Chemical Inhibitors of Epigenetic Methyllysine Reader Proteins

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Chemical Inhibitors of Epigenetic Methyllysine Reader Proteins Natalia Milosevich and Fraser Hof* Department of Chemistry, University of Victoria, Victoria, British Columbia V8W 3V6, Canada ABSTRACT: Protein methylation is a common post-translational modification with diverse biological functions. Methyllysine reader proteins are increasingly a focus of epigenetics research and play important roles in regulating many cellular processes. These reader proteins are vital players in development, cell cycle regulation, stress responses, oncogenesis, and other disease pathways. The recent emergence of a small number of chemical inhibitors for methyllysine reader proteins supports the viability of these proteins as targets for drug development. This article introduces the biochemistry and biology of methyllysine reader proteins, provides an overview of functions for those families of readers that have been targeted to date (MBT, PHD, tudor, and chromodomains), and reviews the development of synthetic agents that directly block their methyllysine reading functions.

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through the formation of multiprotein complexes.5,18 The major families of methyl reader domains are the plant homeodomain (PHD) fingers, WD40 repeat domains, chromatin organization modifier domains (chromodomains), Tudor domains, Agenet domains, proline-tryptophan-tryptophan-proline (PWWP) domains, and malignant brain tumor (MBT) domains.19 The latter five domains in this list are also referred to as the Royal Family domains. Methyllysine and methylarginine readers occur sometimes in the same families. Chemical inhibitors have not been reported that directly target the methylarginine reading function of any protein, so methyllysine readers are the exclusive focus of this review. Methyllysine readers typically use multiple aromatic residues to make up an “aromatic cage” for recognition of the methylated side chain cations (Figure 2).18 The recognition event in the aromatic cage is predominantly mediated through cation-pi interactions between aromatic residues in the protein and the methylammonium group of the side chain.20 Most methyllysine readers are specific for the recognition of a methylation mark of a certain size and shape. Methylated lysines Kme1, Kme2, and Kme3 vary in their size, distribution of positive charge, hydrophobicity, and ability to donate hydrogen bonds. These differences allow for specific methyllysine reader proteins to differentially recognize methylated lysine. For example, Kme1 and Kme2, which retain NH hydrogen bond donors, are often complemented by H-bond acceptor residues integrated into the aromatic cage pocket to provide methylation state specificity (Figure 2B,D,E).18 In addition to their methyllysine recognizing motif, which dictates selectivity for different degrees of methylation, most methyllysine reader proteins have evolved to recognize

ost translational modifications (PTMs) are chemical marks that increase proteomic diversity and play important roles in almost all cellular signaling processes.1,2 The roles of PTMs in epigenetic pathways were first discovered among the large number of PTMs that occur on unstructured protruding Nterminal tails of histone proteins, where the most prominent examples are acetylation, phosphorylation, ubiquitination, and methylation.1,3 Post-translational methylation is a term that most often describes side-chain methylations. “Methylation” in this context actually describes a family of related PTMs, including mainly mono-, di-, and trimethyllysine (Kme, Kme2, Kme3; Figure 1), monomethylarginine (MMA), and asymmetric and symmetric isomers of dimethylarginine (aDMA and sDMA). Diverse downstream biological effects are driven by both the location of these modifications and the degree to which the residues are methylated. Although the canonical “histone code” methylation sites were the first discovered and most heavily studied,3−6 it is increasingly clear that methylation occurs on many non-histone proteins that participate in epigenetic and gene regulation pathways.7 There are three families of “writer” methyltransferases that add methyl groups from S-adenosylmethionine to their targets. Members of two of these families are known to methylate lysinesSET-domain-containing proteins and DOT1-like proteins.8−10 Protein methylation is a dynamic modification, with lysine demethylases first discovered in 2004 and many others since discovered.11−13 Inhibitors of the enzymatic action of methyltransferases and demethylases are proceeding through preclinical and clinical trials for many indications, but are outside of the scope of this review.14−16 Methyllysine and methylarginine “readers” are proteins with domains that recognize and bind to methylation marks (Figure 2).17 They are responsible for conveying the methylation signal downstream, and they do so either by having their own catalytic functions or by recruiting other proteins to sites of action © XXXX American Chemical Society

Special Issue: Epigenetics Received: October 1, 2015 Revised: December 7, 2015

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DOI: 10.1021/acs.biochem.5b01073 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Methylation states of lysine.

