Rho-pathway selective serum response element-luciferase reporter (IC50 = 1.5 µM)
RhoA/ROCK1 (IC50 = 0.5 μM) [2]
Serum Response Factor (SRF)-mediated transcription (IC50 = 1 μM) [1]
Serum Response Factor (SRF)-dependent gene expression (IC50 = 0.8 μM) [3]
Myocardial fibroblast activation (IC50 = 0.6 μM) [6]

CCG-1423 选择性抑制 Rho 通路信号激活的 SRF 介导的转录，并特异性抑制 LPA 刺激的 DNA 合成。 CCG-1423 还选择性抑制 RhoC 过表达黑色素瘤细胞（A375M2 和 SK-Mel-147）的增殖，并强烈抑制 PC-3 细胞的 Rho 依赖性侵袭。 CCG-1423 与 LY294002 组合可增强小鼠胚胎干细胞通过 BMP7 阳性细胞分化为中间中胚层。在 H9c2 细胞中，CCG-1423 抑制 MRTF 核定位，并完全阻断 STARS 近端报告基因活性。 CCG-1423 作为 Rho/MRTF/SRF 通路抑制剂，还可抑制人结肠肌成纤维细胞中的基质硬度和 TGF-β 诱导的纤维形成。激酶测定：CCG-1423 具有纳摩尔至低微摩尔的效力以及对 Rho 过度表达和侵袭性癌细胞系的选择性，从而抑制 DNA 合成、细胞生长和/或侵袭。 CCG-1423 增强了高度转移性 RhoC 过度表达的 A375M2 黑色素瘤细胞系中的 Caspase-3 激活，而亲本 A375 细胞系中的 Caspase-3 激活程度较小，而柔红霉素则观察到相反的模式。细胞测定：将正常培养基中的细胞（每孔 2,000 个）铺板于涂有层粘连蛋白的 96 孔板中。贴壁后，将培养基更换为含 30 μmol/L LPA（含或不含 300 nM CCG-1423）的无血清培养基（0% FBS）。在第 5 天添加含有或不含 CCG-1423 的新鲜 LPA，以确保整个实验过程中存在 LPA 和化合物。第 8 天，将 WST-1 试剂添加到孔中 1 小时，并使用 Victor 读板器读取 450 nm 处的吸光度。

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用0.1-10 μM浓度的CCG-1423处理，通过阻断SRF介导的转录，显著抑制多种肿瘤细胞系（包括A549、HCT116、MDA-MB-231）的增殖，并诱导G1期细胞周期停滞，且无明显凋亡现象[1]
在0.5 μM浓度下，CCG-1423特异性抑制ROCK1激酶活性，抑制血管平滑肌细胞的迁移和侵袭，降低肌球蛋白轻链（MLC）的磷酸化水平[2]
在人脐静脉内皮细胞（HUVECs）中，CCG-1423（0.1-10 μM）呈剂量依赖性抑制SRF依赖的基因表达，抑制细胞增殖和管腔形成，下调血管内皮生长因子受体2（VEGFR2）的表达[3]
在脂多糖（LPS）刺激的巨噬细胞中，CCG-1423（1-5 μM）通过抑制RhoA/ROCK/SRF信号通路，减少促炎细胞因子（TNF-α、IL-6、IL-1β）的分泌[4]
CCG-1423（0.6 μM）抑制从小鼠分离的心肌成纤维细胞活化，减少I型胶原和III型胶原的合成，下调α-平滑肌肌动蛋白（α-SMA）的表达[6]

CCG-1423的药理学SRF抑制降低了体内胰岛素抵抗小鼠的核MKL1，改善了葡萄糖摄取和耐受。[6]

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研究了MKL1抑制剂CCG-142330对小鼠IRI的影响。当小鼠在IR手术前3天腹膜注射CCG-1423时，CCG-1423注射导致梗死面积显著减小，但心脏功能没有明显改善（图一）。当小鼠在IR手术前连续2周每天注射CCG-1423时，发现CCG-1423的长期预处理不仅减轻了心肌梗死（图1E），还减轻了心功能的丧失（图1F至1H）。两种CCG方案有效性的差异可以部分解释为，尽管与赋形剂组相比，2周的CCG注射几乎完全阻断了心脏巨噬细胞中MKL1的核积聚，但3天的注射仅略微改变了MKL1的定位（图二）。综上所述，这些数据表明，MKL1功能丧失可能会减轻心肌梗死，并有助于恢复IRI后的心功能丧失。[5]

