GANT61

GLI1 ( IC50 = 5 μM )

体外活性：GANT61 是 GLI1 以及 GLI2 诱导转录的抑制剂。 GANT61 抑制 GLI1 的 DNA 结合能力。 GANT61 抑制 Hedgehog 信号传导，IC50 为 5 μM，对其他途径表现出选择性，例如 TNF 信号传导/NFκB 激活、糖皮质激素受体基因反式激活和 Ras-Raf-Mek-Mapk 级联。 GANT61 以 GLI 依赖性方式有效抑制体外肿瘤细胞增殖。 GANT61 诱导慢性淋巴细胞白血病细胞 (CLL) 凋亡，但不诱导正常 B 淋巴细胞凋亡。 GANT61 在人结肠癌细胞系中诱导强大的细胞毒性并消除克隆形成。 GANT61 诱导人结肠癌细胞系早期 S 期 DNA 复制的抑制，导致涉及 ATM-Chk2 轴的 DNA 损伤信号传导并诱导细胞死亡。 GANT61 (30 μM) 导致急性髓系白血病 (AML) 细胞生长停滞和凋亡。激酶测定：在 10 cm 平板上用 GLI1 表达质粒以及报告质粒 12×GliBSLuc 和 R-Luc 转染 HEK293 细胞（第 0 天）。 24小时后，将细胞以每孔15,000个细胞的密度接种到底部透明的白色96孔板中。让细胞贴壁，并以终浓度为 10 μM 的 DMSO（0.5% DMSO 最终浓度）添加化合物（第 1.5 天）。细胞再生长 24 小时，随后裂解，然后使用双荧光素酶试剂盒进行分析。细胞测定：BrdU 掺入测定。在 5 μM 测试化合物（或 DMSO）存在下，在透明底部的白色 96 孔板中，将亚汇合细胞在减少的 FBS (2.5%) 中生长 48 小时。随后，用 BrdU 标记细胞 2 小时，固定并分析。

在注射 GLI1 阳性 22Rv1 前列腺癌细胞的裸鼠中，GANT61 会诱导生长衰退，直到摸不到肿瘤为止。在携带 SK-N-AS 神经母细胞瘤异种移植物的裸鼠中，GANT61 治疗（口服灌胃，50 mg/kg）在第 12 天显着抑制肿瘤生长，与对照组相比，肿瘤体积减少至 63%。

ChIP分析[4]
HT29细胞用GANT61（20µM）处理1小时或24小时，根据制造商的方案，使用Gli1或Gli2抗体和Abcam ChIP试剂盒进行ChIP分析。详细信息见补充材料和方法。

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萤光素酶报告物测定[4]
Gli荧光素酶报告构建体之前已经描述过。NF-κB-萤光素酶质粒p5XIP10κB之前已有报道。AP1萤光素酶包含一个与AP1结合元件的串联重复连接的基本启动子元件（TATA盒）。如所述，在GANT61（20µM）处理后24小时，用萤光素酶报告基因进行了24小时的瞬时转染。

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RT-PCR[4]
HT29细胞用GANT61（20µM）处理1小时，然后分离RNA长达4小时。转化为cDNA后，样品用于qPCR，如前所述。

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在10cm平板上（第0天），用GLI1表达质粒以及报告质粒12×GliBSLuc和R-Luc转染HEK293细胞。24小时后，将细胞以每孔15000个的密度接种在白色透明96孔板上。在允许细胞附着后（第1.5天），将化合物以10μM的DMSO终浓度（0.5%DMSO终浓度）加入细胞中。在细胞再生长24小时后，将其裂解，并使用双荧光素酶试剂盒进行分析。

克隆形成试验[3]
将细胞以1500个（HT29、HCT8、HCT116）和3000个（SW480、GC3/c1、VRC5/c1）细胞/孔的密度铺在6孔板中。贴壁过夜后，用不同浓度的GANT61（0-20μM）处理细胞，一式三份，持续72小时。取出药物，用含有dThd（20μM）的新鲜培养基替换，持续时间相当于7次细胞倍增（HT29、SW480、GC3/c1和VRC5/c1为7天；HCT8和HCT116为5天）。用1X Dulbecco's PBS（不含Ca++或Mg++）洗涤细胞，并使其干燥过夜。第二天，用结晶紫对细胞进行染色，并使用Alpha Innotech成像仪分析菌落。

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细胞周期分布、双变量流式细胞术分析和BrdU掺入[4]
对于细胞周期分布和双变量流式细胞术，如前所述分析细胞。为了分析BrdU掺入，将细胞以6孔格式铺板（50000个细胞/孔），并用GANT61（20µM）或环胺（20µM）处理长达48小时。用BrdU（10µM）脉冲细胞30-45分钟，并根据制造商的方案通过流式细胞术分析细胞周期内的分布。

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共聚焦显微镜[4]
将细胞（50000/孔）镀在6孔板的盖玻片上。用GANT61（20µM）或环胺（20µM）处理细胞长达48小时，并进行显微镜处理。显微镜的细节在补充材料和方法中进行了描述。

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BrdU掺入试验。在底部透明的白色96孔板上，亚融合细胞在还原的FBS（2.5%）中生长48小时，同时暴露于5μM的测试化合物（或DMSO）中。然后固定细胞，用BrdU标记两个小时，并进行检查。

[1]. Proc Natl Acad Sci U S A . 2007 May 15;104(20):8455-60.

