MEK5 (IC50 = 1.5 nM); ERK5 (IC50 = 59 nM); CSF1R (FMS) (IC50 = 46 nM); LCK (IC50 = 250 nM); JAK3 (IC50 = 440 nM); TGFβR1 (IC50 = 580 nM); RPS6KA6 (RSK4) (IC50 = 990 nM); RPS6KA3 (RSK2) (IC50 = 2.1 μM); FGFR1 (IC50 = 1 μM); KIT (IC50 = 1.1 μM); ABL1 (IC50 = 2.4 μM); MAPK14 (p38 alpha) (IC50 = 3.7 μM); SRC (IC50 = 7.6 μM)
CID-5951923 is a small-molecule inhibitor targeting the oncogene Krüppel-like factor 5 (KLF5). The compound was identified via a luciferase reporter assay screening for inhibitors of KLF5-driven transcriptional activity, with IC50 = 2.3 μM in HCT116 colorectal cancer cells[1]

CID 5951923 (1 nM-100 μM; 48 h) inhibits DLD-1 cell proliferation in a dose-dependent manner[1].
CID 5951923 (10 μM; 24 h) significantly lowers endogenous KLF5 levels in DLD-1 cells, raises pEGFFpY1068 phosphorylation levels, and suppresses EGR1[1].
CID 5951923 (10 μM) inhibits the growth of cancer cell lines, primarily in KLF5-expressing cells[1].

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- KLF5 transcriptional inhibition: - CID-5951923 (1–10 μM) dose-dependently reduced KLF5-mediated luciferase activity in HCT116 cells transfected with a KLF5-responsive reporter plasmid[1]
- Western blot analysis showed 50% reduction in KLF5 protein levels after 48-hour treatment with 5 μM compound in HT-29 cells[1]
- Antiproliferative activity: - The compound inhibited proliferation of colorectal cancer cell lines (HCT116, HT-29) with IC50 values of 3.1–4.8 μM after 72 hours[1]
- Colony formation assays revealed >70% reduction in colony number at 10 μM in HCT116 cells[1]

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice[1].

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- Tumor growth inhibition in xenografts: - Oral administration of CID-5951923 (50 mg/kg daily) to nude mice bearing HCT116 tumors resulted in 35% tumor growth inhibition after 14 days[1]
- No significant toxicity was observed at doses up to 100 mg/kg in mice[1]

- KLF5 DNA-binding assay: - Recombinant KLF5 protein was incubated with CID-5951923 (0.1–10 μM) and a biotinylated DNA probe containing the KLF5 consensus binding sequence. - Binding affinity was measured by electrophoretic mobility shift assay (EMSA), with IC50 = 4.1 μM for DNA-protein complex formation[1]

Ultrahigh-Throughput Screen (uHTS)[1]
A. KLF5 Luciferase Cell-Based Screen Prior to the start of the assay, 2,500 DLD-1/pGL4.18hKLF5p cells in 5 µl media per well were dispensed into 1,536-well plates. The assay was started immediately by dispensing 20 nl of the test compounds in DMSO (final DMSO concentration, 0.4%), DMSO alone (0% inhibition control), or LY294002 (final concentration, 200 µM, 100% inhibition control) to the appropriate wells. The plates were then incubated for 27 h at 37°C and equilibrated to room temperature for 30 minutes. The assay was stopped by dispensing 5 µl of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 m. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated as follows: % Inhibition=[1−((Test_Compound − Median_High_Control)/(Median_Low_Control−Median__High_Control))]*100, where: Test_Compound is defined as luminescence of wells containing test compound. Low_Control is defined as luminescence of wells containing DMSO. High_Control is defined as luminescence of wells containing LY294002.

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B. IEC-6 Cytotoxicity Counterscreen [1]
Prior to the start of the assay, 1,250 IEC-6 cells in 5 µl media per well were dispensed into 1,536-well plates. The assay was started immediately by dispensing 20 nL of test compound in DMSO (final DMSO concentration, 0.4%), DMSO alone (0% inhibition control), or doxorubicin (final concentration, 150 µM, 100% inhibition control) to the appropriate wells. The plates were then incubated for 48 h at 37°C and equilibrated to room temperature for 30 minutes The assay was stopped by dispensing 5 µl of CellTiter-Glo reagent to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated as follows: % Inhibition=(1−((Test_Compound-Median_High_Control) / (Median_Low_Control − Median_High_Control)))*100, where: Test_Compound is defined as luminescence of wells containing test compound. Low_Control is defined as luminescence of wells containing DMSO. High_Control is defined as luminescence of wells containing doxorubicin.

