Search

Increased expression and hyperactivation of the methyltransferase SETDB1 are commonly observed in cancer and central nervous system disorders. However, there are currently no reported SETDB1-specific methyltransferase inhibitors in the literature, suggesting this is a challenging target. Here, we disclose that the previously reported small-molecule ligand for SETDB1’s Triple Tudor Domain, (R,R)-59, is unexpectedly able to increase SETDB1 methyltransferase activity bothin vitroand in cells. Specifically, (R,R)-59 promotesin vitroSETDB1-mediated methylation of lysine 64 of the protein kinase Akt1. Treatment with (R,R)-59 also increased Akt1 threonine 308 phosphorylation and activation, a known consequence of Akt1 methylation, resulting in stimulated cell proliferation in a dose-dependent manner. (R,R)-59 is the first SETDB1 small-molecule positive activator for the methyltransferase activity of this protein. Mechanism of action studies show that full-length SETDB1 is required for significantin vitromethylation of an Akt1-K64 peptide, and that this activity is stimulated by (R,R)-59 primarily through an increase in catalytic activity rather than a change in SAM binding.Abstract Figure

Quantification of IHC staining

Extended methods and supplementary figures

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss.Significance:The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer.See related commentary by Bakouny and Barbie, p. 348.This article is highlighted in the In This Issue feature, p. 327

STING acts as a cytosolic nucleotide sensor to trigger host defense upon viral or bacterial infection. While STING hyperactivation can exert anti-tumor effects by increasing T-cell filtrates, in other contexts hyperactivation of STING can contribute to autoimmune and neuroinflammatory diseases. Several STING targeting agonists and a smaller subset of antagonists have been developed, yet STING targeted degraders, or PROTACs, remain underexplored. Here, we report a series of STING-agonist derived PROTACs that promote STING degradation in renal cell carcinoma (RCC) cells. We show that our STING PROTACs activate STING and target activated/phospho-STING for degradation. Locking STING on the endoplasmic reticulum via site-directed mutagenesis disables STING translocation to the proteasome and resultingly blocks STING degradation. We also demonstrate that PROTAC treatment blocks downstream signaling events and attenuates the anti-viral response. Interestingly, we find that VHL acts as a bona fide E3 ligase for STING in RCC; thus, VHL-recruiting STING PROTACs further promote VHL-dependent STING degradation. Our study reveals the design and biological assessment of VHL-recruiting agonist-derived STING PROTACs, as well as demonstrates an example of hijacking a physiological E3 ligase to enhance target protein degradation.

CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FK506-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.

Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.

The heterochromatin protein 1 (HP1) sub-family of CBX chromodomains are responsible for the recognition of histone H3 lysine 9 tri-methyl (H3K9me3)-marked nucleosomal substrates through binding of the N-terminal chromodomain. These HP1 proteins, namely, CBX1 (HP1β), CBX3 (HP1γ), and CBX5 (HP1α), are commonly associated with regions of pericentric heterochromatin, but recent literature studies suggest that regulation by these proteins is likely more dynamic and includes other loci. Importantly, there are no chemical tools toward HP1 chromodomains to spatiotemporally explore the effects of HP1-mediated processes, underscoring the need for novel HP1 chemical probes. Here, we report the discovery of HP1 targeting peptidomimetic compounds, UNC7047 and UNC7560, and a biotinylated derivative tool compound, UNC7565. These compounds represent an important milestone, as they possess nanomolar affinity for the CBX5 chromodomain by isothermal titration calorimetry (ITC) and bind HP1-containing complexes in cell lysates. These chemical tools provide a starting point for further optimization and the study of CBX5-mediated processes.

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

Methyl-lysine (Kme) reader domains are prevalent in chromatin regulatory proteins which bind post-translational modification sites to recruit repressive and activating factors; therefore, these proteins play crucial roles in cellular signaling and epigenetic regulation. Proteins that contain Kme domains are implicated in various diseases, including cancer, making them attractive therapeutic targets for drug and chemical probe discovery. Herein, we report on expanding the utility of a previously reported, Kme-focused DNA-encoded library (DEL), UNCDEL003, as a screening tool for hit discovery through the specific targeting of Kme reader proteins. As an efficient method for library generation, focused DELs are designed based on structural and functional features of a specific class of proteins with the intent of novel hit discovery. To broadly assess the applicability of our library, UNCDEL003 was screened against five diverse Kme reader protein domains (53BP1 TTD, KDM7B JmjC-PHD, CDYL2 CD, CBX2 CD, and LEDGF PWWP) with varying structures and functions. From these screening efforts, we identified hit compounds which contain unique chemical scaffolds distinct from previously reported ligands. The selected hit compounds were synthesized off-DNA and confirmed using primary and secondary assays and assessed for binding selectivity. Hit compounds from these efforts can serve as starting points for additional development and optimization into chemical probes to aid in further understanding the functionality of these therapeutically relevant proteins.

