Counterscreen for inhibitors of 5-meCpG-binding domain protein 2 (MBD2): TRFRET-based biochemical high throughput dose response assay to identify inhibitors of binding of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) to methylated oligonucleotide
Name: Counterscreen for inhibitors of 5-meCpG-binding domain protein 2 (MBD2): TRFRET-based biochemical high throughput dose response assay to identify inhibitors of binding of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) to methylated oligonucleotide ..more
BioActive Compounds: 4
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Bill Nelson
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH098712-01
Grant Proposal PI: Bill Nelson
External Assay: UHRF1-CPGDNA_INH_TRFRET_1536_3XIC50 DCSRUN for MBD2-CPGDNA INH
Name: Counterscreen for inhibitors of 5-meCpG-binding domain protein 2 (MBD2): TRFRET-based biochemical high throughput dose response assay to identify inhibitors of binding of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) to methylated oligonucleotide
Of all the somatic genome changes that accumulate during the pathogenesis of human cancers, only changes in DNA methylation appear to occur consistently (virtually all cases), to arise early (first appearing in preneoplastic lesions), and to be potentially reversible (the DNA sequence remains intact) (1-4). One such change in DNA methylation, increased CpG dinucleotide methylation at CpG islands encompassing the transcriptional regulatory regions of many genes, leads to the transcriptional "silencing" of critical cancer genes (2, 5-6). CpG island hypermethylation has been reported to inhibit gene transcription by interfering with the binding and/or function of transcriptional trans-activators, or by recruiting 5-meCpG-binding domain (MBD) family proteins capable of mediating transcriptional repression (7). As many as 500 or more genes are epigenetically "silenced" in most human cancers. Two MBD family proteins have been implicated in the silencing of genes carrying abnormally hypermethylated CpG island sequences, MECP2 and MBD2. MBD2 binds 5-meCpG-DNA and is a component of a 1 MD transcription repression complex that also contains a chromatin remodeling complex subunits, histone-binding proteins, and a helicase/ATPase domain (8). Evidence suggests that MBD2-containing complexes are responsible for transcriptional repression accompanying somatic hypermethylation at pi-class glutathione S-transferase 1 (GSTP1), the most common genome change yet reported for prostate cancer, and a common alteration in breast and liver cancers (9-11). The goal of this project is the discovery and characterization of small molecule inhibitors of epigenetic gene silencing mediated by MBD2 and thus to identify compounds that will be able to reactivate the silenced genes in cancer cells, restoring gene function.
1. Brooks, J. D., Weinstein, M., Lin, X., Sun, Y., Pin, S. S., Bova, G. S., Epstein, J. I., Isaacs, W. B., and Nelson, W. G. (1998) CG island methylation changes near the GSTP1 gene in prostatic intraepithelial neoplasia, Cancer Epidemiol Biomarkers Prev 7, 531-536.
2. Herman, J. G., and Baylin, S. B. (2003) Gene silencing in cancer in association with promoter hypermethylation, N Engl J Med 349, 2042-2054.
3. Lin, X., Tascilar, M., Lee, W. H., Vles, W. J., Lee, B. H., Veeraswamy, R., Asgari, K., Freije, D., van Rees, B., Gage, W. R., Bova, G. S., Isaacs, W. B., Brooks, J. D., DeWeese, T. L., De Marzo, A. M., and Nelson, W. G. (2001) GSTP1 CpG island hypermethylation is responsible for the absence of GSTP1 expression in human prostate cancer cells, Am J Pathol 159, 1815-1826.
4. Nakayama, M., Bennett, C. J., Hicks, J. L., Epstein, J. I., Platz, E. A., Nelson, W. G., and De Marzo, A. M. (2003) Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a detailed study using laser-capture microdissection, Am J Pathol 163, 923-933.
5. Antequera, F., and Bird, A. (1993) Number of CpG islands and genes in human and mouse, Proc Natl Acad Sci U S A 90, 11995-11999.