Figure 2. Examples of methyl reader proteins bound to native ligands alongside a close up of the aromatic cage motif. (A) PWWP domain of BRPF1 with H3K36me3 (pdb code: 2X4W), (B) PHD finger Pygo2 with H3K4me2 (pdb code: 4UP0), (C) chromodomain CBX7 with H3K27me3 (pdb code: 4X3K), (D) MBT domain L3MBTL1 with H4K20me2 (pdb code: 2RJF), (E) Tudor domain 53BP1 with p53k382me2 peptide (2MWP).

methylated at many positions, including lysine 4 (H3K4), H3K9, H3K27, H3K36, and H3K79. The position and degree of methylation is important for signaling, and reader proteins execute their signals faithfully by recognizing and binding to residues in the sequences surrounding each methylated lysine.18,24,29−32 Methyllysine reader proteins vary in their biological roles, and many are implicated in a broad range of disease states. In general, these proteins play important biological roles in development, differentiation, and regulating cellular processes such as cell cycle regulation, DNA damage, and stress responses.4,32,33 Evidence suggests that protein methylation can be co-opted in cancer and that specific methylation marks are linked to more aggressive cancers with poor survival outcomes.4,34−36 Changes in expression of many methyl reader proteins are seen in a broad range of cancers.4,37−42 Their involvement in disease is increasingly making them targets for

additional motifs beyond the methylated lysine itself. The degree to which this impacts binding is influenced by the mode of methyllysine recognition. There are two categories of recognition, surface groove and cavity insertion. The cavity insertion recognition mode has the methylated lysine buried deep within the aromatic cage. This is commonly seen in recognition of lower methylated states, for example, in complexes with MBT domains and the Tudor domain 53BP1 (see L3MBTL1 and 53BP1 protein−ligand complexes below).18,21,22 Surface groove recognition is common for reader proteins of higher methylated states, where the binding pockets are shallow, and a larger surface is employed in recognition. This mode of binding is seen in chromodomains and PHD fingers (see CBX7-ligand complex below).23−28 In proteins utilizing the surface groove recognition motif, residues neighboring the methylated lysine can be key to molecular recognition and selectivity. Histone 3, for example, can be B


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Biochemistry Table 1. Selected Inhibitors of Methyllysine Reader Proteins

with many inhibitors currently in clinical trials.44−51 Inhibitors of methyl reader proteins are relatively far behind in their development but have increasingly been suggested as druggable targets.52 Despite their potential for modulating disease, the creation of methyllysine reader protein antagonists presents a large challenge in molecular recognition. Protein−protein interactions with shallow interaction surfaces are challenging targets in general,53,54 and many methyl reader proteins form complexes with shallower binding sites than, for example, the bromodomains that are readers of acetyllysine marks. The inhibition of methyllysine readers is further complicated by the fact that many methyllysine readers bind to their native

the development of chemical antagonists. Only six of the hundreds of known methyllysine reader proteins have been targeted. A table of the inhibitors known to bind directly to the Kme recognition motif of the six methyllysine reader proteins are shown in Table 1. New chemical tools and inhibitors for these proteins are becoming more sought after as more research links these proteins to cancer progression and other diseases.43 Other PTM-related epigenetic proteins have been shown to be valid drug targets for diverse indications such as immune, pulmonary, and neurological disorders as well as various types of cancer. Methyltransferases, deacetylases, acetyltransferases, and acetyllysine readers have been targeted for drug discovery C


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Biochemistry

Figure 3. First inhibitor UNC669 for L3MBTL1, and optimized inhibitor UNC926. (A) Inhibitor UNC669 and UNC926, (B) Cocrystal structure of UNC669 bound to L3MBTL1 (PDB code: 3P8H).67,68

functional sequence specificity. The methylation mark H4K20me1 recruits L3MBTL1 to chromatin but H3K9me1/ 2 does not.60 In vitro studies of the same histone peptides with MBT domains showed they both had similar affinities illustrating that importance of context specific binding in vivo.60 MBT domains are named after a family of tumor suppressor genes, l(3)mbt,62 and are found in L3MBT proteins as well as polycomb group proteins.40 These domains play important roles in tumor suppression via L3MBT proteins and development through PcG proteins.40 Much of the research into the biology of these proteins has focused on L3MBTL1 and L3MBTL3 as both members are important in oncogenesis. L3MBTL1 plays important roles in DNA stability and is a reader of methylated lysine on histones as well as other chromatin regulating proteins.40 L3MBTL1 features three MBT domains, and recognizes mono- and dimethyllysine marks on H1.4K26, H3K4, H3K9, H3K27, and H4K20 in vitro.21 Research showing L3MBTL1 to be important player in several gene repression pathways has made it a promising target of chemical antagonism. L3MBTL1 compacts chromatin bearing H4K20me1/2 and H1K26me1/2 and represses expression of E2F regulated genes such as c-myc.63 L3MBTL1 also binds to tumor suppressor p53 following methylation of lysine 382, repressing p53 target genes.64 Depletion of L3MBTL1 contributes to (20q-) myeloproliferative neoplasms, such as polycythermia vera by promoting erythroid differentiation.65 In general, depletion of L3MBTL1 causes DNA damage, replicative stress, and genomic instability.66 The first chemical antagonist of any methyl reader protein was developed through the design of a small molecule based on the natural histone peptide for L3MBTL1.67 Peptide fragment HFK was used in an Alphascreen assay to determine effects of modifying the lysine amine on binding affinity (verified through ITC). A pyrrolidine modified lysine was found to bind with improved affinity to L3MBTL1 compared to mono- and dimethylated lysine. This led to the examination of pyrrolidine lysine mimics as small molecule inhibitors (Figure 3). The lead compound from this work, UNC669, was initially shown to have low μM binding to L3MBTL1 with 6-fold and 10-fold selectivity over related domains L3MBTL3 and L3MBTL4.67 Follow up studies on SAR for UNC669 reported equipotent affinity for L3MBTL1 and L3MBTL3. This work highlighted an expanded set of inhibitors and a further understanding of selectivity for L3MBTL1, L3MBTL3, and L3MBTL4. UNC926 was identified to bind with a slight increase in affinity to

