Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis

Design and Characterization of Chemical Probes Targeting the Chromodomains of Polycomb Repressive Complex I

Eukaryotic DNA is wound around histone proteins to form repeating units of nucleosomes that are packaged into higher order structures to form chromosomes. Nucleosomal packaging is dynamically regulated to control the expression of the genome through regulating access of transcription factors to specific genes in response to stimuli. Critical for these processes are modifications to the N-terminal tails of histones. In particular, methylation of specific histone lysine residues has a profound impact on transcription. This “mark” recruits protein complexes that contain methyl-lysine “reader” modules that bind to the mark and facilitate transcriptional silencing or activation of the underlying gene. Mutation and misregulation of proteins that “write,” “read” and “erase” these marks play a foundational role in the development and progression of nearly all cancers. Chemical tools that antagonize these writers, readers and erasers have been developed in recent years. These tools have been valuable in explicating the biology of these proteins, and in some cases, as therapeutic agents. Development of antagonists of methyl-lysine readers has lagged compared to both methyl-lysine writers and erasers. Herein we describe the design, development and characterization of antagonists of the chromodomain reader modules contained in Polycomb Repressive Complex I (PRC1). PRC1 contains one of five chromodomains; CBX2, -4, -6, -7 or -8. PRC1 complexes containing any one of these proteins play critical roles in normal, growth and development. Accordingly, misregulation of PRC1 chromodomains contributes to the initiation and development of numerous cancers. We developed a series of potent, peptidic antagonists of PRC1 chromodomains and an expanded set of chemical tools around one of these molecules, UNC3866, which has the highest affinity for the chromodomains of CBX4 and -7. We demonstrated the ability of UNC3866 to inhibit proliferation in a metastatic prostate cancer cells through the induction of a senescent-like state. Further optimization of UNC3866 led to a second-generation antagonist of PRC1 chromodomains with a dramatically improved residence time for CBX7 and increased cellular potency. This work has produced a valuable tool set for studying CBX biology and laid the ground work for continued efforts to evaluate and improve CBX antagonists as potential therapeutic agents.Doctor of Philosoph

DEVELOPMENT OF SMALL MOLECULES AND PEPTIDOMIMETIC LIGANDS TARGETING EPIGENETIC READER PROTEINS

The eukaryotic genome is assembled into chromatin, the complex of DNA/RNA, and histones that occupies the nucleus, and structural changes in chromatin are considered to regulate transcription from the genome by controlling the accessibility of the underlying DNA. The post-translational modifications (PTMs) on histones, including acetylation and methylation, are controlled by writers, erasers, and readers, and their dysregulation is implicated in a variety of disease states such as cancer, developmental disorders, and neurological illnesses. Epigenetic readers recognize these histone modifications, which have been deposited by the epigenetic writers, and recruit additional multi-subunit protein complexes which in turn regulate chromatin accessibility and gene activity. For example, Polycomb group (PcG) complexes form a transcriptionally repressive system by modifying chromatin structure at target genes. PcG complexes are responsive to local chromatin structure in eukaryotes, however the details of regulation by PcG complexes is still not fully understood. Several chromatin readers have multiple domains that can together recognize more than one PTM, and their multivalent interactions are considered an integral feature of chromatin regulation. Our lab has hypothesized that the relationship between the distance of two specific PTM marks and the spacing of the individual reader domains within the protein are important for these multivalent interactions. Additionally, our lab has recently discovered several peptide-derived chemical probes for PcG subunit proteins such as an in vitro ligand for embryonic ectoderm development (EED) in Polycomb Repressive Complex 2 (PRC2), and a cellular chemical probe for the chromobox homolg (CBX) readers in Polycomb Repressive Complex 1 (PRC1) through both ligand-based and structure-based approaches. Here, we used three different approaches to explore chromatin regulation: development of multivalent inhibitors to address the biophysical basis for chromatin multivalent regulation; mutant-specific allosteric activators to demonstrate the feasibility of targeted therapeutics which could potentially correct the mutant phenotype of an epigenetic regulator; and selective chemical probes for methyl-lysine reader domains to assess the roles of specific reader proteins in cancer.Doctor of Philosoph