A molecular toolbox to accelerate drug development for histone lysine methylation regulators

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PROJECT SUMMARY Nucleosomes (Nucs) are the repeating unit of chromatin structure and are decorated with diverse post- translational modifications (PTMs) to regulate gene expression. The enzymes that add and remove lysine methylation (KMTs & KDMs) on Nucs play driving roles in many diseases and are important targets for cancer therapy. However, the complexity of chromatin structure has greatly challenged the accurate characterization of these enzymes for drug development. Indeed, many KMTs & KDMs contain multiple domains that engage distinct Nuc features in a multivalent manner, underscoring the need to use full-length enzymes and Nucs to define their activity. Notably, these reagents are difficult / costly to produce, require extensive / customized assay optimization, and are not widely supported by existing high-throughput screening (HTS) platforms, driving the use of protein domains and non-physiological histone peptide-based assays. Further, the field has been stalled by the lack of target-focused chemical compound sets, which are crucial to KMT & KDM inhibitor development, mechanistic analysis, and drug design. Better tools are needed to support this key area of biomedical research. Here, EpiCypher® is directly addressing these problems with the development of EpiVance™, a comprehensive toolbox comprising dNuc substrates, full-length enzymes, validated assays, user-friendly protocols, and a KMT & KDM focused chemical library to advance chromatin research. This innovative, integrated system will enable reliable and sensitive characterization of diverse KMTs & KDMs, which will improve our understanding of these enzymes for therapeutic development. For Phase I proof-of-concept, we developed HTS-compatible enzyme assays for nine KMTs & KDMs using dNuc (or peptide) substrates. We then worked with expert medicinal chemists Drs. Jian Jin and H. Ümit Kaniskan to apply an innovative structure-based optimization strategy, expanding existing KMT & KDM inhibitors into a 200-compound set for HTS. We discovered several compounds with novel target selectivity and found that a defined G9a inhibitor also displayed potent activity towards KDM7A, illustrating the importance of rigorous counter-screening to related enzymes and demonstrating strong feasibility for our approach. In Phase II, we are partnering with Cayman Chemical to exclusively develop and commercialize the EpiVance toolbox and services for drug discovery research. In Aim 1, we will develop a series of HTS assays using full-length KMT & KDM enzymes, dNuc substrates, and highly specific antibodies. In Aim 2, we will work with Drs. Jin / Kaniskan and Cayman Chemical to expand open-source KMT & KDM inhibitors, generating an ~500 compound set. In Aim 3, we will complete end-to-end validation of this system, performing HTS using select enzyme assays from Aim 1 and the chemical set from Aim 2. This project will demonstrate the power of EpiVance for accurate enzyme characterization and inhibitor development, thereby fulfilling a major need in the chromatin field. Our team’s expertise in assay development and medicinal chemistry uniquely positions us to deliver this system, which is expected to have significant market impact.