The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of “reader” proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein–protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.

Responsible for interpreting histone post-translational modifications (PTMs), epigenetic reader proteins have emerged as novel therapeutic targets for a wide range of diseases. Chemical probes have been critical in enabling target validation studies and have led to translational advances in cancer and inflammation-related pathologies. Here, we present the most recently reported probes of reader proteins that recognize acylated and methylated lysine. We will discuss challenges associated with achieving potent antagonism of reader domains and review ongoing efforts to overcome these hurdles, focusing on targeting strategies including the use of peptidomimetic ligands, allosteric modulators, and protein degraders.

The dynamic nature of histone post-translational modifications such as methylation or acetylation makes possible the alteration of disease associated epigenetic states through the manipulation of the associated epigenetic machinery. One approach is through small molecule perturbation. Chemical probes of epigenetic reader domains have been critical in improving our understanding of the biological consequences of modulating their targets, while also enabling the development of novel probe-based reagents. By appending a functional handle to a reader domain probe, a chemical toolbox of reagents can be created to facilitate chemiprecipitation of epigenetic complexes, evaluate probe selectivity, develop in vitro screening assays, visualize cellular target localization, enable target degradation and recruit epigenetic machinery to a site within the genome in a highly controlled fashion.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein-methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure-activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation-π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends.

Bivalent chemical degraders provide a catalytic route to selectively degrade disease-associated proteins. By linking target-specific ligands with E3 ubiquitin ligase recruiting ligands, these compounds facilitate targeted protein ubiquitination and degradation by the proteasome. Due to the complexity of this multistep mechanism, the development of effective degrader molecules remains a difficult, lengthy, and unpredictable process. Since degraders are large heterobifunctional molecules, the efficacy of these compounds may be limited by poor cell permeability, and an efficient and reliable method to quantify the cell permeability of these compounds is lacking. Herein, we demonstrate that by the addition of a chloroalkane tag on the BRD4 specific degrader, MZ1, cell permeability can be quantified via the Chloroalkane Penetration Assay. By extending this analysis to individual components of the degrader molecule, we have obtained structure-permeability relationships that will be informative for future degrader development, particularly as degraders move into the clinic as potential therapeutics.

Plant homeodomain finger protein 1 (PHF1) is an accessory component of the gene silencing complex polycomb repressive complex 2 and recognizes the active chromatin mark, trimethylated lysine 36 of histone H3 (H3K36me3). In addition to its role in transcriptional regulation, PHF1 has been implicated as a driver of endometrial stromal sarcoma and fibromyxoid tumors. We report the discovery and characterization of UNC6641, a peptidomimetic antagonist of the PHF1 Tudor domain which was optimized through in silico modeling and incorporation of non-natural amino acids. UNC6641 binds the PHF1 Tudor domain with a K(d) value of 0.96 ± 0.03 μM while also binding the related protein PHF19 with similar potency. A crystal structure of PHF1 in complex with UNC6641, along with NMR and site-directed mutagenesis data, provided insight into the binding mechanism and requirements for binding. Additionally, UNC6641 enabled the development of a high-throughput assay to identify small molecule binders of PHF1.

NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 ± 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

Canonical targeting of Polycomb Repressive Complex 1 (PRC1) to repress developmental genes is mediated by cell type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7 and 8). Based on their central role in silencing and their misregulation associated with human disease including cancer, CBX proteins are attractive targets for small molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids and participation in variant PRC1. We show that treatment with UNC7040 leads to efficient PRC1 chromatin eviction, loss of silencing and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only revealed the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of polycomb regulation by non-histone lysine methylated interaction partners.

Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein–protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a K(d) of 3.4 μM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.

SummaryTranscriptional silencing of HIV generates a reservoir of latently infected cells, but the mechanisms that lead to this outcome are not well understood. We characterized a primary cell model of HIV latency, and observed that latency is a stable, heritable viral state that is rapidly reestablished after stimulation. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit reduced proviral accessibility, elevated activity of Forkead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs. Latency reversing agents caused distinct patterns of chromatin reopening across the provirus. Furthermore, depletion of a chromatin domain insulator, CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression.

