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.

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.

Improved methods for targeting epigenetic reader domains of acetylated and methylated lysine.

Responsible for interpreting histone post-translational modifications, 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 histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells.

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.