substrates with weak (low-to-mid micromolar) affinities. In addition to creating worries about the feasibility of making high affinity, low molecular weight antagonists, this complicates in vitro assays and high-throughput screening. Many methyllysine reader proteins bind using an induced fit mechanism, making structure-based approaches to inhibitor development challenging. The specific induced fit mechanism is not well understood for many methyllysine reader proteins, and structures of apo states may not be a realistic depiction of the binding conformation. These multiple challenges, along with the promise for reader protein inhibitors to drive new medicine, motivate this area of research.

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MBT INHIBITORS Malignant brain tumor (MBT) domains are chromatin-binding domains, which recognize mono- and dimethylated lysines at numerous H3 and H4 positions as well as on other regulatory proteins. MBT domain recognition of Kme1 or Kme2 results in repression of gene expression, and their misregulation is involved in several disease states (see below).40,55 MBT proteins complex with their native ligands in arrangements of 2−4 MBT repeats. There are nine human members of the MBT family, and each member contains MBT subdomains that occur in tandem repeats.56 The packing of repeat units can form different geometric shapes each with different functions.57,58 In most cases, only one of the MBT repeats in the array is known to bind methylated lysine.40 MBT domains consist of ca. 100 amino acid residues, and all have narrow and deep (“cavity insertion mode”) methyllysine binding pockets. In addition to their hydrophobic aromatic cage, there is a critical hydrogen bond between a remaining N−H group on mono- or dimethylated lysines and an aspartic acid residue in the reader protein. Their selectivity for Kme1 and Kme2 arises from the fact that the narrow aromatic cage pocket is too small to fit Kme3, and the hydrogen bond between the aspartate residue and methylated lysine is possible only with Kme1 and Kme2.59 Additionally, the binding pocket is too hydrophobic to bind unmethylated lysine. They are considered among the more druggable methyllysine reader proteins.52 Unlike many other methyllysine reader domains such as PHD fingers, chromodomains, and Tudor domains, many MBT domains show minimal sequence specificity in vitro due to their localized interaction in the hydrophobic aromatic cage.21,60,61 Binding studies in vivo have found that MBT domains do have D


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Biochemistry

Figure 4. Co-crystal structures of inhibitors bound to L3MBTL3. (A) L3MBTL3 with UNC2533 (4L59), (B) L3MBTL3 with UNC1215 (4FL6).69,72

Figure 5. Inhibitors UNC1679 and compound 56 for L3MBTL3 based on previous inhibitor UNC1215.75

L3MBTL1 (IC50 = 3.9 μM) through Alphascreen assay and ITC. Pull down assays with L3MBTL1 and optimized inhibitor UNC926 confirmed functional antagonism by inhibiting the association of L3MBTL1 with histone peptides.68 The discovery of the UNC669, the first inhibitor for L3MBTL1, initiated the development of the first inhibitor for a closely related MBT domain, L3MBTL3.69 There is currently little known on the role of L3MBTL3 in epigenetic regulation, but there has been research linking L3MBTL3, along with L3MBTL1, to hematopoiesis.65,66,70,71 UNC1215 was discovered as a potent and selective inhibitor for L3MBLT3 through a target-class cross-screening approach.69 The work was motivated in part by the unique structure and polyvalency of MBT domain proteins. MBT domains have multiple repeat units, and the goal was to create inhibitors with multiple Kme mimics to achieve higher affinity binding. This approach was also used with the intent to target a Tudor domain that recognizes Kme2 with adjacent basic residues. To achieve these goals, a series of dibasic compounds were screened against a family of methyllysine reader proteins including MBT domains. The dibasic lead compound from this work, UNC1215, binds to L3MBTL3 with a Kd of 120 nM determined through ITC and was shown to competitively displace mono- or dimethyllysine containing peptides. Co-crystal structures of UNC1215 and of the related inhibitor UNC2533 were solved demonstrating the unique binding in the Kme pocket of L3MBTL3 (Figure 4).69,72 UNC1215 displayed greater than 50 fold-selectivity over other MBT domains and is selective over other methyl reader proteins, as well as an array of epigenetic

