Rational Adaptation of L3MBTL1 Inhibitors to Create Small-Molecule Cbx7 Antagonists.

Chromobox homolog 7 (Cbx7) is an epigenetic modulator that is an important driver of multiple cancers. It is a methyl reader protein that operates by recognizing and binding to methylated lysine residues on specific partners. Herein we report our efforts to create low-molecular-weight inhibitors of Cbx7 by making rational structural adaptations to inhibitors of a different methyl reader protein, L3MBTL1, inhibitors that had previously been reported to be inactive against Cbx7. We evaluated each new inhibitor for Cbx7 inhibition by fluorescence polarization assay, and also confirmed the binding of selected inhibitors to Cbx7 by saturation-transfer difference NMR spectroscopy. This work identified multiple small-molecule inhibitors with modest (IC50 : 257-500 µm) potency.

Selective Inhibition of CBX6: A Methyllysine Reader Protein in the Polycomb Family.

The polycomb paralogs CBX2, CBX4, CBX6, CBX7, and CBX8 are epigenetic readers that rely on "aromatic cage" motifs to engage their partners' methyllysine side chains. Each CBX carries out distinct functions, yet each includes a highly similar methyllysine-reading chromodomain as a key element. CBX7 is the only chromodomain that has yet been targeted by chemical inhibition. We report a small set of peptidomimetic agents in which a simple chemical modification switches the ligands from one with promiscuity across all polycomb paralogs to one that provides selective inhibition of CBX6. The structural basis for this selectivity, which involves occupancy of a small hydrophobic pocket adjacent to the aromatic cage, was confirmed through molecular dynamics simulations. Our results demonstrate the increases in affinity and selectivity generated by ligands that engage extended regions of chromodomain binding surfaces.

Attraction by repulsion: compounds with like charges undergo self-assembly in water that improves in high salt and persists in real biological fluids.

We report a family of highly anionic calixarenes that form discrete homo-dimeric assemblies in pure water, that get stronger in high salt solutions, and that remain assembled in complex, denaturing solutions like real urine. The results reveal the potential of like-charged subunits for self-assembly in high-salt solutions and biological fluids.

Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes.

Plant homeodomain (PHD) finger-containing proteins are implicated in fundamental biological processes, including transcriptional activation and repression, DNA damage repair, cell differentiation, and survival. The PHD finger functions as an epigenetic reader that binds to posttranslationally modified or unmodified histone H3 tails, recruiting catalytic writers and erasers and other components of the epigenetic machinery to chromatin. Despite the critical role of the histone-PHD interaction in normal and pathological processes, selective inhibitors of this association have not been well developed. Here we demonstrate that macrocyclic calixarenes can disrupt binding of PHD fingers to methylated lysine 4 of histone H3 in vitro and in vivo. The inhibitory activity relies on differences in binding affinities of the PHD fingers for H3K4me and the methylation state of the histone ligand, whereas the composition of the aromatic H3K4me-binding site of the PHD fingers appears to have no effect. Our approach provides a novel tool for studying the biological roles of methyllysine readers in epigenetic signaling.

Inhibition of histone binding by supramolecular hosts.

The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys9). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms.

Chromodomain antagonists that target the polycomb-group methyllysine reader protein chromobox homolog 7 (CBX7).

We report here a peptide-driven approach to create first inhibitors of the chromobox homolog 7 (CBX7), a methyllysine reader protein. CBX7 uses its chromodomain to bind histone 3, lysine 27 trimethylated (H3K27me3), and this recognition event is implicated in silencing multiple tumor suppressors. Small trimethyllysine containing peptides were used as the basic scaffold from which potent ligands for disruption of CBX7-H3K27me3 complex were developed. Potency of ligands was determined by fluorescence polarization and/or isothermal titration calorimetry. Binding of one ligand was characterized in detail using 2D NMR and X-ray crystallography, revealing a structural motif unique among human CBX proteins. Inhibitors with a ∼200 nM potency for CBX7 binding and 10-fold/400-fold selectivity over related CBX8/CBX1 proteins were identified. These are the first reported inhibitors of any chromodomain.

Synthetic trimethyllysine receptors that bind histone 3, trimethyllysine 27 (H3K27me3) and disrupt its interaction with the epigenetic reader protein CBX7.

