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 (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].

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].

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.

---

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.

[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.

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. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC(50) < 10 μmol/L for KLF5 inhibition and EC(50) > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.[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.)