and nonepigenetic targets.69 The protein and inhibitor display unique 2:2 polyvalent mode of binding. The amine meta to the aniline substituent binds in the Kme binding pocket of MBT domain 2 of one L3MBTL3, and the amine ortho to the aniline binds to MBT domain 1 of a second L3MBTL3. Cellular studies with UNC1215 showed it binds to the Kme binding pocket of L3MBTL3 in vivo.69 Studies were carried out alongside UNC1079, a negative control compound that does not bind the Kme pocket of L3MBTL3. Cellular effects observed from treatment with UNC1215 that are not present with treatment of UNC1079 are presumed to be due to inhibition of the Kme binding pocket of L3MBTL3.69 UNC1215 was used in cells to find a novel interacting partner BCLAF1, which is a protein involved in DNA damage repair and apoptosis. Recent follow-up work expands on the cellular mode of action of L3MBTL3 using UNC1215. L3MBTL3 was shown to have functional roles as a dimer in cellular studies yielding insight into potential mechanisms of action.73 UNC1215 was used in studies with the PHD finger protein 20-like 1 (PHF20L1), which is a known methyllysine reader.74 The MBT domain of PHF20L1 binds to methylated lysine 142 on DNA (cytosine-5) methyltransferase 1 (DNMT1) and regulates its turnover. Inhibition of PHF20L1 by UNC1215 in mammalian cells increased the proteasomal degradation of DNMT1, demonstrating the importance of the MBT domain of PHF20L1 in regulating levels of DNMT1.74 UNC1215 is the first inhibitor for a methyllysine reader protein meeting the criteria for a chemical probe, demonstrating potent in vitro binding and showing evidence of intracellular activity.69 E


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Biochemistry

Figure 6. Weak inhibitors of JARID1A PHD3.90 (A) Small molecules that disrupt JARID1A PHD3 binding to H3K4me3 (identified through a HaloTag-dependent assay), (B) improved amiodarone analogues that inhibit JARID1A PHD3 (identified by fluorescence polarization).

methyl-reader pocket and contain different combinations of between 2−4 aromatic and hydrophobic residues.18,27,31,76,77 Despite the variability, each domain binds to their target modification with a high degree of specificity. The PHD domain of ING2, for example, binds to H3K4me3 with significantly greater affinity than the lower methylation states Kme2 and Kme1, and does not bind to the unmodified lysine.27 There are also examples that bind similar targets bearing either unmodified lysines or methyllysines with little discrimination on the basis of the methylation state.82−85 PHD fingers are involved in many different diseases, including immunodeficiency syndromes, cancer, and neurological disorders.86−88 The first set of PHD fingers identified as histone code readers within the family were the PHD fingers of BPTF (bromodomain and PHD domain transcription factor) and those of the protein ING2 (inhibitor of growth 2). Both were discovered to bind to H3K4me3 and H3R2 concurrently in two neighboring channels that are divided by a conserved tryptophan in the PHD finger.77,79 ING2 belongs to the ING family of tumor suppressors and is important in regulating control of cell growth, DNA damage repair and apoptosis.89 Research is still uncovering the connections between dysregulation of PHD fingers and disease states, and aiding in that goal is the development of chemical probes to study the proteins role in transcriptional regulation. Currently there are two reports of inhibitors for PHD fingers in the literature.90,91 The first targets the third PHD finger of JARID1A, a protein implicated in acute myeloid leukemia (AML).37,90,92 To find novel small molecules that disrupt the binding of JARID1A PHD finger, the authors developed a novel small molecule screening strategy using HaloTag technology.93−95 96-well plates activated with HaloTag ligand captured JARID1A PHD3 HaloTag fusion. Disruption of biotin labeled H3K4me3 binding to the PHD finger by small molecule ligands was detected in an ELISA-like assay using streptavidin HRP. The NIH Clinical Collection 1 was screened using this assay, and hits were further studied using Alphascreen, affinity pulldown, and competitive fluorescence polarization assays. Disulfiram (a chelator that acts by ejecting zinc from the PHD finger), amiodarone, and tegaserod were identified as weak

