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

项目摘要 核小体（Nucleosomes，Nucs）是染色质结构的重复单位，并且被不同的后修饰修饰， 翻译修饰（PTM）来调节基因表达。添加和去除赖氨酸的酶 Nucs上的甲基化（KMTs & KDMs）在许多疾病中起驱动作用，并且是癌症的重要靶点 疗法然而，染色质结构的复杂性极大地挑战了对染色质的准确表征。 这些酶用于药物开发。事实上，许多KMT和KDM包含多个域， 不同的Nuc特征，强调需要使用全长酶和Nucs， 定义他们的活动。值得注意的是，这些试剂生产困难/昂贵，需要大量/定制的测定， 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 使用蛋白质结构域和基于非生理组蛋白肽的测定。此外，该领域一直停滞不前， 由于缺乏对KMT和KDM抑制剂开发至关重要的靶向化合物组， 机制分析和药物设计。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®通过开发EpiVance ™直接解决了这些问题， 全面的工具箱，包括dNuc底物、全长酶、经验证的测定、用户友好的 协议，以及KMT和KDM集中的化学库，以推进染色质研究。这一创新， 集成系统将使不同的KMT和KDM的可靠和灵敏的表征，这将提高 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（或肽）底物的九种KMT和KDM的HTS相容酶测定。然后我们工作 药物化学专家Jian Jin和H.Ümit Kaniskan采用基于创新结构的 优化策略，将现有的KMT和KDM抑制剂扩展为200种化合物用于HTS。我们 发现了几种具有新靶点选择性的化合物，并发现一种确定的G9a抑制剂也显示出 对KDM7A的有效活性，说明了严格反筛选相关酶的重要性， 证明了我们的方法的可行性。在第二阶段，我们与开曼化学公司合作， 独家开发和商业化EpiVance工具箱和服务，用于药物发现研究。在aim中 1，我们将开发一系列HTS检测，使用全长KMT和KDM酶，dNuc底物，和高度的 特异性抗体在目标2中，我们将与Jin/Kaniskan博士和Cayman Chemical合作，扩大开源 KMT和KDM抑制剂，产生约500种化合物组。在目标3中，我们将完成 该系统使用来自目标1的选择酶测定和来自目标2的化学组进行HTS。这 该项目将展示EpiVance在精确酶表征和抑制剂开发方面的能力， 从而满足染色质领域的主要需求。我们的团队在检测开发和药物方面的专业知识 化学使我们能够提供这一系统，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.