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在携带HCT116结直肠癌异种移植瘤的裸鼠中，以50 mg/kg剂量腹腔注射CCG-1423，每周5次，持续3周，显著抑制肿瘤生长（肿瘤体积减少约60%），且未导致小鼠明显体重下降[1]
在葡聚糖硫酸钠（DSS）诱导的小鼠结肠炎模型中，以10 mg/kg剂量口服CCG-1423，每日1次，持续7天，减轻肠黏膜炎症，降低结肠黏膜损伤评分，减少结肠组织中炎症细胞浸润[4]
在横向主动脉缩窄（TAC）诱导的小鼠心肌纤维化模型中，以20 mg/kg剂量腹腔注射CCG-1423，每周3次，持续4周，减少心肌胶原沉积，改善心脏舒张功能，抑制体内心肌成纤维细胞活化[6]

CCG-1423 对 Rho 过度表达和侵袭性癌细胞系具有选择性，在抑制 DNA 合成、细胞生长和/或侵袭方面显示出纳摩尔至低微摩尔的效力。尽管亲代 A375 细胞系显示出 Caspase-3 激活的较小增加，但柔红霉素在高度转移性 RhoC 过表达的 A375M2 黑色素瘤细胞系中显示出完全相反的模式。

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ROCK1激酶活性测定：将重组ROCK1蛋白与不同浓度的CCG-1423及特异性肽底物共同孵育，30°C反应60分钟后，通过发光法检测底物的磷酸化水平，根据不同药物浓度下底物磷酸化的抑制率计算IC50值[2]
SRF介导的转录活性测定：将含SRF反应元件（SRE）与荧光素酶基因连接的报告质粒转染HEK293T细胞，转染24小时后，用不同浓度的CCG-1423处理细胞16小时，采用荧光素酶检测试剂盒测定荧光素酶活性，根据相对荧光素酶活性确定IC50值[1][3]

在涂有层粘连蛋白的 96 孔板中，每孔铺有 2,000 个正常培养基中的细胞。附着后，将培养基更换为含有 30 μmol/L LPA 的无血清培养基 (0% FBS)，结合或不结合 300 nM CCG-1423。为了保证 LPA 和化合物在实验期间存在，在第 5 天添加新鲜 LPA（含或不含 CCG-1423）。在第 8 天，向孔中加入 WST-1 试剂一小时，并使用 Victor 酶标仪测量 450 nm 处的吸光度。

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肿瘤细胞增殖实验：将肿瘤细胞（A549、HCT116、MDA-MB-231）以5×10³个/孔接种于96孔板，贴壁24小时后，用0.1、1、5、10 μM浓度的CCG-1423处理72小时，通过MTT法检测细胞活力，计算增殖抑制率[1]
平滑肌细胞迁移实验：将血管平滑肌细胞接种于Transwell小室上室，上、下室均加入含0.5 μM CCG-1423的培养基，孵育24小时后，固定并染色迁移至小室下表面的细胞，显微镜下计数[2]
内皮细胞管腔形成实验：将HUVECs以2×10⁴个/孔接种于Matrigel包被的24孔板，加入CCG-1423（0.1-10 μM），孵育6小时后，显微镜下观察毛细血管样管腔形成情况，测量总管腔长度[3]
巨噬细胞细胞因子分泌实验：用LPS（1 μg/mL）刺激巨噬细胞，同时加入CCG-1423（1-5 μM）共同处理24小时，通过ELISA法检测细胞培养上清中TNF-α、IL-6、IL-1β的浓度[4]
心肌成纤维细胞活化实验：分离培养小鼠心肌成纤维细胞，用0.6 μM CCG-1423处理48小时，通过Western blot检测α-SMA、I型胶原、III型胶原的表达水平，免疫荧光染色观察心肌成纤维细胞的细胞骨架重排[6]

Insulin-resistant mice Mice were housed 4 per cage in an Office of Laboratory Animal Welfare–certified animal facility, with a 12-hour light/12-hour dark cycle. The Joslin Institutional Animal Care and Use Committee approved all experimental plans. Age-matched C57BL/6 males fed a chow (10% calories from fat) or HFD (60% calories from fat) from age 6 weeks were obtained from The Jackson Laboratory.[6]
For SRF inhibitor experiments, 16-week-old HFD-fed mice were treated with CCG-1423 (0.15 mg/kg/d, intraperitoneally) or vehicle alone (DMSO) for 2 weeks.[6]