Follow-up studies focused on optimization of UNC1215 to improve selectivity and cellular potency.75 Extensive SAR was conducted leading to the development of the improved inhibitors UNC1679 and 56 (Figure 5). UNC1679 binds L3MBTL3 with a Kd of 0.47 μM using ITC and showed improved selectivity for L3MBTL3 over other MBT domains including closely related L3MBTL1 (150-fold selective). Compound 56 was reported to have a Kd of 0.35 μM with even greater selectivity for L3MBTL3 over L3MBTL1 (380fold selective). An Alphascreen assay demonstrated the optimized inhibitors to be selective against 11 other methyllysine readers. Trends in binding were confirmed using an orthogonal LANCE time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Pull down studies showed 56 interacts with unlabeled L3MBTL3 in cells. The work toward improving inhibitors for L3MBTL3 demonstrated the potential to improve selectivity by modifying the amines that bind to both domain 1 and domain 2. The potential for novel Kme mimics designed on these inhibitors could allow for other methyllysine reader proteins to be targeted using similar scaffolds and strategies.

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PHD FINGER INHIBITORS PHD fingers are a large family of cysteine-rich zinc-binding modules that can selectively bind unmethylated, methylated, and acetylated lysine residues. 76 They are known to predominantly recognize methylated H3K4, with some subsets binding to H3K9me3 and/or unmodified H3 tails.27,28,77−80 There are 99 PHD fingers as reported in ChromoHub, and a subset are known to be functional methyllysine readers.56 PHD fingers are found in many proteins involved in chromatin remodeling through the recruitment of multiprotein complexes. The PHD finger itself consists of ca. 50−80 amino acid residues and is characterized by a Cys4-His-Cys3 motif that coordinates two zinc ions.76,81 PHD fingers have only recently been targeted by chemical approaches, and much is still unknown about their biological roles and interacting protein partners. Crystal structures of PHD fingers in complex with methylated histones demonstrate the versatility and variability of their aromatic cages. Many of the PHD fingers differ in their F


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Biochemistry

Figure 7. Chemical fragment inhibitors identified through NMR fragment based screen with Pygo PHD finger. (A) Chemical fragment hits CF4 and CF16 showing benzothiazole and benzimidazole scaffolds, (B) cocrystal structure of CF4 bound to the rear of the Pygo PHD finger (4UP5).91

observed by crystallography to bind the rear surface of the PHD finger, not directly in the Kme pocket. CF16 demonstrated competitive binding to Pygo with an H3K4me2-derived pentapeptide ARTKme2Q, and NMR binding studies show the ethyl group of CF16 occupying the Kme binding pocket. The fragments discovered demonstrate the ability to inhibit the Pygo PHD finger and could lead to future small molecule inhibitors of the PHD−HD1 complex and other PHD containing proteins. A recently published approach to studying PHD fingers utilized a macrocyclic calixarene, which binds to methyllysines and not directly to reader proteins, to inhibit the binding of PHD fingers to H3K4me3 in test tube and cellular experiments.100 Amino acids and post-translationally modified amino acids are known to interact with a diverse set of supramolecular hosts, and these tool compounds can provide new ways to study epigenetic pathways.101−105 Several substituted calixarenes were tested for binding and selectivity with different H3Kme3 peptides using a dye displacement assay. A parasulfonato calixarene (among others) was found to inhibit the interaction between the PHD finger of ING2 and H3K4me3. Additionally, the same calixarene was found to disrupt the binding of the PHD finger of MLL5 to H3K4me3 in cells.100

inhibitors of the interaction between JARID1A PHD3 and H3K4me3. Amiodorane analogues with varying methylation states on the ammonium ion, and with extended methylene carbon chain lengths, were synthesized (Figure 6). Unmethylated di-N-desethylamiodarone and the trimethyl variants (WAG-003 and WAG-005) were shown to inhibit JARID1A PHD3 by fluorescence polarization (IC50 values between 26 and 41 μM). Both methylated and unmethylated analogues were shown to bind 10-fold stronger to JARID1A PHD3 compared to the parent compound, amiodorane.90 The authors suggest that the amiodorane analogues were inhibiting the PHD finger independently of the Kme binding pocket. Modeling studies showed that amiodarone analogues docked into the peptide binding groove and the H3R2 binding pocket. Site-directed mutagenesis with D1629, a key residue that interacts with H3R2, supports the idea that amiodarone analogs bind in this adjacent pocket.90 In 2014 Miller et al. reported an NMR fragment screen used to identify small molecules that bound to the pocket of the Pygo PHD finger.91 Their choice of target was motivated by the interaction between Pygo-BCL9 complex and oncogenic βcatenin. Inhibition of β-catenin has proved challenging;96 however the armadillo repeat domain (ARD) of β-catenin interacts with BCL9 adaptor proteins, which in turn interact with the rear of Pygo PHD fingers through a BCL9 adapter domain called HD1. This interaction induces a change in the binding module of the PHD finger, which promotes binding to methylated H3K4.97−99 The Pygo PHD finger reader function binds H3K4me and promotes active transcription. Binding is mediated by two deep binding pockets. One pocket anchors the N-terminal alanine residue (H3A1), and the second pocket binds to the monomethylated side chain of lysine 4 (H3K4me). A library of commercially available compounds was screened in silico, and 313 identified hits were tested by NMR spectroscopy using a Pygo-HD1 construct.91 To identify compounds with improved solubility and ligand efficiency, a fragment-based NMR screen was also conducted. Following identification of benzothiazoles that bind to the PHD−HD1 interface, analogue testing resulted in the discovery of a benzimidazole that bound the K4me binding pocket of the PHD−HD1 protein (Figure 7). The mode of binding was determined using NMR in combination with docking simulations. Fragments CF4 and CF16 bind with weak affinity (2.5 mM and 7.3 mM, respectively) to the PHD− HD1 complex as determined through NMR titrations. CF4 was

