Fraser Hof

Anion–π interaction augments halide binding in solution

1H NMR spectroscopic data and complementary theoretical predictions suggest that a designed receptor exhibits the anion-pi interaction in solution.

Molecules within molecules: recognition through self-assembly.

Synthetic molecular receptors that completely surround their target molecules can be created through the use of noncovalent interactions. These molecular capsules selectively sequester guest molecules from the influence of bulk solvent and other molecules on the basis of size, shape, and chemical complementarity. This reversible isolation spawns unique behavior within the confines of the host; the catalysis of chemical reactions and the stabilization of reactive species are possible outcomes that have been recently demonstrated. Compartmentalization of reagents can also have a dramatic effect on reactions that take place outside of the capsule, producing nonlinear kinetics in relatively simple reaction systems.

A simple calixarene recognizes post-translationally methylated lysine.

The methylation of amino acid side chains is a posttranslational modification (PTM) important for several gene regulation and developmental signaling pathways. Lysine residues that are specifically mono-, di-, and trimethylated by methyltransferase enzymes carry out their signaling function by acting as recruitment points for new protein–protein interactions. Histone methylation, in particular, is controlled by histone lysine methyl transferases (HKMTs) and lysine-specific demethylases (LSDs). Growing interest in methylation pathways has recently driven efforts at therapeutic intervention through the discovery and synthesis of HKMT and LSD inhibitors. The first members of these enzyme inhibitor families are being pursued as novel cancer therapies and as chemical agents for the generation of pluripotent cells from differentiated cell lines. The various effector domains that recognize and bind to diand trimethylated lysine residues share a common recognition motif referred to as an “aromatic cage”. These motifs consist of a highly preorganized cluster of aromatic side chains that bind the cationic Lys(Me2/3) side chains through cation–p interactions. Aromatic cage residues are often accompanied by carboxylate side chains that provide an additional electrostatic driving force for binding (Figure 1 A). 15] There exist no synthetic molecules that can distinguish between post-translational methylation states. We hypothesized that Lys(Me3) sites might be selectively recognized on the basis of their methylation state by an appropriate concave host molecule. The well-known host p-sulfonatocalix[4]arene (1, Figure 1 B) imitates the rigid multiaromatic cavity and charge complementarity of natural aromatic cages, and has been reported to bind ammonium ions in pure water. We first examined its affinity for the free amino acid lysine with all possible degrees of side-chain methylation (Lys, Lys(Me), Lys(Me2), and Lys(Me3)) using NMR titrations in D2O (40 mm Na2HPO4/ NaH2PO4, pD 7.0 = pH 7.4 ). Fitting the chemical shift data to 1:1 binding isotherms provided Kassoc values for each host– guest pair, and the 1:1 stoichiometry was confirmed by Job plot (Figure S1 in the Supporting Information). The affinity of 1 for lysine derivatives increases with increasing methylation, with an overall 70-fold selectivity for Lys(Me3) versus Lys (Table 1). Isothermal titration calorimetry (ITC) was also carried out in order to confirm Kassoc values and to determine thermodynamic parameters for the binding events. The Kassoc values and stoichiometries (N = 0.67–1.33 across all guests studied) determined by ITC agree well with the NMR spectroscopy results (Table 1). The complexation of each lysine derivative by 1, as measured by ITC at 303 K, has a large favorable enthalpic component and a smaller favorable entropic component. Increasing methylation of lysine is accompanied by significant increases in enthalpic driving force and smaller favorable changes in entropy. The binding of host 1 to other free amino acids in water has been previously studied. 22, 24, 25] The maximum reported affinity of 1 for any free amino acid under similar buffered conditions is for Arg (Kassoc = 1520 m , pH 8, 10 mm phosphate buffer). Whether comparing to this literature value or to the value we observe under our slightly more competitive experimental conditions (Kassoc = 330 m , pH 7.4, 40 mm phosphate buffer), the affinity of 1 for Lys(Me3) is far higher than for any other amino acid. We further explored this selectivity by comparison to other post-translationally modified lysines and arginines. We found, unsurprisingly, that the biologically important product of post-translational lysine acetylation (Lys(Ac)) displays only very weak binding to 1 (Table 1). The weakness of this interaction is almost certainly due to the fact that this modification renders the side chain neutral—prior studies of 1 have generally demonstrated weak binding of neutral amino acids in phosphate buffer. Figure 1. A) The aromatic cage binding motif of heterochromatin protein 1 (HP1) chromodomain bound to a trimethylated lysine side chain from histone 3. B) Host p-sulfonatocalix[4]arene (1). C) Methylated lysine derivatives used in this study. D) Other amino acids used in this study; MMA: monomethylarginine; aDMA: asymmetric dimethylarginine; sDMA: symmetric dimethylarginine.