[Image: see text] A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.

Summary The CDY (chromodomain on the Y) proteins play an essential role in normal spermatogenesis and brain development. Dysregulation of their expression has been linked to male infertility and various neurological diseases. Like the chromodomains of HP1 and Polycomb, the CDY chromodomains also recognize the lysine-methylated ARKS motif embedded in histone and non-histone proteins. Interestingly, the CDY chromodomains exhibit different binding preferences for the lysine-methylated ARKS motif in different sequence contexts. Here, we present the structural basis for selective binding of CDY1 to H3K9me3 and preferential binding of CDYL2 to H3tK27me3 over H3K27me3. In addition, we use a CDYL1/2-selective compound, UNC4850, to gain further insight into the molecular mechanisms underlying CDYL2 binding specificity. Our work also provides critical implications that CDYL1b's role in the regulation of neural development is dependent on its recognition of the lysine-methylated ARKS motif.

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss. Significance: The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer. See related commentary by Bakouny and Barbie, p. 348. This article is highlighted in the In This Issue feature, p. 327

Microbial β-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS–GlcA catalytic intermediate as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm the GUS-dependent formation of inhibitor–glucuronide conjugates by LC–MS and show that methylated piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved piperazine- and piperidine-containing drugs from many classes, including those for the treatment of depression, infection, and cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of therapeutics may impact GUS activities in the human gut., Piperazine-containing compounds are substrate-dependent inhibitors of bacterial β-glucuronidases that intercept the glucuronic-acid-linked catalytic intermediate.

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure–activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.

Preclinical Research Discerning a mechanistic understanding of the cause-and-effect relationships between chromatin post-translational modifications (PTMs) and DNA accessibility for replication, transcription, and repair is an elusive goal being pursued using molecular and cellular biology, biochemistry, and more recently chemical inhibition. Chemical intervention of the chromatin-associated complexes that regulate PTM maintenance and chromatin structure faces numerous challenges due to the broad surface-groove interactions between many of these proteins and histones; yet, the increasing interest in understanding chromatin-modifying complexes suggests tractable lead compounds will be critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains. Peptides and peptidomimetics afford several advantages to efficient inhibitor development including a rational starting point, modular assembly, and retention of secondary structure. Numerous peptide technologies have been employed in the chromatin field to characterize substrate interactions, evaluate ligand selectivity, and optimize potent peptidomimetic inhibitors. We describe the progress and advantages of these efforts, and provide a perspective on their implications for future chemical probe and drug discovery efforts. Drug Dev Res, 2017. © 2017 Wiley Periodicals, Inc.

The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.

The CDY (Chromodomain on the Y) family is a small family of chromodomain containing proteins, whose chromodomains closely resemble those in HP1 and Polycomb. The CDY proteins play an essential role in normal spermatogenesis and brain development. Dysregulation of their expression has been linked to male infertility and various neurological diseases. Like the chromodomains of HP1 and Polycomb, the CDY chromodomains also recognize the lysine-methylated ARKS motif embedded in histone and non-histone proteins. Interestingly, the CDY chromodomains exhibit different binding preferences for the lysine-methylated ARKS motif in different sequence contexts. Here, we present the structural basis for selective binding of CDY1 to H3K9me3 and preferential binding of CDYL2 to H3tK27me3 over H3K27me3. Based on our structural, binding and mutagenesis data, we synthesized a more CDYL1/2 selective peptidic ligand UNC4850. Our work provides critical implications that CDYL1b’s role in the regulation of neural development is dependent on its recognition of lysine-methylated ARKS motifs.

Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates., Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to promote gene silencing. Here the authors use reversible tethering of Polycomb proteins to ectopic sites in mouse ESCs and find that canonical but not variant PRC1 can trigger sequence-independent propagation of Polycomb-mediated transcriptional repression.

Many common disease-causing mutations result in loss-of-function (LOF) of the proteins in which they occur. LOF mutations have proven recalcitrant to pharmacologic intervention, presenting a challenge for the development of targeted therapeutics. Polycomb repressive complex 2 (PRC2), which contains core subunits (EZH2, EED, and SUZ12), regulates gene activity by trimethylation of histone 3 lysine 27. The dysregulation of PRC2 catalytic activity by mutations has been implicated in cancer and other diseases. Among the mutations that cause PRC2 malfunction, an I363M LOF mutation of EED has been identified in myeloid disorders, where it prevents allosteric activation of EZH2 catalysis. We describe structure-based design and computational simulations of ligands created to ameliorate this LOF. Notably, these compounds selectively stimulate the catalytic activity of PRC2-EED-I363M over wildtype-PRC2. Overall, this work demonstrates the feasibility of developing targeted therapeutics for PRC2-EED-I363M that act as allosteric agonists, potentially correcting this LOF mutant phenotype.

