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.

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.

Use of AD Informer Set compounds to explore validity of novel targets in Alzheimer's disease pathology.

Introduction: A chemogenomic set of small molecules with annotated activities and implicated roles in Alzheimer's disease (AD) called the AD Informer Set was recently developed and made available to the AD research community: https://treatad.org/data-tools/ad-informer-set/. Methods: Small subsets of AD Informer Set compounds were selected for AD-relevant profiling. Nine compounds targeting proteins expressed by six AD-implicated genes prioritized for study by Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) teams were selected for G-protein coupled receptor (GPCR), amyloid beta (Aß) and tau, and pharmacokinetic (PK) studies. Four non-overlapping compounds were analyzed in microglial cytotoxicity and phagocytosis assays. Results: The nine compounds targeting CAPN2, EPHX2, MDK, MerTK/FLT3, or SYK proteins were profiled in 46 to 47 primary GPCR binding assays. Human induced pluripotent stem cell (iPSC)-derived neurons were treated with the same nine compounds and secretion of Aß peptides (Aß40 and Aß42) as well as levels of phosphophorylated tau (p-tau, Thr231) and total tau (t-tau) peptides measured at two concentrations and two timepoints. Finally, CD1 mice were dosed intravenously to determine preliminary PK and/or brain-specific penetrance values for these compounds. As a final cell-based study, a non-overlapping subset of four compounds was selected based on single-concentration screening for analysis of both cytotoxicity and phagocytosis in murine and human microglia cells. Discussion: We have demonstrated the utility of the AD Informer Set in the validation of novel AD hypotheses using biochemical, cellular (primary and immortalized), and in vivo studies. The selectivity for their primary targets versus essential GPCRs in the brain was established for our compounds. Statistical changes in tau, p-tau, Aß40, and/or Aß42 and blood-brain barrier penetrance were observed, solidifying the utility of specific compounds for AD. Single-concentration phagocytosis results were validated as predictive of dose-response findings. These studies established workflows, validated assays, and illuminated next steps for protein targets and compounds.

AD Informer Set: Chemical tools to facilitate Alzheimer's disease drug discovery.

Introduction: The portfolio of novel targets to treat Alzheimer's disease (AD) has been enriched by the Accelerating Medicines Partnership Program for Alzheimer's Disease (AMP AD) program. Methods: Publicly available resources, such as literature and databases, enabled a data-driven effort to identify existing small molecule modulators for many protein products expressed by the genes nominated by AMP AD and suitable positive control compounds to be included in the set. Compounds contained within the set were manually selected and annotated with associated published, predicted, and/or experimental data. Results: We built an annotated set of 171 small molecule modulators targeting 98 unique proteins that have been nominated by AMP AD consortium members as novel targets for the treatment of AD. The majority of compounds included in the set are inhibitors. These small molecules vary in their quality and should be considered chemical tools that can be used in efforts to validate therapeutic hypotheses, but which will require further optimization. A physical copy of the AD Informer Set can be requested on the Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) website. Discussion: Small molecules that enable target validation are important tools for the translation of novel hypotheses into viable therapeutic strategies for AD.

Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery.

Despite the established roles of the epigenetic factor UHRF1 in oncogenesis, no UHRF1-targeting therapeutics have been reported to date. In this study, we use fragment-based ligand discovery to identify novel scaffolds for targeting the isolated UHRF1 tandem Tudor domain (TTD), which recognizes the heterochromatin-associated histone mark H3K9me3 and supports intramolecular contacts with other regions of UHRF1. Using both binding-based and function-based screens of a ~ 2300-fragment library in parallel, we identified 2,4-lutidine as a hit for follow-up NMR and X-ray crystallography studies. Unlike previous reported ligands, 2,4-lutidine binds to two binding pockets that are in close proximity on TTD and so has the potential to be evolved into more potent inhibitors using a fragment-linking strategy. Our study provides a useful starting point for developing potent chemical probes against UHRF1.