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CHROMODOMAIN INHIBITORS Chromodomains (chromatin-organization-modifier domains) are a prominent family of methyllysine readers. Chromohub reports 29 chromodomains in the human genome. 56 Chromodomains consists of ca. 40−60 amino acid residues and are named after their connection to chromatin remodeling processes. The two most studied families of chromodomains are the heterochromatin (HP1) family and the polycomb (Pc) family, which are typically thought of as binders of H3K9me3 and H3K27me3, respectively.106 There are also many families of enzymes that possess chromodomains as target recruitment elements such as chromo helicase DNA-binding (CHD) proteins and chromomethylases. CHD proteins are tandem chromdomain containing proteins and interact with methylated H3K4.107 The human paralogs of HP1 and Pc are collectively called the chromobox (CBX) proteins. There are eight human CBX proteins, with CBX1/3/5 belonging to the HP1 family and CBX2/4/6/7/8 to the polycomb family. All CBX chromodomains share a conserved aromatic cage for the recognition of Kme3.24 Their aromatic cages are shallower surface recognition G


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Biochemistry

Figure 8. CBX7 cocrystal structures with peptidic and small molecule inhibitors. (A) CBX7 bound to peptide inhibitor Ac-FAYKme3S-NH2 (pdb code: 4MN3), (B) CBX7 bound to small molecule inhibitor MS37452 (pdb code: 4X3T), (C) inhibitors Ac-FAYKme3S-NH2 and MS37452.121,122

Figure 9. Lead peptidic CBX7/CBX4 inhibitors.121

pockets than those of other methyllysine reader families.23,24 Each of their aromatic cages consists of 3−4 aromatic residues that make multiple cation-pi contacts. Despite their high degree of structural similarity, there are significant differences between the two families in the residues that line the binding groove adjacent to the aromatic cages.23−26 CBX proteins use a surface recognition mode of binding and may form multiple hydrogen bond contacts with the histone tail backbone. There is

increasing evidence linking CBX proteins to stem cell regulation, development, and disease.39,108 The five human paralogs of Drosophila polycomb read trimethylated lysine 27 on histone 3 (H3K27me3) and bind with significantly reduced binding to lower methylated states.24 They are responsible for cellular differentiation during development and are involved in cancer progression and stem cell maintenance via transcriptional repression.42 The CBX polycombs can each participate in different versions of the H


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Biochemistry

Figure 10. 53BP1 cocrystal structures with small molecule inhibitor. (A) Structure of UNC2170, and (B) its cocrystal structure with the 53BP1 tandem Tudor domain (pdb code: 4RG2).136

pyruvate kinase and promote protein dimerization.123,124 These early hit compounds were not reported in further testing. MS37452 was the solitary hit confirmed from the L1 library with a Kd of 29 μM determined by NMR titration, and similar values obtained by fluorescence anisotropy. MS37452 is selective for the polycomb paralogs CBX2/4/6/7/8 over the related chromodomain subgroup heterochromatin 1 (HP1) consisting of CBX1/3/5. Within the polycomb paralogs, MS37452 is 3-fold selective for CBX7 over CBX4 and 10fold selective over CBX2/6/8. It contains a dimethoxybenzene that occupies the aromatic cage of CBX7 (Figure 8B,C). The ligand adopts two distinct conformations, as observed in two different crystal forms for the complex.122 To study the effects of CBX7 inhibition in cells, chromatin immunoprecipitation (ChIP) was used to measure CBX7 occupancy across the INK4A/ARF locus (a known genetic target of CBX7 repression)125 with and without MS37452 treatment. Human PC3 prostate cancer cells treated with MS37452 showed reduced CBX7 occupancy at INK4A/ARF. The authors also report transcriptional de-repression of the gene products p14/ARF and p16/INK4a, showing that the chromodomain inhibitors have a functional effect in cells.122 The above reports of CBX7 inhibitors are significant steps toward learning about the chemical antagonism of CBX7. The peptidomimetic inhibitors, although potent, have liabilities in the form of expected low cell permeability and questionable in vivo stability. The small molecule inhibitors have relatively modest potency, necessitating the use of high inhibitor concentrations in cellular studies. 126 Further medicinal chemistry is expected to help optimize both compound classes.

