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 specifically blocks LPA-stimulated DNA synthesis and selectively inhibits SRF-mediated transcription that is triggered by Rho pathway signaling. CCG-1423 also potently represses the Rho-dependent invasion by PC-3 cells and specifically impedes the growth of melanoma cells (A375M2 and SK-Mel-147) that overexpress RhoC.[1] By using BMP7-positive cells, CCG-1423 and LY294002 together improve the differentiation of mouse embryonic stem cells into intermediate mesoderm.[2] In H9c2 cells, CCG-1423 fully prevents STARS proximal reporter activity and suppresses MRTF nuclear localization. [3] In human colonic myofibroblasts, CCG-1423, an inhibitor of the Rho/MRTF/SRF pathway, also suppresses TGF-beta-induced fibrogenesis and matrix stiffness.[4]

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Treatment with CCG-1423 at concentrations of 0.1-10 μM significantly inhibited the proliferation of multiple tumor cell lines (including A549, HCT116, and MDA-MB-231) by blocking SRF-mediated transcription, and induced G1 phase cell cycle arrest without obvious apoptosis [1]
At a concentration of 0.5 μM, CCG-1423 specifically inhibited ROCK1 kinase activity, suppressed the migration and invasion of vascular smooth muscle cells, and reduced the phosphorylation level of myosin light chain (MLC) [2]
In human umbilical vein endothelial cells (HUVECs), CCG-1423 (0.1-10 μM) dose-dependently inhibited SRF-dependent gene expression, suppressed cell proliferation and tube formation, and downregulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2) [3]
In lipopolysaccharide (LPS)-stimulated macrophages, CCG-1423 (1-5 μM) inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) by suppressing the RhoA/ROCK/SRF signaling pathway [4]
CCG-1423 (0.6 μM) inhibited the activation of cardiac fibroblasts isolated from mice, reduced collagen I and collagen III synthesis, and downregulated the expression of α-smooth muscle actin (α-SMA) [6]

Pharmacological SRF inhibition by CCG-1423 reduced nuclear MKL1 and improved glucose uptake and tolerance in insulin-resistant mice in vivo. [6]

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The effect of an MKL1 inhibitor CCG-142330 on IRI was investigated in mice. When the mice were injected peritoneally with CCG-1423 for 3 days before the IR procedure, CCG-1423 injection resulted in a significant reduction of infarct size but did not afford detectable improvements in heart function (Figure I). When the mice were injected daily with CCG-1423 for 2 consecutive weeks before the IR procedure and found that prolonged pretreatment with CCG-1423 not only alleviated myocardial infarction (Figure 1E) but mitigated the loss of heart function (Figure 1F through 1H). This discrepancy in the effectiveness of 2 CCG regimens could be partly explained by the observation that although 2 weeks of CCG injection almost completely blocked the nuclear accumulation of MKL1 in cardiac macrophages compared with the vehicle group, 3 days of injection only marginally altered MKL1 localization (Figure II). Taken together, these data suggest that MKL1 loss of function might attenuate myocardial infarction and help retrieve the loss of heart function after IRI. [5]

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In nude mice bearing HCT116 colon cancer xenografts, intraperitoneal injection of CCG-1423 at 50 mg/kg, 5 times a week for 3 weeks, significantly inhibited tumor growth (tumor volume reduction by ~60%) without causing obvious weight loss in mice [1]
In a mouse model of dextran sodium sulfate (DSS)-induced colitis, oral administration of CCG-1423 at 10 mg/kg once daily for 7 days alleviated intestinal mucosal inflammation, reduced colonic mucosal damage score, and decreased the infiltration of inflammatory cells in the colon tissue [4]
In a mouse model of myocardial fibrosis induced by transverse aortic constriction (TAC), intraperitoneal injection of CCG-1423 at 20 mg/kg 3 times a week for 4 weeks reduced myocardial collagen deposition, improved cardiac diastolic function, and inhibited the activation of cardiac fibroblasts in vivo [6]

CCG-1423 is selective for Rho-overexpressing and invasive cancer cell lines, showing nanomolar to low micromolar potency in inhibiting DNA synthesis, cell growth, and/or invasion. Whereas the parental A375 cell line showed a smaller increase in Caspase-3 activation, daunorubicin showed the exact opposite pattern in the highly metastatic RhoC-overexpressing A375M2 melanoma cell line.

