS的DMEM中培养，其中一种化合物存在。使用细胞计数试剂盒8通过形成甲醛来测定活细胞数。采用原位细胞检测试剂盒，采用标准TUNEL法检测凋亡细胞。

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细胞周期分析[3]
A549细胞与一定浓度的GA、Kobe0065和Tip孵育24 h。然后，收获细胞，在- 20 °C的低温条件下，将细胞固定在70% （v/v）乙醇中过夜。将细胞颗粒重悬于PBS中，用含有0.5% Triton X-100的柠檬酸钠中RNase（10 μg/mL）和PI（25 mg/mL）的混合物在黑暗中染色30 min。用流式细胞仪测定荧光

Xenografts in nude mice[3]
Six-week-old female BALB/c nude mice (specific pathogen-free [SPF]) were usedStudies using experimental animals were performed according to protocols approved by Nankai University Ethics Committee on Pre-Clinical Studies. A549 cells (1 × 107) were subcutaneously (s.c.) injected into the right flanks of the mice. The mice were randomly assigned to 4 groups (n = 6 mice per group): two groups were intragastrically (i.g.) administered GA (50 or 100 mg/kg) daily for 40 days, and one group was i.g. administered Kobe0065 (80 mg/kg) daily. Tumour volumes (V) were calculated using previously described methods.

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Tumor Xenografts. [1]
Cells (5 × 106 ) were implanted into the right flanks of female athymic nude mice (6–8 wk old). After tumor sizes reached ∼50 mm3 on average, compounds (e.g. Kobe0065) suspended in Cremophor:ethanol:water (1:1:6) were administered orally for five consecutive days per week for 17 d. Tumor volumes (V) were calculated with the following formula: V = A × B2 /2, where A is the largest diameter and B is the perpendicular diameter. Dissected tumors after 17-d administration of the 80 mg/ kg compounds were fixed in 4% (wt/vol) paraformaldehyde and embedded in paraffin. Their sections were subjected to immunohistochemistry with an anti-ERK1/2 antibody or an anti-CD31 antibody using a HISTMOUSE-PLUS kit. Apoptotic cells were detected by a TUNEL assay. Statistical significance for groups of three or more was determined by one-way ANOVA with Tukey’s test for post hoc analysis.

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Formulated in Cremophor:ethanol:water (1:1:6); 80 mg/kg and 160 mg/kg; oral administration

A xenograft of SW480 cells in nude mice.

[1]. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proceedings of the National Academy of Sciences of the United States of America (2013), 110(20), 8182-8187,.

[2]. Discovery of small-molecule Ras inhibitors that display antitumor activity by interfering with Ras·GTP-effector interaction. Enzymes. 2013;34 Pt. B:1-23.

[3].Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins. Acta Pharm Sin B. 2018 Nov 27;9(2):294–303.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.[1]

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Ras proteins, particularly their active GTP-bound forms (Ras·GTP), were thought "undruggable" owing to the absence of apparent drug-accepting pockets in their crystal structures. Only recently, such pockets have been found in the crystal structures representing a novel Ras·GTP conformation. We have conducted an in silico docking screen targeting a pocket in the crystal structure of M-Ras(P40D)·GTP and obtained Kobe0065, which, along with its analogue Kobe2602, inhibits binding of H-Ras·GTP to c-Raf-1. They inhibit the growth of H-rasG12V-transformed NIH3T3 cells, which are accompanied by downregulation of not only MEK/ERK but also Akt, RalA, and Sos, indicating the blockade of interaction with multiple effectors. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma carrying K-rasG12V. The nuclear magnetic resonance structure of a complex of the compound with H-Ras(T35S)·GTP confirms its insertion into the surface pocket. Thus, these compounds may serve as a novel scaffold for the development of Ras inhibitors with higher potency and specificity.[2]

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Members of the RAS proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of Glycyrrhiza plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.[3]

C15H11CLF3N5O4S

449.79

449.017

C, 40.05; H, 2.46; Cl, 7.88; F, 12.67; N, 15.57; O, 14.23; S, 7.13

436133-68-5

3827663

Light yellow to yellow solid powder

1.7±0.1 g/cm3

485.1±55.0 °C at 760 mmHg

247.2±31.5 °C

0.0±1.2 mmHg at 25°C

1.688

6.16

166.86

3

9

3

29

609

0

KSJVAYBCXSURMQ-UHFFFAOYSA-N

InChI=1S/C15H11ClF3N5O4S/c1-7-2-3-9(6-10(7)16)20-14(29)22-21-13-11(23(25)26)4-8(15(17,18)19)5-12(13)24(27)28/h2-6,21H,1H3,(H2,20,22,29)

N-(3-chloro-4-methylphenyl)-2-(2,6-dinitro-4-(trifluoromethyl)phenyl)hydrazinecarbothioamide.

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.56 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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));
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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；
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