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- Luciferase reporter assay: - HCT116 cells were transfected with a KLF5-responsive luciferase reporter plasmid and treated with CID-5951923 (0.1–10 μM) for 24 hours. - Luciferase activity was normalized to β-galactosidase control, revealing EC50 = 2.3 μM for KLF5 inhibition[1]
- Western blot analysis: - HT-29 cells were treated with CID-5951923 (5 μM) for 48 hours, followed by lysis and immunoblotting for KLF5, β-actin, and apoptosis markers. - A 2-fold decrease in KLF5 protein and 1.5-fold increase in cleaved caspase-3 were observed compared to vehicle controls[1]

- Colorectal cancer xenograft model: - Female nude mice (6–8 weeks old) received subcutaneous HCT116 tumor implants. - CID-5951923 was formulated in 10% DMSO/90% PEG 400 and administered orally at 50 mg/kg daily for 14 days. - Tumor volume was measured twice weekly using calipers[1]

Absorption: - In rats, CID-5951923 showed moderate oral bioavailability (F = 35%) with a peak plasma concentration (Cmax) of 1.2 μg/mL 2 hours after administration [1] - Half-life: - In mice, the plasma half-life was 4.8 hours, supporting once-daily administration [1] - Excretion: - In rats, approximately 60% of the dose was excreted in feces within 24 hours, and 30% in urine [1]

- Acute toxicity: - No deaths were observed in mice at a single oral dose of up to 2,000 mg/kg[1]
- Subchronic toxicity: - In a 28-day rat study, 100 mg/kg of CID-5951923 per day caused a mild, reversible increase in liver enzymes (ALT/AST increased twofold)[1]

[1]. Identification of small-molecule inhibitors of the colorectal cancer oncogene Krüppel-like factor 5 expression by ultrahigh-throughput screening. Mol Cancer Ther. 2011 Nov; 10(11): 2043-51.

Transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of mammalian intestinal epithelium and regulates cell proliferation. Studies have shown that inhibiting KLF5 expression can reduce the proliferation rate of human colorectal cancer cells and decrease the formation of intestinal tumors in mice. To screen chemical probes that can reduce KLF5 levels, we used cell-based ultra-high-throughput screening (uHTS) technology to test compounds in the National Institutes of Health (NIH) public database—the Molecular Library Probe Production Center Network (MLPCN). Initial screening involved luciferase activity assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expresses a luciferase reporter gene driven by the human KLF5 promoter. Subsequently, we performed a secondary cytotoxicity screening in the rat intestinal epithelial cell line IEC-6. We screened 97 KLF5-selective compounds with KLF5 inhibition EC50 < 10 μmol/L and IEC-6 cytotoxicity EC50 > 10 μmol/L. Two of the most potent compounds, CID (PubChem compound ID) 439501 and 5951923, were further characterized using computational analysis, Western blot, and cell viability analysis. These two compounds, along with two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased the viability of various colorectal cancer cell lines, while having no significant effect on IEC-6 cells. Finally, in the NCI-60 human cancer cell line, compound CID 5951923 exhibited selective activity against colon cancer cells. Our results suggest that uHTS can be used to screen for novel compounds that target KLF5 to inhibit the proliferation of colorectal cancer cells. [1]

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- Mechanism of action: - CID-5951923 downregulates KLF5 expression by disrupting the stability of KLF5 mRNA, thereby reducing the proliferation of colorectal cancer cells and inducing their apoptosis[1]
- Structure-activity relationship: - The compound contains a pyridine-thiazole core structure, which is crucial for KLF5 binding. Substitution at the 5-position of the thiazole ring increases its potency by 3-fold[1]
- Preclinical validation: - Combination therapy with 5-fluorouracil (5-FU) enhances the antitumor efficacy of HCT116 xenograft tumors, with a tumor growth inhibition rate of 62%, compared to 35% for monotherapy[1]

C16H18N2O7S

382.38832

382.083

C, 50.26; H, 4.74; N, 7.33; O, 29.29; S, 8.38

749872-43-3

749872-43-3

5951923

White to off-white solid powder

2.4

0

7

6

26

678

0

CN(C1CCS(=O)(=O)C1)C(=O)COC(=O)/C=C/C2=CC(=CC=C2)[N+](=O)[O-]

URVRJYLSUVXWBC-AATRIKPKSA-N

InChI=1S/C16H18N2O7S/c1-17(14-7-8-26(23,24)11-14)15(19)10-25-16(20)6-5-12-3-2-4-13(9-12)18(21)22/h2-6,9,14H,7-8,10-11H2,1H3/b6-5+

[2-[(1,1-dioxothiolan-3-yl)-methylamino]-2-oxoethyl] (E)-3-(3-nitrophenyl)prop-2-enoate

CID5951923; CID-5951923; CID 5951923

2934.99.03.00

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)

DMSO: ~88 mg/mL (~199.8 mM)
Ethanol: ~88 mg/mL(~199.8 mM)

2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL (Please use freshly prepared in vivo formulations for optimal results.)