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.

Supramolecular hosts that recognize methyllysines and disrupt the interaction between a modified histone tail and its epigenetic reader protein

Post-translational modifications of proteins (including phosphorylation, acetylation and methylation, among others) frequently carry out their biological functions by serving as ‘on’ switches for protein–protein interactions. As highly localized and perfectly defined hot-spots for protein–protein binding, they are a diverse set of motifs that collectively offer great promise as targets for therapeutic intervention and fundamental studies of chemical biology. Recent years have seen the discovery of a very large number of such modification sites on the unstructured tails of proteins, including histones and the tumor suppressor p53. These unstructured protein elements do not present concave binding pockets, and as such cannot be targeted by the conventional small-molecule agents of chemical biology and medicinal chemistry. We report here a family of calixarene-based supramolecular hosts that bind selectively and with high affinity to histone trimethyllysine motifs that are relevant to gene regulation and oncogenesis. We show that these compounds constitute a novel class of protein–protein interaction disruptors and that they can operate selectively against their targeted trimethyllysine sites even in highly complex protein substrates bearing a background of many unmethylated lysines and arginines.

Tetrazoles are potent anion recognition elements that emulate the disfavored anti conformations of carboxylic acids.

We report here the first study of the protonated, neutral form of tetrazoles as anion binding functional groups. Our studies reveal them to be capable of binding anions with extremely high potency in polar solutions. In studying carboxylic acid-containing congeners, we find a remarkable discrepancy: a strictly analogous acid-containing host binds anions > or = 50,000-fold more weakly than the tetrazole under study. We can explain this functional difference by considering tetrazole tautomerization equilibria and carboxylic acid conformational preferences.

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.

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.

Recognition properties of carboxylic acid bioisosteres: anion binding by tetrazoles, aryl sulfonamides, and acyl sulfonamides on a calix[4]arene scaffold.

Tetrazoles and acyl sulfonamides are functional groups that are common in medicinal chemistry but virtually unexplored as recognition elements in supramolecular chemistry. We report here on the anion binding properties of these highly acidic N-H functional groups. We have prepared two new calixarene-based tetrazole-containing hosts, as well as new acetyl sulfonamide and benzoyl sulfonamide hosts. We also report on analogous hosts bearing the better-known aryl sulfonamide functional group as a point of comparison. We find that these hosts are competent anion binders and that the recognition of anions by these groups is highly dependent on their conformational preferences. We also report in detail on the preferred molecular shape of each acid bioisostere as determined by calculations and structural database surveys, and discuss how these shapes impact binding in the context of the reported hosts.

(How) does 1,3,5-triethylbenzene scaffolding work? Analyzing the abilities of 1,3,5-triethylbenzene- and 1,3,5-trimethylbenzene-based scaffolds to preorganize the binding elements of supramolecular hosts and to improve binding of targets

Summary 1,3,5-triethylbenzenes have been widely used as supramolecular templates to organize molecular-recognition elements. It is believed that the steric-gearing effect of the 1,3,5-triethylbenzene template directs the binding elements toward the same face of the central ring, hence increasing the binding affinity. At the same time the 1,3,5-trimethylbenzene scaffold, without steric-gearing effects, has also been found to improve the binding affinities of hosts compared to the unsubstituted analogues. By studying experimental data from the literature and the Cambridge Structural Database, as well as by conducting computational studies of representative structures, we concluded that the steric gearing offered by the ethyl groups confers some energetic advantage over the methyl groups, but the size of this advantage can be small and is dependent on the groups involved.