[2]. Oncogene . 2010 Sep 2;29(35):4885-95.

[3]. Cancer Res . 2011 Feb 1;71(3):1092-102.

[4]. Cancer Res . 2011 Sep 1;71(17):5904-14.

[5]. Leuk Res . 2012 Jun;36(6):742-8.

[6]. Int J Cancer . 2013 Apr 1;132(7):1516-24.

GANT61 is an aminal that is hexahydropyrimidine which is substituted on each nitrogen by a 2-(dimethylamino)benzyl group, and at the aminal carbon by a pyridin-4-yl group. A Hedgehog signaling pathway and Gli protein inhibitor. It has a role as a Hedgehog signaling pathway inhibitor, a glioma-associated oncogene inhibitor, an antineoplastic agent and an apoptosis inducer. It is a tertiary amino compound, a member of pyridines, a substituted aniline and an aminal.

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The developmentally important Hedgehog (Hh) signaling pathway has recently been implicated in several forms of solid cancer. Current drug development programs focus on targeting the protooncogene Smoothened, a key transmembrane pathway member. These drug candidates, albeit promising, do not address the scenario in which pathway activation occurs downstream of Smoothened, as observed in cases of medulloblastoma, glioma, pericytoma, breast cancer, and prostate cancer. A cellular screen for small-molecule antagonists of GLI-mediated transcription, which constitutes the final step in the Hh pathway, revealed two molecules that are able to selectively inhibit GLI-mediated gene transactivation. We provide genetic evidence of downstream pathway blockade by these compounds and demonstrate the ineffectiveness of upstream antagonists such as cyclopamine in such situations. Mechanistically, both inhibitors act in the nucleus to block GLI function, and one of them interferes with GLI1 DNA binding in living cells. Importantly, the discovered compounds efficiently inhibited in vitro tumor cell proliferation in a GLI-dependent manner and successfully blocked cell growth in an in vivo xenograft model using human prostate cancer cells harboring downstream activation of the Hh pathway.[1]

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The Hedgehog (Hh) pathway regulates cell proliferation and survival and contributes to tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and in healthy B lymphocytes. Profiling of cognate Hh pathway members revealed reduced expression of two key Hh signaling effectors, Smoothened (SMOH) and GLI, in CLL cells, whereas transcription levels of other investigated members resembled normal B-lymphocyte levels. Examining the functional role of SMOH and GLI in cell survival, we found that CLL cells were hardly sensitive toward specific SMOH inhibition, but showed an unspecific decline in cell viability in response to high concentrations of the SMOH antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT61 reduced expression of the target gene Patched and preferentially decreased the viability of malignant cells. Specific RNA interference knockdown experiments in a CLL-derived cell line confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemic cells was partly attenuated by protective stromal cells, but not soluble sonic hedgehog ligand. In summary, our data show a downregulation of the classical Hh pathway in CLL and suggest an intrinsic SMOH-independent role of GLI in the ex vivo survival of CLL cells.[2]

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Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.[3]

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Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.[4]

C27H35N5

429.6

429.289

C, 75.49; H, 8.21; N, 16.30

500579-04-4

421610

White to off-white solid powder

1.1±0.1 g/cm3

549.0±50.0 °C at 760 mmHg

285.8±30.1 °C

0.0±1.5 mmHg at 25°C

1.632

3.53

25.85

0

5

7

32

508

0

N1(C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])[H])C([H])([H])[H])C1([H])C1C([H])=C([H])N=C([H])C=1[H]

KVQOGDQTWWCZFX-UHFFFAOYSA-N

InChI=1S/C27H35N5/c1-29(2)25-12-7-5-10-23(25)20-31-18-9-19-32(27(31)22-14-16-28-17-15-22)21-24-11-6-8-13-26(24)30(3)4/h5-8,10-17,27H,9,18-21H2,1-4H3

2-[[3-[[2-(dimethylamino)phenyl]methyl]-2-pyridin-4-yl-1,3-diazinan-1-yl]methyl]-N,N-dimethylaniline

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: ≥ 5 mg/mL (11.64 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 50.0 mg/mL 澄清乙醇储备液加入到 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: 5 mg/mL (11.64 mM) in 10% EtOH + 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 50.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 5 mg/mL (11.64 mM) (饱和度未知) in 10% EtOH + 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 50.0 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (5.82 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 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 5 中的溶解度: 2.5 mg/mL (5.82 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100μL 25.0mg/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 6 中的溶解度: ≥ 2.5 mg/mL (5.82 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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配方 7 中的溶解度: ≥ 2.5 mg/mL (5.82 mM) (饱和度未知) 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), 澄清溶液。
*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 8 中的溶解度: 2.5 mg/mL (5.82 mM) in 5% DMSO + 95% (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。
*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 9 中的溶解度: 5% DMSO+95% Corn oil: 30 mg/mL

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配方 10 中的溶解度: 8 mg/mL (18.62 mM) in Cremophor EL (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.

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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。