6. Bird, A. P. (1986) CpG-rich islands and the function of DNA methylation, Nature 321, 209-213.
7. Bird, A. P., and Wolffe, A. P. (1999) Methylation-induced repression--belts, braces, and chromatin, Cell 99, 451-454.
8. Feng, Q., and Zhang, Y. (2001) The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes, Genes Dev 15, 827-832.
9. Bakker, J., Lin, X., and Nelson, W. G. (2002) Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells, J Biol Chem 277, 22573-22580.
10. Lin, X., and Nelson, W. G. (2003) Methyl-CpG-binding domain protein-2 mediates transcriptional repression associated with hypermethylated GSTP1 CpG islands in MCF-7 breast cancer cells, Cancer Res 63, 498-504.
11. Singal, R., van Wert, J., and Bashambu, M. (2001) Cytosine methylation represses glutathione S-transferase P1 (GSTP1) gene expression in human prostate cancer cells, Cancer Res 61, 4820-4826.
DCSRUN, counterscreen, secondary, triplicate, dose, dose response, titration, dilution, Ubiquitin-like containing PHD and RING finger domains 1, UHRF1, UHRF1-CpGDNA, UHRF1-CPGDNA, MBD2, MBD2-CpGDNA, Set-and RING-Associated, SRA, DNA replication, DNA methylation maintenance, DNA binding domain, cancer, singlicate, TRFRET, biochemical, Scripps, FRET, fluorescence, high throughput screen, 1536, HTS, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, "TRFRET-based biochemical primary high throughput screening assay to identify inhibitors of 5-meCpG-binding domain protein 2 (MBD2)-DBD binding to methylated oligonucleotide" (PubChem AID 686964), and that confirmed activity in a set of experiments entitled, "TRFRET-based biochemical high throughput confirmation assay to identify inhibitors of 5-meCpG-binding domain protein 2 (MBD2)-DBD binding to methylated oligonucleotide" (PubChem AID 720531) are nonselective, as determined by inhibition of methylated DNA-binding activity of the SRA domain polypeptide of ubiquitin-like with PHD and ring finger domains 1 (UHRF1).
The assay was started by dispensing 5 microliters of Control Mix in assay buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 125 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.2% Tween-20) containing 125 nanomolar of UHRF1 protein, 5 micromolar of FAM labeled methylated CpG DNA oligo and 5 nanomolar of Terbium labeled anti-his antibody into columns 1 thru columns 2 of 1536 microtiter plates. Next, 5 microliters of Experimental Mix containing 125 nanomolar of UHRF1 protein, 100 nanomolar FAM labeled Hemimethylated CpG DNA oligo and 5 nanomolar Terbium labeled anti-his antibody in assay buffer were dispensed into columns 3 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO, Mitoxantrone (1.3 micromolar final concentration) in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 60 minutes at 25 degrees Celsius and TR-FRET was measured on a Viewlux microplate reader (Perkin Elmer, Turku, Finland). After excitation at 340 nm, well fluorescence was monitored at 495 nm (Tb) and 525 nm (FAM). For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I525nm / I495nm
I525nm represents the measured fluorescence emission at 525 nm.
I495nm represents the measured fluorescence emission at 495 nm.
The percent inhibition for each compound was calculated using as follows:
%_Inhibition = 100 * ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) )
Test_Compound is defined as wells containing the Experimental Mix in the presence of test compound.
High_Control is defined as wells containing the Experimental Mix, Mitoxantrone and DMSO.
Low_Control is defined as wells containing the Experimental Mix and DMSO.
PubChem Activity Outcome and Score:
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. 43.8 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 43.8 uM.
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.
The PubChem Activity Score range for active compounds is 100-79, and for inactive compounds 76-0.
List of Reagents:
UHRF1_SRA protein (supplied by Assay Provider)
FAM labeled Hemimethylated CpG DNA Oligo (IDT-custom order)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
Lanthascreen Tb Anti-His Antibody (Invitrogen, part PV5895)
MgCl2 (Fisher, part BP214)
NaCl (Sigma, part S6546)
DTT (Fisher, part BP172)
EDTA (Sigma, part E7889)
Tris (Amresco, part 0497)
Tween 20 (Sigma, part P9416)
Glycerol (Sigma, part G7893)
1536-well plates (Corning, part 7261)
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. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)