multiprotein complex called polycomb repressive complex-1 (PRC1), but each is functionally distinct and plays different roles in development, stem cell regulation, and cancer.109−111 Inhibition of these proteins is very challenging due to their shallow aromatic cages that do not participate in hydrogen bonding and because of the high degree of sequence and structural similarity of the proteins within the family.24 CBX7 is the only chromodomain that has been targeted in drug design efforts. Recent literatures supports CBX7 as a potential therapeutic target for prostate cancer, lymphoma, and other aggressive stem-like cancers.110−114 CBX7 has been targeted because of the large body of evidence linking it to regulating aggressive cancer, and also because it binds with greater affinity to its native histone tail ligand compared to other polycomb paralogs, making it an easier target for chemical inhibition.24 Although CBX7’s role in cancer is the most wellknown, each CBX protein has distinct roles at different stages of various aggressive cancers.39,115−119 Activity of CBX proteins, like other epigenetic reader proteins, is tissue- and context-dependent.120 The first antagonists of CBX7 were developed using a peptide-driven approach.121 The low affinity H3K27me3 peptide was discarded in favor of a higher affinity peptide sequence from the methyltransferase SETDB1.24 Optimization was carried out by substituting each amino acid in the parent pentapeptide (FALKme3S) for both natural and un-natural modifications. Substitutions were guided by an X-ray cocrystal structure with an early pentapeptide analogue (Figure 8A,C). Several iterations of targeted library synthesis and testing provided multiple peptidomimetic CBX7 inhibitors with Kd values between 0.2 μM and 4.1 μM as determined through ITC and FP assays (Figure 9). The best leads showed selectivity for CBX7 over other CBX proteins tested (CBX8, and the more distantly related HP1 homologue CBX1), but not over the very similar chromodomain of CBX4. Subsequently, high-throughput screens of a library of FDA approved agents and an L1 library of drug-like small molecules were carried out using a fluorescence polarization assay.122 The FDA library screen resulted in several confirmed hits, which were sennoside A, suramin, aurin tricarboxylic acid, trypan blue, and Evans blue with IC50 values between 4−33 μM. These hits were all symmetric, contained polyhydroxyl groups, and had molecular masses between 420 and 1300 g/mol. Suramin is known to bind to many different proteins such as thrombin and

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TUDOR DOMAIN INHIBITORS Tudor domain proteins are a structurally diverse family of proteins that can recognize several states of methylated lysine and methylated arginine. There are 41 reported tudor domain containing proteins in the human genome,56 and little is known about their role as methyllysine reader proteins. Those that are known to be methyllysine reader proteins contain tandem Tudor domains, with the most well-known being that of the demethylase JMJD2A,127 methyllysine reader proteins p53binding protein-1 53BP1, and ubiquitin-like with PHD and ring finger domains 1 UHRF1.128−131 Tudor domains are best known for their ability to regulate RNA pathways and metabolism, and for their importance in development.132,133 I


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Biochemistry

is a fundamental difference between biological studies that rely on gene knockout or knockdown (which removes an entire multidomain reader protein from the cell) and inhibition of a reader domain with a chemical agent. A protein whose reader domain is inhibited, but whose other domains remain functional, becomes disengaged from its methyllysine targets but might retain its other functions in the cell. Inhibiting a reader protein might be better thought of as “detargeting” the protein’s other f unctions, rather than as a direct, complete inhibition of every f unction of the protein. The biological ramifications of these differences are exciting, but unknown for this class of proteins. The results reported here show that many of the above problems are surmountable for at least some methyllysine readers. But as of now, very few cell-based studies and zero animal-based studies exist. The biology of methyllysine readers is being studied intensely and is increasingly supporting the idea that their inhibitors will be useful on their own or as combination epigenetic therapies in the future. The onus now is on chemists to create chemical tool compounds that will help to unravel further the basic biology driven by methyllysine recognition and to realize the therapeutic potential of reader protein inhibitors.

