Late stage results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for inhibitors of KLF5 (Round 1)
Name: Late stage results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for inhibitors of KLF5 (Round 1). ..more
BioActive Compounds: 13
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: KLF5_INH_LUMI_1536_3X1C50 MDRUN SAR_Round_1
Name: Late stage results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for inhibitors of KLF5 (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. Epub 2001 Jan 10. Intestinal-enriched Kruppel-like factor (Kruppel-like factor 5) is a positive regulator of cellular proliferation.
Late stage, powders, purchased, synthesized, KLF5, BTEB2, kruppel-like factor 5, cancer, dose response, Round 1, SAR, 1536, inhibitor, inhibition, luciferase, reporter, luminescence, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for Round 1 powder samples of compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)" (AID 1700), "Luminescence-based confirmation cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)" (AID 1834), "Luminescence-based dose response cell-based high throughput screening assay for inhibitors of kruppel-like factor 5 (KLF5)" (AID 1973), and that were inactive in a set of experiments entitled, "Luminescence-based counterscreen assay for KLF5 inhibitors: cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line." (AID 1825), and "Luminescence-based counterscreen assay for KLF5 inhibitors: dose response cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line)" (AID 1975).
This cell-based reporter assay employs a human DLD-1 colorectal cancer cell line that is stably transfected with a plasmid containing 2.0 kb of the human KLF5 promoter used to drive expression of the firefly luciferase reporter gene (DLD-1/pGL4.18hKLF5p). Cells are seeded in medium supplemented with 10% FBS, followed by immediate incubation with test compounds for one day. Luciferase activity is monitored using a luminescent detection reagent, which contains the luciferase substrate D-luciferin that releases light energy upon conversion to oxyluciferin in the presence of ATP, Mg2+, and oxygen. As designed, compounds that inhibit KLF5 expression will reduce the activity of the KLF5 promoter, thereby reducing transcription of the luciferase reporter gene and luciferase protein levels, resulting in decreased conversion of luciferin to oxyluciferin, and reduced well luminescence. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 40 uM.
The DLD-1/pGL4.18hKLF5p cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 supplemented with 10% v/v certified fetal bovine serum, 800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay 2500 cells in a 5 microliter volume of assay media (growth media as above except without geneticin) were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 20 nL of test compound in DMSO (0.4 % final DMSO concentration), DMSO alone, or LY294002 (200 uM final concentration) to the appropriate wells. Next, the plates were incubated for 27 hours at 37 degrees C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 30 minutes, the assay was stopped by dispensing 5 microliters of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 5 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
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing LY294002.
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 Explorer software (Symyx Technologies 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. In cases where the highest concentration tested (i.e. 40 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 40 uM.
PubChem Activity Outcome Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50/ equal to or less than 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, 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 54-0.
List of Reagents:
DLD-1/pGL4.18hKLF5p cell line (provided by Assay Provider)
RPMI-1640 medium (Invitrogen, 11875-119)
Geneticin (Invitrogen, part 10131-035)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16000-044)
SteadyLite HTS Assay Kit (PerkinElmer, part 6016989)
LY294002 Calbiochem, part 440202)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789173)
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this assay LY294002 had an IC50 of approximately 20 uM. 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)