Phase I Amount
$1,025,085
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. Ãmit 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.
Public Health Relevance Statement: PROJECT NARRATIVE Histone lysine methyltransferases and demethylases (KMTs & KDMs) play important roles in regulating chromatin structure and gene expression, and are widely indicated in disease, making them key therapeutic targets. However, the study of KMTs & KDMs for drug development has been challenged by the lack of reliable, physiological enzyme assays and targeted chemical libraries - crucial to define enzyme activity and identify selective inhibitors. Here, EpiCypher® is developing EpiVance, a comprehensive system that will accelerate drug discovery research for high value histone lysine methylation regulators that remain largely undruggable using current technologies.
Project Terms: inhibitor; Antibodies; Automobile Driving; driving; Biological Assay; Assay; Bioassay; Biologic Assays; Biomedical Research; Pharmaceutical Chemistry; Medicinal Chemistry; Pharmaceutic Chemistry; Chromatin; High Pressure Liquid Chromatography; HPLC; High Performance Liquid Chromatography; High Speed Liquid Chromatography; Disease; Disorder; Drug Design; Engineering; Enzymes; Enzyme Gene; Gene Expression; Histones; Libraries; Lysine; L-Lysine; Methylation; Noise; Nucleosomes; Parents; Peptides; Kinases; Phosphotransferase Gene; Transphosphorylases; Phosphotransferases; Play; Histone-Lysine Methyltransferase; Protein Lysine Methyltransferase; Protein Methylase III; Protein Methyltransferase III; Histone-Lysine N-Methyltransferase; Post-Translational Modification Protein/Amino Acid Biochemistry; Post-Translational Modifications; Post-Translational Protein Modification; Posttranslational Modifications; Posttranslational Protein Processing; Protein Modification; Post-Translational Protein Processing; Reagent; Research; social role; Role; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Specificity; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Mass Spectrum Analysis; Technology; Work; Caymans; Custom; Tertiary Protein Structure; Peptide Domain; Protein Domains; base; improved; Area; Phase; Physiological; Physiologic; Series; Chemicals; enzyme activity; Letters; tool; Protocol; Protocols documentation; System; Services; preference; chemical library; small molecule libraries; Structure; novel; G Protein-Complex Receptor; G Protein-Coupled Receptor Genes; GPCR; G-Protein-Coupled Receptors; Position; Positioning Attribute; assay development; drug development; High Throughput Assay; high throughput screening; LC/MS; liquid chromatography mass spectrometry; therapeutic enzyme; MLL gene; ALL1; ALL1 gene; Acute Lymphoblastic Leukemia Protein 1; CXXC7; Drosophila Homolog of Trithorax; HRX; KMT2A; Lysine-Specific Methyltransferase 2A; MLL1; Mixed Lineage Leukemia Gene; Mixed-Lineage Leukemia Protein; Multiple lineage leukemia 1; Myeloid-Lymphoid Leukemia Gene; Myeloid-Lymphoid Leukemia Protein; Myeloid/Lymphoid Leukemia Gene; Myeloid/Lymphoid Or Mixed Lineage Leukemia Protein; Myeloid/Lymphoid or Mixed Lineage Leukemia Gene; Proto Oncogene Proteins MLL; Zinc Finger Protein HRX; drug discovery; Molecular Interaction; Binding; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; JARID1B; Jumonji, AT-Rich Interactive Domain 1B; KDM5B; Lysine-Specific Demethylase 5B; PLU-1 Protein; PLU1; PUT1; Putative DNA/Chromatin-Binding Motif 1; RBBP2H1A; Retinoblastoma-Binding Protein 2 Homolog 1A; Retinoblastoma-Binding Protein 2, Homolog 1A; KDM5B gene; Address; Length; Affinity; Resolution; Chromatin Structure; Epigenetic Process; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Validation; Molecular; Process; Development; developmental; cost; design; designing; innovation; innovate; innovative; user-friendly; open source; therapeutic target; commercialization; therapeutic development; therapeutic agent development; screening; targeted cancer therapy; ASH1L gene; ASH1L
