Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the DLD-1 cell line (Round 1)
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the DLD-1 cell line (Round 1). ..more
BioActive Compounds: 2
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Vincent Yang, Emory University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-R03-DA026215-01
Grant Proposal PI: Vincent Yang
External Assay ID: DLD1CYTOX_INH_LUMI_3XIC50 CSDRUN SAR_Round 1
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the DLD-1 cell line (Round 1).
Transcription factors are essential regulators of transcription that bind DNA to control both the rate and frequency of gene expression (1). Many diseases of cell homeostasis are associated with aberrant transcription factor activity (2). Colon cancer, in particular, is a disease of uncontrolled proliferation of the epithelial cells that line the intestinal crypts. Kruppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that binds to GC-rich sequences in promoters of numerous genes (3) including cyclin D1 (4), cyclin B1/Cdc2 (4), and integrin-linked kinase (5). KLF5 is highly expressed in rapidly dividing epithelial cells in intestinal crypts (6). This expression pattern of KLF5, along with studies demonstrating that KLF5 mediates the transforming effects of oncogenic H-Ras (7), and that ectopic expression of KLF5 leads to increased cell proliferation and anchorage-independent growth of cultured intestinal epithelial cells (8, 9), suggest that KLF5 may be involved in colon cancer pathogenesis. Therefore, the identification of selective inhibitors of KLF5 may provide useful tools to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium.
1. Ptashne M. Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci. 2005 Jun;30(6):275-9.
2. Fre S, Vignjevic D, Schoumacher M, Duffy SL, Janssen KP, Robine S, Louvard D. Adv Cancer Res. 2008;100:85-111. Epithelial morphogenesis and intestinal cancer: new insights in signaling mechanisms.
3. Goldstein BG, Chao HH, Yang Y, Yermolina YA, Tobias JW, Katz JP. Am J Physiol Gastrointest Liver Physiol. 2007 Jun;292(6):G1784-92. Overexpression of Kruppel-like factor 5 in esophageal epithelia in vivo leads to increased proliferation in basal but not suprabasal cells.
4. Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Kruppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell Res. 2005 Feb;15(2):92-6.
5. Yang Y, Tetreault MP, Yermolina YA, Goldstein BG, Katz JP. Kruppel-like factor 5 controls keratinocyte migration via the integrin-linked kinase. J Biol Chem. 2008 Jul 4;283(27):18812-20.
6. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays. 2007 Jun;29(6):549-57. Erratum in: Bioessays. 2007 Sep;29(9):946.
7. Nandan MO, Yoon HS, Zhao W, Ouko LA, Chanchevalap S, Yang VW. Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras. Oncogene. 2004 Apr 22;23(19):3404-13.
8. Chanchevalap S, Nandan MO, Merlin D, Yang VW. FEBS Lett. 2004 Dec 3;578(1-2):99-105. All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Kruppel-like factor 5.
9. Sun R, Chen X, Yang VW. J Biol Chem. 2001 Mar 9;276(10):6897-900. Intestinal-enriched Kruppel-like factor (Kruppel-like factor 5) is a positive regulator of cellular proliferation.
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The purpose of this assay is to determine whether powder samples of compounds identified as possible KLF5 inhibitor probe candidates decrease proliferation of DLD-1 cell line. In this assay, DLD-1 cells are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in triplicate using a 6-point titration series with final concentrations: 20, 10, 1, 0.1, 0.01, 0.001 uM.
The parental DLD-1 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 supplemented with 10% v/v certified fetal bovine serum and 1X antibiotic mix (penicillin and streptomycin).
Prior to the start of the assay 104 cells in a 100 uL volume of growth media were dispensed into each well of 96-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 0.5 uL of test compound in DMSO (0.4 % final DMSO concentration) or DMSO alone to the appropriate wells. Next, the plates were incubated for 48 hours at 37 C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 30 minutes, the assay was stopped by dispensing 100 uL of CellTiter-Glo reagent to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the Biotek Synergy 2 plate reader.
The percent inhibition for each compound was calculated as follows:
% Inhibition = ( 1 - ( Test_Compound - Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay software (GraphPad Prism software Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value.
PubChem Activity Outcome and Score:
Compounds with an average percent inhibition value < 40 at 10 uM were considered inactive. Compounds with an average percent inhibition value >= 40 at 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-81, and for inactive compounds 0-0.
List of Reagents:
DLD-1 cell line (ATCC, CCL-221),
RPMI640 medium (Invitrogen, part 11875-093)
100X Penicillin-Streptomycin mix (Invitrogen, part 15140-122)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16000-044)
Cell Titer Glo (Promega, part G7571)
T-175 tissue culture flasks (Corning, part 431080)
96-well plates (Costar, part 3917)
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided. The activity cutoff was 40% inhibition at 10 uM. This assay was performed by the Assay Provider.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)