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

项目总结 核小体是染色质结构的重复单位，具有不同的后染色质。 翻译修饰(PTM)调节基因表达。添加和去除赖氨酸的酶 Nucs上的甲基化(KMTs和KDms)在许多疾病中起驱动作用，是癌症的重要靶点 心理治疗。然而，染色质结构的复杂性极大地挑战了对其准确的表征。 这些酶用于药物开发。事实上，许多KMT和KDM包含多个域 以多价方式提供独特的Nuc功能，强调需要使用全长酶和NuC来 定义他们的活动。值得注意的是，这些试剂很难生产/成本很高，需要广泛/定制的分析 优化，并且没有得到现有高通量筛选(HTS)平台的广泛支持，从而推动了 使用基于蛋白质结构域和非生理性的组蛋白多肽的分析。此外，该油田已经停滞不前 由于缺乏对KMT和KDM缓蚀剂开发至关重要的靶向化合物组， 机械分析和药物设计。需要更好的工具来支持生物医学研究的这一关键领域。 在这里，EpiCypher®正在通过EpiVance™的开发直接解决这些问题， 全面的工具箱，包括dNuc底物、全长酶、有效检测、用户友好 协议，以及以KMT和KDM为重点的化学库，以促进染色质研究。这一创新的， 集成系统将能够可靠和敏感地描述不同的KMT和KDM，这将改善 我们对这些酶的理解用于治疗开发。对于第一阶段的概念验证，我们开发了 使用dNuc(或多肽)底物对九种KMT和KDM进行HTS相容酶分析。然后我们就开始工作 与专家药用化学家金健博士和HüMIT Kaniskan博士一起应用创新的结构为基础 优化策略，将现有的KMT和KDM缓蚀剂扩大为200组化合物，用于高温超导。我们 发现了几种具有新的靶标选择性的化合物，并发现定义的G9a抑制剂也显示了 对KDM7A的有效活性，说明了严格的反筛选对相关酶和 证明我们的方法具有很强的可行性。在第二阶段，我们正在与开曼化学公司合作 独家开发和商业化用于药物发现研究的EpiVance工具箱和服务。在AIM 1，我们将开发一系列使用全长KMT和KDM酶、dNuc底物和高度 特定的抗体。在目标2中，我们将与金/卡尼斯坎博士和开曼化学公司合作，扩大开源 KMT和KDM抑制剂，生成~500个化合物集。在目标3中，我们将完成对 该系统使用来自Aim 1的精选酶分析和来自Aim 2的化学试剂集进行HTS。这 该项目将展示EpiVance在准确的酶特性和抑制剂开发方面的力量， 从而满足染色质领域的主要需求。我们团队在化验开发和药物方面的专业知识 化学为我们提供这一系统提供了独特的定位，预计将产生重大的市场影响。

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.