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BioAssay: AID 540317

HTS for Inhibitors of HP1-beta Chromodomain Interactions with Methylated Histone Tails

As our understanding of epigenetics expands, it is becoming clear that players in this field are potential targets for drug development. This area of epigenetic therapy has established applications for the treatment of cancer and neurological diseases. We have been involved in this field for a number of years and have performed high-throughput screens to identify the first small molecule more ..
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 Tested Compounds
 Tested Compounds
All(383355)
 
 
Active(2142)
 
 
Inactive(367962)
 
 
Inconclusive(13366)
 
 
 Tested Substances
 Tested Substances
All(387034)
 
 
Active(2150)
 
 
Inactive(371468)
 
 
Inconclusive(13416)
 
 
AID: 540317
Data Source: NCGC (HP1b001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-28

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: chromobox protein homolog 1 [Homo sapiens]
Description ..   
Protein Family: ChSh

Gene:CBX1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2142
Related Experiments
AIDNameTypeComment
488962Probe Summary Assay for Inhibitors of HP1-beta Chromodomain Interactions with Methylated Histone TailsSummarydepositor-specified cross reference: Project summary AID
488953qHTS Validation Assay for Inhibitors of HP1-beta Chromodomain Interactions with Methylated Histone TailsConfirmatorysame project related to Summary assay
Description:
As our understanding of epigenetics expands, it is becoming clear that players in this field are potential targets for drug development. This area of epigenetic therapy has established applications for the treatment of cancer and neurological diseases. We have been involved in this field for a number of years and have performed high-throughput screens to identify the first small molecule inhibitors of protein methyltransferases. These methyltransferases target multiple substrates, and thus regulate many arms of an epigenetic pathway. The methylation of different substrates often generates docking sites for effector proteins that harbor binding domains (chromo, tudor, PHD, MBT and ANK repeats).

It has been proposed that the binding sites generated for different proteins by these post-translational modifcations mediate downstream effects on chromatin. Consistent with this theory, di- and tri-methylation of histone H3K9 facilitates the binding of heterochromatin protein HP1. The HP1 protein harbors a chromodomain that is responsible for the methyl-dependent interaction between HP1 and lysine 9 of histone H3. HP1 was first described in flies as a heterochromatin associated protein that can regulate gene silencing. It harbors a chromodomain that binds H3K9me3. In addition, it harbors a chromoshadow domain that interacts with a large number of proteins that are involved in heterochromatin formation and gene repression. There are three highly related HP1 isoforms. All three variants are associated with pericentric heterochromatin and it is not clear whether they have specific or redundant functions. MPP8 is also involved in the repression of gene in euchromatic regions (unpublished data) and like HP1 its chromodomain binds H3K9me3. MPP8 harbors an ankyrin repeat region that is likely involved in binding and recruiting interacting proteins with transcriptional repressive function, much like the chromoshadow domain of HP1 does.

We hypothesize that by inhibiting specific protein-protein interactions we will be able to develop compounds that block a single arm of an epigenetic pathway. The specificity attained by blocking a single protein-protein interaction will be far greater than that attained by blocking the enzyme that regulates one of many interactions.

Low-affinity protein-protein interactions (Kd in the single- to double-digit micromolar range) such as those discussed here have been very difficult to assay by traditional techniques such as fluorescence polarization or fluorescence resonance energy transfer. We have developed a sensitive chemiluminescence-based assay (Quinn, et al. 2009), which serves as the starting point for this project.

The following is a validation assay to identify lead compounds that can block methyl-dependent protein-protein interactions (specifically HP1-beta), which can be used as probes to analyze these methyl-driven interactions and perhaps even developed into targeted epigenetic therapies.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: DA031087
Assay Submitter (PI): Mark Bedford, University of Texas, Medical Anderson Cancer Center, TX
Protocol
To each well, 2 uL of HP1-beta protein (final 50 nM) and 1 uL biotinylated histone peptide trimethylated at Lys9 [b-H3(1-18)K9me3] (final 50 nM) was added in 1x PBS buffer, pH 7.4, containing 0.01% Tween-20 using a nanoliter dispenser. Formation of a protein-peptide complex proceeded at room temperature for 30 min. A Kalypsys pin-tool was employed to transfer 23 nL of library compound solution in DMSO to each well. Aurintricarboxylic acid (ATA) was used as an intraplate control with a 12-point titration in duplicate. Following a 30 min incubation of the protein-peptide complex with compounds at room temperature, a mixture of 40 ug/mL each streptavidin-coated donor and nickel chelate acceptor AlphaScreen beads (final 10 ug/mL) was added in a 1 uL dispense for a final volume of 4 uL. Plates were incubated protected from the light for 30 min at room temperature. Microplates were then read on an EnVision multilabel plate reader using the 1,536 plate HTS AlphaScreen aperture (excitation time 80 ms, measurement time 240 ms).
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Assay Cell Type: NULL
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
15Activity at 0.458 uM (0.458μM**)% Activity at given concentration.Float%
16Activity at 2.291 uM (2.291μM**)% Activity at given concentration.Float%
17Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
18Activity at 57.01 uM (57.01μM**)% Activity at given concentration.Float%
19Activity at 113.8 uM (113.8μM**)% Activity at given concentration.Float%
20Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA031087

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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