p53-binding protein-1 (53BP1) is involved in DNA repair and in modulating the function of p53, the best-known human tumor suppressor. It contains a tandem Tudor domain that has been reported to bind to two different methyllysine targets. 53BP1 forms foci at sites of DNA damage in the literature that implicates the tandem Tudor domain binding H4K20me2,134 and can also bind to p53 by engaging p53K382me2.135 Both of these interactions are proposed to be mediated by a single methyllysine binding site on the tandem Tudor domain (although presumably both partners cannot bind to the reader domain simultaneously). It has been hypothesized that a 53BP1 inhibitor could rescue the deadly phenotypes associated with BRCA1 mutations, providing strong motivation for making and testing inhibitors. The UNC group again used their cross-screening approach as the basis for lead identification for this, the first Tudor domain targeted for chemical inhibition.136 Alphascreen was used to identify UNC2170 (Figure 10), a small fragment-like inhibitor of 53BP1 with Kd of 22 μM and ≥17-fold selectivity over other methyllysine readers in the cross-screening panel. This inhibitor is notable for its use of a t-butylammonium side chain to occupy the aromatic cage. A survey of many similar bulky groups on the same fragment scaffold showed that the tbutylammonium group is uniquely able to provide 53BP1 selectivity. All variations of linker chemistry between the bromoarene and t-butylammonium binding element resulted in compounds with no measurable affinity for any methyl reader protein tested, while replacement of the bromine atom with isosteres provided minor improvements in 53BP1 affinity. The inhibitory action of UNC2170 on 53BP1 was demonstrated in cell lysates. Interestingly, cell-based studies showed that UNC2170 did not reduce formation of 53BP1 loci in cells with DNA damage. This is counter to the aforementioned hypothesis that 53BP1’s methyl reader function is involved in 53BP1 recruitment to sites of damage, but leaves intact the idea that 53BP1’s other, ubiquitin-reading function is responsible for this recruitment.136 This study of competing hypotheses is one of the first examples of a chemical biology lesson derived from methyllysine reader antagonists.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Author Contributions

N.M. conceived of and wrote the manuscript. F.H. defined the scope and provided input for revisions. All authors have given approval to the final version of the manuscript. Funding

N.M. was supported by an award from WestCoast Ride to Live (#2014-01) and a Banting and Best Canada Graduate Scholarship-M from CIHR. F.H. is supported by a Canada Research Chair (#950224104). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank all coauthors and collaborators, past and present, for their contributions to our understanding of reader protein epigenetics.

CONCLUSIONS AND OUTLOOK The examples above show that inhibition of methyllysine reader proteins is a relatively new area of research, with only a handful of proteins having had inhibitors reported. Chromohub, an online database of chromatin modifers and their inhibitors maintained by the Structural Genomics Consortium, now includes many records of recently released X-ray structures of small-molecule ligands bound to several previously untargeted methyl reader proteins, suggesting that an explosion of progress is nigh.56 In spite of this progress, there remain significant challenges for the creation of inhibitors with the potencies and selectivities needed to power new chemical biology and medicinal chemistry. Methyllysine reader proteins have very low in vitro affinities for native ligands that hamper high-throughput screening. There are relatively few pharmacologically compatible methyllysine isosteres known to engage aromatic cage pockets. While selectivity between families of readers seems straightforward at this point, there remains a significant challenge in achieving selectivity among structurally similar readers within certain families. Many functional roles of methyllysine reader proteins have been elucidated and tied to disease pathology. However, there

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ABBREVIATIONS PTM, post-translational modification; MBT, malignant brain tumour; PHD, plant homeodomain; Kme, monomethylated lysine; Kme2, dimethyl lysine; Kme3, trimethyl lysine; MMA, monomethyl arginine; aDMA, asymmetric dimethyl arginine; sDMA, symmetric dimethyl arginine; CBX, chromobox; ITC, isothermal titration calorimetry; SET, Su(var)3-9 and enhancer of zeste proteins; PWWP, proline tryptophan tryptophan proline; DOT1, disrupter of telomeric silencing-1; ING2, inhibitor of growth factor 2; MLL5, mixed lineage leukemia 5; SAR, structure activity relationship; LANCE, lanthanide chelate excite; TR-FRET, time resolved fluorescence resonance energy transfer; NMR, nuclear magnetic resonance; FP, fluorescence polarization; CHD, chromodomain helicase DNA binding protein; FDA, Food and Drug Administration; IC50, inhibitory concentration that reduces effect by 50%; Kd, dissociation constant; JMJD2A, jumonji domain containing protein 2A; DNMT1, DNA (cytosine-5) methyl transferase 1; PHF20L1, J


Current Topic

Biochemistry PHD finger protein 20-like 1; 53BP1, p-53 binding protein-1; BRCA1, breast cancer 1; UHRF1, ubiquitin-like with PHD and ring finger domains 1; HP1, heterochromatin protein 1; PRC1, polycomb repressive complex 1

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Biochemistry

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Chemical Inhibitors of Epigenetic Methyllysine Reader Proteins

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