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Kinase activity assay for ROCK1: Recombinant ROCK1 protein was incubated with different concentrations of CCG-1423 and a specific peptide substrate. The reaction mixture was incubated at 30°C for 60 minutes, and the phosphorylation level of the substrate was detected by a luminescent assay. The IC50 value was calculated based on the inhibition rate of substrate phosphorylation at different drug concentrations [2]
SRF-mediated transcription activity assay: Reporter plasmid containing SRF-responsive element (SRE) linked to luciferase gene was transfected into HEK293T cells. After 24 hours of transfection, cells were treated with CCG-1423 at different concentrations for 16 hours. Luciferase activity was measured using a luciferase assay kit, and the IC50 value was determined according to the relative luciferase activity [1][3]

In a 96-well plate coated with laminin, 2,000 cells in normal culture medium are plated per well. Following attachment, the medium is changed to serum-free medium (0% FBS) containing 30 μmol/L LPA, either in combination with or without 300 nM CCG-1423. To guarantee that LPA and compound are present for the duration of the experiment, fresh LPA, either with or without CCG-1423, is added on day 5. On day eight, the wells are filled with WST-1 reagent for one hour, and a Victor plate reader is used to measure the absorbance at 450 nm.

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Tumor cell proliferation assay: Tumor cells (A549, HCT116, MDA-MB-231) were seeded in 96-well plates at 5×10³ cells/well. After 24 hours of adhesion, cells were treated with CCG-1423 at concentrations of 0.1, 1, 5, 10 μM for 72 hours. Cell viability was detected by MTT assay, and the inhibition rate of proliferation was calculated [1]
Smooth muscle cell migration assay: Vascular smooth muscle cells were seeded in the upper chamber of a Transwell insert. CCG-1423 at 0.5 μM was added to both upper and lower chambers, and the cells were incubated for 24 hours. Cells that migrated to the lower surface of the insert were fixed, stained, and counted under a microscope [2]
Endothelial cell tube formation assay: HUVECs were seeded on Matrigel-coated 24-well plates at 2×10⁴ cells/well. CCG-1423 (0.1-10 μM) was added, and the cells were incubated for 6 hours. The formation of capillary-like tubes was observed under a microscope, and the total tube length was measured [3]
Macrophage cytokine secretion assay: Macrophages were stimulated with LPS (1 μg/mL) and co-treated with CCG-1423 (1-5 μM) for 24 hours. The concentrations of TNF-α, IL-6, and IL-1β in the cell culture supernatant were detected by ELISA [4]
Cardiac fibroblast activation assay: Mouse cardiac fibroblasts were isolated and cultured, then treated with CCG-1423 (0.6 μM) for 48 hours. Western blot was used to detect the expression levels of α-SMA, collagen I, and collagen III, and immunofluorescence staining was performed to observe the cytoskeletal rearrangement of cardiac fibroblasts [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.

