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.

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.

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.

Build-Couple-Transform: A Paradigm for Lead-like Library Synthesis with Scaffold Diversity.

High-throughput screening provides one of the most common ways of finding hit compounds. Lead-like libraries, in particular, provide hits with compatible functional groups and vectors for structural elaboration and physical properties suitable for optimization. Library synthesis approaches can lead to a lack of chemical diversity because they employ parallel derivatization of common building blocks using single reaction types. We address this problem through a "build-couple-transform" paradigm for the generation of lead-like libraries with scaffold diversity. Nineteen transformations of a 4-oxo-2-butenamide scaffold template were optimized, including 1,4-cyclizations, 3,4-cyclizations, reductions, and 1,4-additions. A pool-transformation approach efficiently explored the scope of these transformations for nine different building blocks and synthesized a >170-member library with enhanced chemical space coverage and favorable drug-like properties. Screening revealed hits against CDK2. This work establishes the build-couple-transform concept for the synthesis of lead-like libraries and provides a differentiated approach to libraries with significantly enhanced scaffold diversity.

Microwave-assisted synthesis of 4-oxo-2-butenoic acids by aldol-condensation of glyoxylic acid.

4-Oxobutenoic acids are useful as biologically active species and as versatile intermediates for further derivatisation. Currently, routes to their synthesis can be problematic and lack generality. Reaction conditions for the synthesis of 4-oxo-2-butenoic acid by microwave-assisted aldol-condensation between methyl ketone derivatives and glyoxylic acid have been developed. They provide the desired products in moderate to excellent yields for a wide range of substrates, by applying a simple procedure to accessible starting materials. The investigation revealed different conditions are required depending on the nature of the methylketone substituent, with aryl derivatives proceeding best using tosic acid and aliphatic substrates reacting best with pyrrolidine and acetic acid. This substituent effect is rationalised by frontier orbital calculations. Overall, this work provides methods for synthesis of 4-oxo-butenoic acids across a broad range of substrates.