Chromatin regulatory complexes localize to specific sites via recognition of posttranslational modifications (PTMs) on N-terminal tails of histone proteins (e.g., methylation, acetylation, and phosphorylation). Molecular recognition of modified histones is mediated by "reader" protein subunits. The recruited complexes govern processes such as gene transcription, DNA replication, and chromatin remodeling. Dysregulation of histone modifications and consequent downstream effects have been associated with a variety of disease states, leading to an interest in developing small-molecule inhibitors of reader proteins. Herein, we describe a generalized time-resolved fluorescence resonance energy transfer (TR-FRET) assay for a panel of methyl-lysine (Kme) reader proteins. These assays are facile, robust, and reproducible. Importantly, this plug-and-play assay can be used for high-throughput screening (HTS) campaigns, generation of structure-activity relationships (SARs), and evaluation of inhibitor selectivity. Successful demonstration of this assay format for compound screening is highlighted with a pilot screen of a focused compound set with CBX2. This assay platform enables the discovery and characterization of chemical probes that can potently and selectively inhibit Kme reader proteins to ultimately accelerate studies of chromatin reader proteins in normal biology and disease states.

While the repressive roles of Polycomb complexes have been well-established with in vitro systems, it remains unclear how subtypes of these complexes function in living cells. Conditional alleles have enabled studies of Polycomb Repressive Complex 1 (PRC1), but the available timescales on which inducible deletion occurs is not sufficient to define its direct regulatory targets. We have used short-term treatments with a new PRC1 probe UNC4976, that antagonizes recognition of H3K27me3 by the chromodomain-containing subunit CBX7. This methyl-lysine binding domain targeted strategy has enabled the discovery that hundreds of chromatin loop and contact domains are repressed by the chromodomain function of PRC1. Repressive chromatin architecture that is dependent specifically upon methyl-lysine recognition by the PRC1 chromodomain is rapidly de-repressed by 4 hours of CBX antagonism. Utilizing Hi-C experiments after compound treatment, and defining UNC4976-sensitive chromatin by ChIP-seq before major transcriptional changes occur, we characterize, for the first time, direct canonical PRC1-dependent chromatin architecture in human T lymphocytes.

Polycomb-directed repression of gene expression is frequently misregulated in human diseases. A quantitative and target-specific cellular assay was utilized to discover the first potent positive allosteric modulator (PAM), UNC4976, of nucleic acid binding by CBX7, a chromodomain methyl-lysine reader of Polycomb Repressive Complex 1. The PAM activity of UNC4976 resulted in enhanced efficacy across three orthogonal cellular assays by simultaneously antagonizing H3K27me3-specific recruitment of CBX7 to target genes while increasing non-specific binding to DNA and RNA. PAM activity thereby reequilibrates PRC1 away from H3K27me3 target regions. Together, our discovery and characterization of UNC4976 not only revealed the most cellularly potent PRC1-specific chemical probe to date, but also uncovers a potential mechanism of Polycomb regulation with implications for non-histone lysine methylated interaction partners.

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency., Author summary HIV is able to persist during antiviral therapy by entering a state of viral latency, in which viral gene expression is greatly reduced. These latently infected cells can re-seed infection if therapy is interrupted, and thus represent a major obstacle to an HIV cure. Identifying the mechanisms that lead to this state will help to identify strategies to block or eliminate HIV latency, leading to a cure for infection. By observing HIV gene expression in infected CD4 T cells, we isolated cells in which HIV has entered latency and identified characteristics that distinguish them from cells with active viral replication. We found that latently infected cells have elevated activity of specific transcription factors including Forkhead TFs and Kruppel-like factors. We also identify CTCF, a protein responsible for mediating insulation of genome domains from each other, as being required for the establishment of HIV latency. Developing agents to target these factors may lead to new strategies to eliminate the HIV reservoir.

To better understand the contribution of methyl-lysine (Kme) binding proteins to various disease states, we recently developed and reported the discovery of 1 (UNC3866), a chemical probe that targets two families of Kme binding proteins, CBX and CDY chromodomains, with selectivity for CBX4 and -7. The discovery of 1 was enabled in part by the use of molecular dynamics simulations performed with CBX7 and its endogenous substrate. Herein, we describe the design, synthesis, and structure-activity relationship studies that led to the development of 1 and provide support for our model of CBX7-ligand recognition by examining the binding kinetics of our antagonists with CBX7 as determined by surface-plasmon resonance.

