TRFRET-based biochemical primary high throughput screening assay to identify inhibitors of 5-meCpG-binding domain protein 2 (MBD2)-DBD binding to methylated oligonucleotide
Name: TRFRET-based biochemical primary high throughput screening assay to identify inhibitors of 5-meCpG-binding domain protein 2 (MBD2)-DBD binding to methylated oligonucleotide. ..more
BioActive Compounds: 1149
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 ID: MBD2-CPGDNA_INH_TRFRET_1536_1X%INH PRUN
Name: TRFRET-based biochemical primary high throughput screening assay to identify inhibitors of 5-meCpG-binding domain protein 2 (MBD2)-DBD binding 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.
PRUN, Primary, Primary screen, Methyl-CpG binding domain, MBD2, DNA binding domain, MBD2-CpGDNA, MBD2-CPGDNA, MBD2-DBD, MBD2-MBD, FAM CpG DNA, Lanthascreen Tb Anti-HIS antibody, gene silencing, epigenetic gene silencing, cancer, GSTP1, glutathione S-transferase 1, pi class glutathione S-transferase 1, FAM, carboxyfluorescene, primary, singlicate, confirmation, triplicate, dose response, biochemical, antagonist, inhibit, inhibitor, inhibition, inh, FRET, fluorescence, HTS, high throughput screen, 1536, Scripps, 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 identify compounds that inhibit the binding of cloned recombinant 6-His-tagged DNA-binding domain of MBD2 (MBD2-DBD) to a FAM-labeled 14 bp hairpin oligonucleotide containing a symmetrically-methylated CpG, whose interaction is monitored using Tb labeled anti-his antibody. Pre-incubation of the MBD2 protein, FAM labeled methylated CpG oligo and Tb-labeled anti-His antibody leads to formation of a stable complex. Anti-his-complexed lanthanide (Tb) attaches to the anti-his-binding peptide tag on MBD2-DBD protein. When terbium donor is excited, Tb transfers energy to a fluorescein tag on the methylated oligo, now complexed with MBD2-DBD, resulting in an increase in TR-FRET. An inhibitor of the MBD2-DBD:FAM methylated CpG interaction will inhibit binding between MBD2 and FAM methylated oligo, leading to reduced energy transfer and reduced well FRET (fluorescein emission / Tb emission). Compounds are tested in singlicate at a final nominal concentration of 4.4 uM.
The assay was started by dispensing 5 uL 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 25 nM of MBD2 protein, 30 nM of FAM labeled Unmethylated CpG DNA oligo and 5 nM of Terbium labeled anti-his antibody into columns 1 thru columns 3 of 1536 microtiter plates. Next, 5 uL of Experimental Mix containing 25 nM of MBD2 protein, 30 nM FAM labeled Methylated CpG DNA oligo and 5 nM Terbium labeled anti-his antibody in assay buffer were dispensed into columns 4 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 30 minutes at 25 C 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 Control Mix and DMSO.
Low_Control is defined as the median of the wells containing test compounds.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported Pubchem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-11, and for inactive compounds 11-0.
List of Reagents:
MBD2-MBD protein (supplied by Assay Provider)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
FAM labeled Unmethylated 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)