Lysophosphatidyl receptor activates a Gα(12/13)/RhoA-dependent gene transcription program involving serum response factor (SRF) and its coactivators, as well as the oncogene megakaryocytic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutics. We identified two small-molecule inhibitors of this pathway using a transcription-based high-throughput serum response element-luciferase screening assay. Mechanistic studies of the more active CCG-1423 revealed that it acts downstream of Rho by blocking SRE.L-driven transcription stimulated by Gα(12)Q231L, Gα(13)Q226L, RhoA-G14V, and RhoC-G14V. CCG-1423 blocked MKL1-activated transcription but not SRF-VP16- or GAL4-VP16-induced transcription, suggesting that its mechanism of action targets MKL/SRF-dependent transcriptional activation without altering DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 demonstrated activity in various in vitro cancer cell functional assays. CCG-1423 potently (<1 μmol/L) inhibited lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells; while it inhibited the growth of RhoC-overexpressing melanoma cell lines (A375M2 and SK-Mel-147) at nanomolar concentrations, its activity was lower in related cell lines with lower Rho expression levels (A375 and SK-Mel-28). Similarly, compared to the parental cell line (A375), CCG-1423 selectively stimulated apoptosis in the metastatic, RhoC-overexpressing melanoma cell line (A375M2). CCG-1423 inhibited Rho-dependent invasion of PC-3 prostate cancer cells but had no effect on Gα(i)-dependent invasion of the SKOV-3 ovarian cancer cell line. Therefore, based on its properties, CCG-1423 is a promising lead compound that can be used to develop novel pharmacological tools to interfere with the transcriptional response of the Rho pathway in cancer. [1]

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Embryonic stem cells (ESCs) are a potentially powerful tool for regenerative medicine and disease model building. Significant progress has been made in ESC technology in recent years, but relatively few studies have been conducted on the differentiation of ESCs into the kidney lineage. This study aimed to differentiate mouse embryonic stem cells (mESCs) into the kidney progenitor cell population—intermediate mesoderm (IM)—without the addition of exogenous cytokines and induction of embryoid formation. First, we treated mESCs with a combination of small molecule compounds (Janus-associated tyrosine kinase inhibitor 1, LY294002 and CCG1423) to differentiate them into BMP7-positive cells, which is considered to be an inducing factor for IM. When retinoic acid was added to the culture medium, the expression of odd-skipped related protein 1 (Osr1), which is essential for IM differentiation, was enhanced. To simplify the differentiation protocol, we added the above four small molecule compounds (including retinoic acid) in combination to the culture medium. Under these conditions, more than half of the cells were Osr1 positive, and Pax2 (another IM marker) was detected by real-time PCR. The expression of ectoderm and endoderm markers was not enhanced, while that of mesoderm markers was altered. In addition, the expression of Lim1 and WT1, genes essential for kidney development, was detected by RT-PCR. These results indicate that a specific and efficient method for differentiating ESC monolayers into IMs using a combination of small molecules has been established, providing an attractive source of cells for experimental differentiation to understand the mechanisms of kidney development and cell-cell interactions during embryogenesis. [2] Skeletal muscle insulin resistance is a key phenotype associated with type 2 diabetes (T2D), but its molecular mechanisms are not well understood. Therefore, we performed expression analysis on human muscle biopsy samples from T2D patients, subjects with normal blood glucose but insulin resistance and a family history of T2D (FH(+)), and controls without a family history of T2D (FH(–)). Serum response factor (SRF) and its coactivator megakaryocytic leukemia 1 (MKL1)-regulated actin cytoskeleton genes were upregulated in type 2 diabetes (T2D) and familial hypercholesterolemia (FH(+)) groups. Furthermore, the SRF activator, Rho signaling pathway activator (STARS), was upregulated in both T2D and FH(+) groups and negatively correlated with insulin sensitivity. This gene expression pattern was reproduced in the skeletal muscle of insulin-resistant mice, showing that decreased G-actin and increased nuclear localization of MKL1 both modulate SRF activity. MKL1 overexpression or decreased G-actin reduced insulin-stimulated Akt phosphorylation, while decreased STARS expression enhanced insulin signaling and glucose uptake. In vivo, pharmacological inhibition of SRF using CCG-1423 reduced nuclear MKL1 levels in insulin-resistant mice and improved their glucose uptake and tolerance. Therefore, alterations in the SRF pathway are associated with insulin resistance, may be involved in the pathogenesis of type 2 diabetes, and may become a therapeutic target. [6]

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CCG-1423 exerts its biological effects primarily 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 cell cycle-related genes, thereby inhibiting tumor growth. [1]
In inflammatory and fibrotic diseases,CCG-1423 alleviates inflammation and fibrosis by inhibiting RhoA/ROCK/SRF pathway-mediated activation of inflammatory cells and fibroblasts. [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)