Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

SETDB1 Triple Tudor Domain Ligand, (R,R)-59, Promotes Methylation of Akt1 in Cells.

Increased expression and hyperactivation of the methyltransferase SET domain bifurcated 1 (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 that 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 both in vitro and in cells. Specifically, (R,R)-59 promotes in vitro SETDB1-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 significant in vitro methylation 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 S-adenosyl methionine binding.

Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display.

Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein-protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.

SETDB1 Triple Tudor Domain Ligand, ( R,R )-59, Promotes Methylation of Akt1 in Cells.

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 both in vitro and in cells. Specifically, ( R,R )-59 promotes in vitro SETDB1-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 significant in vitro methylation 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.

PROTAC Linkerology Leads to an Optimized Bivalent Chemical Degrader of Polycomb Repressive Complex 2 (PRC2) Components.

Bivalent chemical degraders, otherwise known as proteolysis-targeting chimeras (PROTACs), have proven to be an efficient strategy for targeting overexpressed or mutated proteins in cancer. PROTACs provide an alternative approach to small-molecule inhibitors, which are restricted by occupancy-driven pharmacology, often resulting in acquired inhibitor resistance via compensatory increases in protein expression. Despite the advantages of bivalent chemical degraders, they often have suboptimal physicochemical properties and optimization for efficient degradation remains highly unpredictable. Herein, we report the development of a potent EED-targeted PRC2 degrader, UNC7700. UNC7700 contains a unique cis-cyclobutane linker and potently degrades PRC2 components EED (DC50 = 111 nM; Dmax = 84%), EZH2WT/EZH2Y641N (DC50 = 275 nM; Dmax = 86%), and to a lesser extent SUZ12 (Dmax = 44%) after 24 h in a diffuse large B-cell lymphoma DB cell line. Characterization of UNC7700 and related compounds for ternary complex formation and cellular permeability to provide a rationale for the observed improvement in degradation efficiency remained challenging. Importantly, UNC7700 dramatically reduces H3K27me3 levels and is anti-proliferative in DB cells (EC50 = 0.79 ± 0.53 µM).

Discovery of hit compounds for methyl-lysine reader proteins from a target class DNA-encoded library.

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.

Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4.

MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.

Cdyl Deficiency Brakes Neuronal Excitability and Nociception through Promoting Kcnb1 Transcription in Peripheral Sensory Neurons.

Epigenetic modifications are involved in the onset, development, and maintenance of pain; however, the precise epigenetic mechanism underlying pain regulation remains elusive. Here it is reported that the epigenetic factor chromodomain Y-like (CDYL) is crucial for pain processing. Selective knockout of CDYL in sensory neurons results in decreased neuronal excitability and nociception. Moreover, CDYL facilitates histone 3 lysine 27 trimethylation (H3K27me3) deposition at the Kcnb1 intron region thus silencing voltage-gated potassium channel (Kv ) subfamily member Kv 2.1 transcription. Loss function of CDYL enhances total Kv and Kv 2.1 current density in dorsal root ganglia and knockdown of Kv 2.1 reverses the pain-related phenotypes of Cdyl deficiency mice. Furthermore, focal administration of a novel potent CDYL antagonist blunts nociception and attenuates neuropathic pain. These findings reveal that CDYL is a critical regulator of pain sensation and shed light on the development of novel analgesics targeting epigenetic mechanisms.

Discovery of Potent Peptidomimetic Antagonists for Heterochromatin Protein 1 Family Proteins.

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 (HP1ß), 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.

A Peptidomimetic Ligand Targeting the Chromodomain of MPP8 Reveals HRP2's Association with the HUSH Complex.

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.

Discovery of an H3K36me3-Derived Peptidomimetic Ligand with Enhanced Affinity for Plant Homeodomain Finger Protein 1 (PHF1).

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 Kd 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.

IRE1α Is a Therapeutic Target for Cystic Fibrosis Airway Inflammation.

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.

Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.

Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.

Design and Construction of a Focused DNA-Encoded Library for Multivalent Chromatin Reader Proteins.

Chromatin structure and function, and consequently cellular phenotype, is regulated in part by a network of chromatin-modifying enzymes that place post-translational modifications (PTMs) on histone tails. These marks serve as recruitment sites for other chromatin regulatory complexes that 'read' these PTMs. High-quality chemical probes that can block reader functions of proteins involved in chromatin regulation are important tools to improve our understanding of pathways involved in chromatin dynamics. Insight into the intricate system of chromatin PTMs and their context within the epigenome is also therapeutically important as misregulation of this complex system is implicated in numerous human diseases. Using computational methods, along with structure-based knowledge, we have designed and constructed a focused DNA-Encoded Library (DEL) containing approximately 60,000 compounds targeting bi-valent methyl-lysine (Kme) reader domains. Additionally, we have constructed DNA-barcoded control compounds to allow optimization of selection conditions using a model Kme reader domain. We anticipate that this target-class focused approach will serve as a new method for rapid discovery of inhibitors for multivalent chromatin reader domains.

Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader.

Protein 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. Here, we report a 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 EZH2 gain-of-function mutations. UNC6852 degrades both wild-type and mutant EZH2, and additionally displays anti-proliferative effects in this cancer model system.

A General TR-FRET Assay Platform for High-Throughput Screening and Characterizing Inhibitors of Methyl-Lysine Reader Proteins.

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.

Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1.

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 bromodomain 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.

Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2.

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 the 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.