Post-translational modifications act as 'on' or 'off' switches causing downstream changes in gene transcription. Modifications such as trimethylation of lysine 27 on histone H3 (H3K27me3) cause repression of transcription and stable gene silencing, and its presence is associated with aggressive cancers of many types. We report here macrocyclic host-type compounds that can bind H3K27me3 preferentially over unmethylated H3K27, and characterize their binding affinities and selectivities using a convenient dye-displacement method. We also show that they can disrupt the protein-protein interaction of H3K27me3 with the chromobox homolog 7 (CBX7), a methyllysine reader protein, using fluorescence polarization. These results show that sub-micromolar potencies are achievable with this family of host compounds, and suggest the possibility of their use as new tools to induce the disruption of methyllysine-mediated protein-protein interactions and to report on lysine methylation in vitro.

The cation-π interaction at protein-protein interaction interfaces: developing and learning from synthetic mimics of proteins that bind methylated lysines.

First discovered over 60 years ago, post-translational methylation was considered an irreversible modification until the initial discoveries of demethylase enzymes in 2004. Now researchers understand that this process serves as a dynamic and complex control mechanism that is misregulated in numerous diseases. Lysine methylation is most often found on histone proteins and can effect gene regulation, epigenetic inheritance, and cancer. Because of this connection to disease, many enzymes responsible for methylation are considered targets for new cancer therapies. Although our understanding of the biology of post-translational methylation has advanced at an astonishing rate within the last 5 years, chemical approaches for studying and disrupting these pathways are only now gaining momentum. In general, enzymes methylate lysine and arginine residues with very high specificity for both the location and methylation state. Each methylated target serves as the focused hot spot for an inducible protein-protein interaction (PPI). Conceptually, lysine or arginine methylation is a subtle modification that leads to no change in charge and small changes in size, but it significantly alters the hydration energies and hydrogen bonding potential of these side chains. Nature has evolved a special motif for recognizing the methylation states of lysine, called the "aromatic cage", a collection of aromatic protein residues, often accompanied by one or more neighboring anionic residues. The combination of favorable cation-π, electrostatic, and van der Waals interactions, as well as size matching, gives these proteins a high degree of specificity for the methylation state. This Account summarizes the development of various supramolecular host system scaffolds developed to recognize and bind to ammonium cations, such as trimethyllysine, on the basis of their methylation state. Early systems bound to their targets in pure, buffered water but failed to achieve biochemically relevant affinities and selectivities. Surprisingly, the use of the simple and very well-known p-sulfonatocalix[4]arene provides protein-like affinities and selectivities for trimethyllysine in water. New analogs, created by synthetic modification of the same scaffold, allow for further tuning of affinities and selectivities for trimethyllysine. Our studies of each family of hosts paint a consistent picture: cation-π interactions and electrostatics are important, and solvation effects are complex. Rigidity is especially important for host-guest systems that function in pure water. Despite their simplicity, synthetic systems that take these lessons into account can achieve affinities that rival or surpass those of their naturally evolved counterparts. The stage is now set for the next act: the use of such compounds as tunable and adaptable tools for modern chemical biology.

Antibody-free reading of the histone code using a simple chemical sensor array.

The histone code refers to the complex network of histone post-translational modifications that control gene expression and are of high interest as drivers of a large number of human diseases. We report here on a mix-and-match toolkit of readily available dyes and calixarene host molecules that can be combined to form dye-displacement sensors that respond to a wide variety of cationic peptides. Using the data from only two or three such simple supramolecular sensors as a chemical sensor array produces fingerprints of data that discriminate robustly among many kinds of histone code elements. "Reads" that are accomplished include the discrimination of unmethylated, mono-, di-, and trimethylated lysines on a single histone tail sequence, identification of different modifications and combinations of modifications on a single histone tail sequence, identification of a single modification type in several different sequence contexts, and identification of isomeric dimethylarginine modifications. Reads that are sometimes troublesome for antibodies are achieved. We also report on the ability of the sensor array to report simultaneously on the concentrations and identities of histone modifications. This sensor array discriminates between post-translationally modified analytes without being limited to partners that contain a single, programmed binding interaction.

Synthesis of new trisulfonated calix[4]arenes functionalized at the upper rim, and their complexation with the trimethyllysine epigenetic mark.

A synthetic route to produce a new family of trisulfonated calix[4]arenes bearing a single group, selectively introduced, that lines the binding pocket is reported. Ten examples, including new sulfonamide and biphenyl-substituted hosts, each with additional binding elements, demonstrate the tuning of guest affinities and selectivities. NMR titrations in phosphate-buffered water show that one of the new hosts binds to the modified amino acid trimethyllysine with the highest affinity and selectivity observed to date.