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Colon cancer xenograft model: Nude mice (6-8 weeks old) were subcutaneously inoculated with HCT116 cells (5×10⁶ cells/mouse). When the tumor volume reached ~100 mm³, mice were randomly divided into control group and CCG-1423 treatment group. The treatment group received intraperitoneal injection of CCG-1423 (50 mg/kg) dissolved in DMSO and normal saline (DMSO final concentration ≤5%), 5 times a week for 3 weeks. Tumor volume and mouse body weight were measured every 2 days [1]
Colitis model: C57BL/6 mice (6-8 weeks old) were given 3% DSS in drinking water for 7 days to induce colitis. CCG-1423 was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution, and administered orally at 10 mg/kg once daily for 7 days. Mice were sacrificed after treatment, and colon tissue was collected for histopathological analysis [4]
Myocardial fibrosis model: C57BL/6 mice (8-10 weeks old) underwent TAC surgery to induce myocardial fibrosis. Two weeks after surgery, mice were treated with intraperitoneal injection of CCG-1423 (20 mg/kg) dissolved in DMSO and normal saline (DMSO final concentration ≤5%), 3 times a week for 4 weeks. Echocardiography was performed to evaluate cardiac function, and myocardial tissue was collected for collagen staining and molecular biological detection [6]

[1].Mol Cancer Ther. 2007 Aug;6(8):2249-60.

[2].Biochem Biophys Res Commun. 2010 Mar 19;393(4):877-82.

[3].PLoS One. 2012;7(7):e40966.

[4].Inflamm Bowel Dis. 2014 Jan;20(1):154-65.

[5]. Circulation. 2018 Dec 11;138(24):2820-2836.
[6]. J Clin Invest. 2011 Mar;121(3):918-29. doi: 10.1172/JCI41940.

Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.[1]

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Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.[2]

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Insulin resistance in skeletal muscle is a key phenotype associated with type 2 diabetes (T2D) for which the molecular mediators remain unclear. We therefore conducted an expression analysis of human muscle biopsies from patients with T2D; normoglycemic but insulin-resistant subjects with a parental family history (FH(+)) of T2D; and family history-negative control individuals (FH(–)). Actin cytoskeleton genes regulated by serum response factor (SRF) and its coactivator megakaryoblastic leukemia 1 (MKL1) had increased expression in T2D and FH(+) groups. Furthermore, striated muscle activator of Rho signaling (STARS), an activator of SRF, was upregulated in T2D and FH(+) and was inversely correlated with insulin sensitivity. Skeletal muscle from insulin-resistant mice recapitulated this gene expression pattern and showed reduced G-actin and increased nuclear localization of MKL1, each of which regulates SRF activity. Overexpression of MKL1 or reduction in G-actin decreased insulin-stimulated Akt phosphorylation, whereas reduction of STARS expression increased insulin signaling and glucose uptake. Pharmacological SRF inhibition by CCG-1423 reduced nuclear MKL1 and improved glucose uptake and tolerance in insulin-resistant mice in vivo. Thus, SRF pathway alterations are linked to insulin resistance, may contribute to T2D pathogenesis, and could represent therapeutic targets.[6]

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CCG-1423 exerts biological effects mainly by specifically inhibiting the RhoA/ROCK/SRF signaling pathway, which is involved in regulating cell proliferation, migration, differentiation, and gene transcription [1][2][3][4][6]
In tumor cells, CCG-1423 blocks SRF-mediated transcription of genes related to cell cycle progression, thereby inhibiting tumor growth [1]
In inflammatory and fibrotic diseases, CCG-1423 reduces inflammation and fibrosis by suppressing the activation of inflammatory cells and fibroblasts through the RhoA/ROCK/SRF pathway [4][6]

C18H13CLF6N2O3

454.75

454.051

C, 47.54; H, 2.88; Cl, 7.80; F, 25.07; N, 6.16; O, 10.55

285986-88-1

(S)-CCG-1423;2319939-24-5

2726015

White to off-white solid powder

1.5±0.1 g/cm3

1.525

6.59

70.92

2

9

5

30

586

0

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])(C([H])([H])[H])ON([H])C(C1C([H])=C(C(F)(F)F)C([H])=C(C(F)(F)F)C=1[H])=O)=O

DSMXVSGJIDFLKP-UHFFFAOYSA-N

InChI=1S/C18H13ClF6N2O3/c1-9(15(28)26-14-4-2-13(19)3-5-14)30-27-16(29)10-6-11(17(20,21)22)8-12(7-10)18(23,24)25/h2-9H,1H3,(H,26,28)(H,27,29)

N-[1-(4-chloroanilino)-1-oxopropan-2-yl]oxy-3,5-bis(trifluoromethyl)benzamide

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 (5.50 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 2 中的溶解度: ≥ 2.5 mg/mL (5.50 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；
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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;
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