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 ..
BioActive Compounds: 2142
Depositor Specified Assays
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 MPP8 is also involved in the repression of gene in euchromatic regions (unpublished data) and like HP1 its chromodomain binds H3K9me3.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
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).
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)