Novel Therapeutics Targeting Epigenetics: New Molecules, New Methods

The scientific community’s interest in epigenetic regulation and medicinal chemists’ ability to design potent and selective small molecule probes and drug leads have intersected in an exciting and productive fashion over the last five years resulting in rapid translation of epigenetic therapeutic hypotheses to clinical interventions

Asymmetric Chemical Synthesis of (R)- and (S)-Citramalate in High Enantiomeric Purity

Both enantiomers of dimethyl 2-acety1citramalate have been asymmetrically synthesized in over 96% enantiomeric excess and good overall chemical yield (500/0)from 2-keto-1,3-oxathianes 1a, and 1b

Chemical probes for methyl lysine reader domains

The primary intent of a chemical probe is to establish the relationship between a molecular target, usually a protein whose function is modulated by the probe, and the biological consequences of that modulation. In order to fulfill this purpose, a chemical probe must be profiled for selectivity, mechanism of action, and cellular activity, as the cell is the minimal system in which ‘biology’ can be explored. This review provides a brief overview of progress toward chemical probes for methyl lysine reader domains with a focus on recent progress targeting chromodomains

Peptide Technologies in the Development of Chemical Tools for Chromatin‐Associated Machinery

Discerning a mechanistic understanding of the cause‐and‐effect relationships between chromatin post‐translational modifications (PTMs) and DNA accessibility for replication, transcription, and repair is an elusive goal being pursued using molecular and cellular biology, biochemistry, and more recently chemical inhibition. Chemical intervention of the chromatin‐associated complexes that regulate PTM maintenance and chromatin structure faces numerous challenges due to the broad surface‐groove interactions between many of these proteins and histones; yet, the increasing interest in understanding chromatin‐modifying complexes suggests tractable lead compounds will be critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains. Peptides and peptidomimetics afford several advantages to efficient inhibitor development including a rational starting point, modular assembly, and retention of secondary structure. Numerous peptide technologies have been employed in the chromatin field to characterize substrate interactions, evaluate ligand selectivity, and optimize potent peptidomimetic inhibitors. We describe the progress and advantages of these efforts, and provide a perspective on their implications for future chemical probe and drug discovery efforts. Drug Dev Res 78 : 300–312, 2017. © 2017 Wiley Periodicals, Inc

Design, synthesis, and protein methyltransferase activity of a unique set of constrained amine containing compounds

Epigenetic alterations relate to various human diseases, and developing inhibitors of Kme regulatory proteins is considered to be a new frontier for drug discovery. We were inspired by the known multicyclic ligands, UNC669 and UNC926, which are the first reported small molecule ligands for a methyl-lysine binding domain. We hypothesized that reducing the conformational flexibility of the key amine moiety of UNC669 would result in a unique set of ligands. Twenty-five novel compounds containing a fused bi- or tricyclic amine or a spirocyclic amine were designed and synthesized. To gauge the potential of these amine-containing compounds to interact with Kme regulatory proteins, the compounds were screened against a panel of 24 protein methyltransferases. Compound 13 was discovered as a novel scaffold that interacts with SETD8 and could serve as a starting point for the future development of PKMT inhibitors

MERTK Is a Potential Therapeutic Target in Ewing Sarcoma

Outcomes are poor in patients with advanced or relapsed Ewing sarcoma (EWS) and current treatments have significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. MER proto-oncogene tyrosine kinase (MERTK) promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies in a variety of cancers. MERTK was ubiquitously expressed in five EWS cell lines and five patient samples. Moreover, data from CRISPR-based library screens indicated that EWS cell lines are particularly dependent on MERTK. Treatment with MRX-2843, a first-in-class, MERTK-selective tyrosine kinase inhibitor currently in clinical trials, decreased the phosphorylation of MERTK and downstream signaling in a dose-dependent manner in A673 and TC106 cells and provided potent anti-tumor activity against all five EWS cell lines, with IC50 values ranging from 178 to 297 nM. Inhibition of MERTK correlated with anti-tumor activity, suggesting MERTK inhibition as a therapeutic mechanism of MRX-2843. Combined treatment with MRX-2843 and BCL-2 inhibitors venetoclax or navitoclax provided enhanced therapeutic activity compared to single agents. These data highlight MERTK as a promising therapeutic target in EWS and provide rationale for the development of MRX-2843 for the treatment of EWS, especially in combination with BCL-2 inhibitors

Discovery of a Selective, Substrate-Competitive Inhibitor of the Lysine Methyltransferase SETD8

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure–activity relationships (SAR) of this series

The Lipid Kinase PIP5K1C Regulates Pain Signaling and Sensitization

SummaryNumerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain

Identification of Non-Peptide Malignant Brain Tumor (MBT) Repeat Antagonists by Virtual Screening of Commercially Available Compounds

The Malignant Brain Tumor (MBT) repeat is an important epigenetic-code “reader” and is functionally associated with differentiation, gene silencing and tumor suppression1–3. Small molecule probes of MBT domains should enable a systematic study of MBT-containing proteins, and potentially reveal novel druggable targets. We designed and applied a virtual screening strategy, which identified potential MBT antagonists in a large database of commercially available compounds. A small set of virtual hits was purchased and submitted to experimental testing. Nineteen of the purchased compounds showed a specific dose-dependent protein binding and will provide critical structure-activity information for subsequent lead generation and optimization

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

Transcriptional regulators lacking enzymatic activity or binding pockets with targetable molecular features have typically been considered “undruggable,” and a reductionist approach based on identification of their molecular targets has largely failed. We have demonstrated that the Ewing sarcoma chimeric transcription factor, EWSR1-FLI1, maintains accessible chromatin at disease-specific regions. We adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), an assay for accessible chromatin, to screen an epigenetically targeted small molecule library for compounds that reverse the disease-associated signature. This approach can be applied broadly for discovery of chromatin-based developmental therapeutics and offers significant advantages because it does not require the selection of a single molecular target. Using this approach, we identified a specific class of compounds with therapeutic potential