Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in DLD1 cells
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in DLD1 cells. ..more
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
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, Emory University
External Assay ID: DLD1-ERK1-2_INH_WESTERN-BLOT_HRP_%INH MCSRUN
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in DLD1 cells.
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.
ERK1/2, map kinase, kinase, mapk1, Late stage, late stage AID, powders, purchased, synthesized, DLD, DLD-1, cells, KLF5, KLF, BTEB2, kruppel-like factor 5, cancer, Western blot, immunoblot, protein, counterscreen, inhibitor, inhibition, assay provider, 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 whether powder samples of compounds identified as possible KLF5 inhibitor probe candidates change protein levels of the Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2). In this assay, DLD-1 cells are seeded in 12-well plates, treated with test compounds for 24 hours, followed by lysis in Laemmli buffer and determination of protein levels using standard western blotting protocols. Actin is used as an internal control. This assay was run in the assay provider's lab. Compounds were tested in singlicate at a nominal concentration of 10 uM.
The parental DLD-1 cell lines were routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 (DLD-1) supplemented with 10% v/v certified fetal bovine serum, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay 2 x 10e5 cells in a 1 mL volume of growth media were dispensed into each well of 12-well tissue culture-treated plates. The assay was started immediately by dispensing 4 mL of test compound in DMSO (0.4 % final DMSO concentration), DMSO alone to the appropriate wells. Next, the plates were incubated for 24 hours at 37 C (5% CO2, 95% RH. After 24 h of incubation, cells were lysed in 200 uL Laemmli buffer and subjected to electrophoresis in 10% polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane and developed with appropriate antibodies. The DMSO treated cells were considered as 100% (control). We did not perform any statistical analysis as this experiment was performed once.
PubChem Activity Outcome and Score:
Compounds were considered active if percent inhibition or activation in protein levels value were greater than or equal to 30%. Compounds were considered inactive if percent inhibition or activation in protein levels value were less than 30%.
The reported PubChem Activity Score has been normalized to 100% observed absolute value of the fold change.
The PubChem Activity Score range for active compounds is 100-97. There are no inactive compounds.
List of Reagents:
DLD-1 cell lines (assay provider);
Laemmli buffer (assay provider)
Rabbit anti-ERK1/2 (Millipore, Part 06-182)
Mouse anti-B-actin (Sigma Aldrich Part A1978)
HRP-conjugated Secondary antibody: goat anti-rabbit and goat anti-mouse (vendor & part information not provided by assay provider)
Chemiluminescent developer (Millipore, part not provided by assay provider)
This assay was run by the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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
From BioAssay Depositor:
BAO: assay design: binding reporter: western blot
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay control: background control: Actin
BAO: bioassay specification: assay measurement throughput quality: single concentration single measurement
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: mmoa characterization
BAO: bioassay specification: bioassay type: functional: process
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: transcription factor
BAO: version: 1.4b1090
Assay Cell Type: DLD-1
** Test Concentration.
Data Table (Concise)