We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K d of â ∼1/4100 nM for each, and is 6-to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.

Purpose: Metastatic urothelial bladder cancer (mUC) is incurable, and options are limited after patients fail 1st and 2nd line treatments. Thus, there is an unmet need to develop novel therapeutic strategies that increase survival in mUC patients. According to data from The Cancer Genome Atlas, UC has the third highest rate of somatic mutations, and mutations in DNA damage repair (DDR) genes are common. This provides rationale for the use of PARP inhibitors in UC patients with DDR mutations. Epigenetic changes are also common in UC, so epigenetic regulator inhibitors in combination with PARP inhibitors could be a viable treatment strategy in mUC. The purpose of this study was to evaluate the effects of combined olaparib plus an epigenetic regulator inhibitor in preclinical models of UC. Methods: BRCA1-competent 5637 cells and BRCA1-mutant J82 cells were screened against 62 compounds in the UNC EpiG Diamond library to identify epigenetic regulator inhibitors that could have synergy with olaparib. Cells were treated with 5 ascending concentrations of each compound (10 nM–10 µM), and cell viability was measured by alamarBlue™. RT-qPCR and western blotting were used to evaluate the effects of birabresib (OTX-015) on MYC and BRCA1 gene and c-MYC and BRCA1 protein expression, respectively. To evaluate birabresib effects on cell cycle, flow cytometry was performed. To evaluate cell viability, cells were treated with 8 ascending concentrations of olaparib and birabresib (0.1nM–100uM), alone and in combination, and then treated with CellTitrGlo™. IC50 values were calculated using a four-parameter non-linear regression model, and synergy estimates were derived by Compusyn software. Results: The EpiG screen revealed that as a class BET inhibitors potently limited cell viability in 5637 and J82 cells. Birabresib was particularly potent (IC50 in 5637 cells=100nM and in J82 cells=330nM; n=2), and selected for further evaluation. After 24h, birabresib significantly inhibited both MYC and BRCA1 expression (P Conclusion: Birabresib represses MYC/c-MYC and BRCA1/BRCA1 expression. Olaparib plus birabresib induces cell cycle arrest, and appears to be synergistic. This provides rationale supporting additional lines of prospective inquiry to evaluate olaparib plus birabresib in UC. Citation Format: Kaitlyn A. Maffuid, Chad D. Torrice, Brian Hardy, William A. Murphy, Lindsey I. James, William Y. Kim, Stephen V. Frye, Kenneth J. Pearce, Daniel J. Crona. Evaluation of the effects of combined chemical inhibition of PARP and BET proteins in preclinical models of urothelial bladder cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1743.

SUMMARYProtein degradation via the use of bivalent chemical degraders provides an alternative strategy to block protein function and assess the biological roles of putative drug targets. This approach capitalizes on the advantages of small molecule inhibitors while moving beyond the restrictions of traditional pharmacology. Herein we report a first-in-class chemical degrader (UNC6852) that targets Polycomb Repressive Complex 2 (PRC2). UNC6852 contains an EED226 derived ligand and a ligand for VHL which bind to the WD40 aromatic cage of EED and CRL2VHL, respectively, to induce proteasomal degradation of PRC2 components, EED, EZH2, and SUZ12. Degradation of PRC2 with UNC6852 blocks the histone methyltransferase activity of EZH2, decreasing H3K27me3 levels in HeLa cells and diffuse large B-cell lymphoma (DLBCL) cells containing an EZH2Y641N gain-of-function mutation. UNC6852 degrades both wild type EZH2 and EZH2Y641N, and additionally displays anti-proliferative effects in this cancer model system.Abstract Figure

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure–affinity relationships for a series of Bromosporine-based bivalent bromodo-main ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques—Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance—were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein–ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

Selection of molecular targets based on disease understanding is a dominant paradigm in drug discovery. We argue that a focus on classes of targets with central roles in biology provides a complementary approach that has higher quality outcomes in early discovery efforts.

The function of EED within Polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein-protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EED’s recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low affinity methylated jumonji, AT rich interactive domain 2 (Jarid2) peptide to a smaller, more potent peptidomimetic ligand (Kd = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EED’s reader function can lead to allosteric inhibition of PRC2 catalytic activity.