Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)
Name: Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5) ..more
BioActive Compounds: 671
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_%INH
Name: Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)
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.
KLF5, BTEB2, kruppel-like factor 5, cancer, primary screen, HTS, high throughput screen, 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 cell-based reporter assay is to identify compounds that inhibit the expression of KLF5. This 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 for one day, followed by incubation with test compounds. 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. Test compounds were assayed in singlicate at a final nominal concentration of 5 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 serum800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay 2500 cells in a 5 ul 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 25 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 ul of SteadyLite HTS luciferase substrate 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-
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.
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary Screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active. The reported PubChem Activity Score has been normalized to 100% of the highest observed primary inhibition value. Negative % inhibition values are reported as activity score zero.
The inactive compounds of this assay have activity score range of 0 to 52 and active compounds range of activity score is 52 to 100.
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)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay LY294002 had an IC50 of approximately 20 uM. 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 non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)