Discovery of Small Molecule Ligands for PHD1 Reader Domain of Histone Demethylase KDM5A

组蛋白去甲基化酶 KDM5A 的 PHD1 阅读器结构域小分子配体的发现

基本信息

批准号：
10183207
负责人：
Michelle Arkin
金额：
$ 57.77万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10183207
关键词：
Active Sites Address Binding Biological Assay Catalytic Domain Cells Chemicals Chromatin Collection Colon Carcinoma Development Disease Dose Drug Tolerance Effectiveness Enzymes Epigenetic Process Evaluation Family Fluorescence Polarization Gene Expression Histone H3 Histones Human Immune Immune Evasion In Vitro KDM5B gene Lead Libraries Ligands Lysine Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of prostate Methylation Modality Neoplasm Metastasis Neuroblastoma Oncogenic Outcome Peptides Pharmaceutical Preparations Phosphotransferases Primary carcinoma of the liver cells Process Property Proteins Quality Control Radiation therapy Reader Reproducibility Research Resistance Series Site Structural Models Structure Surface Plasmon Resonance Tail Testing Therapeutic Up-Regulation Validation Work alpha ketoglutarate base cancer cell cancer heterogeneity cancer therapy cell growth cheminformatics chromatin modification counterscreen demethylation enzyme activity high throughput screening histone demethylase hormone therapy improved inhibitor/antagonist interest knock-down malignant breast neoplasm malignant stomach neoplasm melanoma member migration novel therapeutic intervention overexpression protein expression response small molecule stoichiometry targeted treatment therapeutic target therapy resistant tumor initiation tumor progression tumorigenesis

项目摘要

PROJECT SUMMARY Misregulation of chromatin-modifying proteins is a common alteration in human cancers, and aberrant activities of these proteins are implicated in various aspects of tumorigenesis and cancer progression, as well as in treatment resistance. Histone demethylases that belong to KDM5 subfamily are epigenetic “eraser” proteins that antagonizes methylation of lysine 4 of histone H3. Two members of KDM5 family, KDM5A and KDM5B, are frequently amplified and overexpressed in cancer. Elevated expression of these demethylases is critical for tumorigenesis, proliferation, migration and metastasis in cancers such as breast, prostate, lung, gastric and colon cancer, as well as hepatocellular carcinoma and neuroblastoma. Furthermore, elevated expression of these proteins promotes resistance to radiation therapy and targeted therapy. Several orthosteric inhibitors have been developed to target the binding pocket of the obligatory co-substrate of this family of enzyme, α-ketoglutarate (α-KG), however the high cellular concentrations of α-KG impede cellular effectiveness of these orthosteric chemical probes. We hypothesize that chemical probes that do not compete with this abundant cellular metabolite can have significant advantages in the context of a cell. Our recent work has identified PHD1 domain, one of the three chromatin reader domains within KDM5A, as an allosteric regulatory site in this demethylase. Here we propose to develop allosteric small molecule modulators of KDM5A by targeting its PHD1 domain. We will address development of PHD1-directed chemical probes using high-throughput screening. Specifically, hits will be identified in a fluorescence polarization (FP)-based high-throughput screen of a structurally diverse 250,000 compounds library available at UCSF's Small Molecule Discovery Center. Hits will be prioritized based on their potency and through cheminformatics filters, and validated by an orthogonal FP-based assay as well as by surface plasmon resonance-based binding assay. Selectivity of prioritized hits and their available derivatives will be assessed in a comprehensive counter-screen against related chromatin reader domains. Using protein NMR, we will determine binding poses of the most potent and selective hits. A series of activity assays will be used to assess activity and mode of action of identified ligands, both in vitro and in cells. The proposed research has a potential to yield chemical probes for PHD1, expanding repertoire of small molecules that target epigenetic reader domains, and to enable allosteric modulation of KDM5A and KDM5B.

项目概要染色质修饰蛋白的失调是人类癌症和异常活性的常见改变这些蛋白质涉及肿瘤发生和癌症进展的各个方面，以及属于 KDM5 亚家族的组蛋白去甲基化酶是表观遗传“擦除”蛋白。拮抗组蛋白 H3 赖氨酸 4 的甲基化，KDM5 家族的两个成员 KDM5A 和 KDM5B。这些去甲基酶的表达升高对于癌症至关重要。乳腺癌、前列腺癌、肺癌、胃癌和结肠癌等癌症的肿瘤发生、增殖、迁移和转移此外，这些基因的表达也升高。蛋白质可促进对放射治疗和靶向治疗的抵抗。开发用于靶向该酶家族的必需辅助底物 α-酮戊二酸的结合口袋 (α-KG)，然而，α-KG 的高细胞浓度阻碍了这些正构蛋白的细胞有效性我们追求的是不与这种丰富的细胞竞争的化学探针。我们最近的工作已经确定了 PHD1 结构域，代谢物在细胞中具有显着的优势。 KDM5A 内的三个染色质阅读器结构域之一，作为该去甲基酶的变构调节位点。在这里，我们建议通过靶向 KDM5A 的 PHD1 结构域来开发 KDM5A 的变构小分子调节剂。我们将利用高通量筛选来开发针对 PHD1 的化学探针。命中将在基于荧光偏振（FP）的结构多样化的高通量筛选中被识别 UCSF 小分子发现中心提供的 250,000 个化合物库将根据命中进行优先排序。通过化学信息学过滤器评估其效力，并通过基于正交 FP 的测定以及通过基于表面等离子共振的结合测定的优先命中及其可用衍生物的选择性。将使用蛋白质在针对相关染色质阅读器域的全面反筛选中进行评估。 NMR，我们将确定最有效和选择性的命中的结合姿势，将进行一系列活性测定。用于评估已识别配体的体外和细胞内活性和作用方式。有潜力产生 PHD1 化学探针，扩大针对表观遗传的小分子库阅读器域，并启用 KDM5A 和 KDM5B 的变构调制。