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

项目总结 染色质修饰蛋白的错误调节是人类癌症中的一种常见变化，并具有异常活动 这些蛋白质与肿瘤发生和癌症进展的各个方面有关，以及在 治疗耐药。组蛋白去甲基酶属于KDM5亚家族，是表观遗传的“橡皮擦”蛋白。 拮抗组蛋白H3的赖氨酸4甲基化。KDM5家族的两个成员KDM5A和KDM5B是 在癌症中经常被扩增和过度表达。这些脱甲基酶的高表达对 乳腺癌、前列腺癌、肺癌、胃癌和结肠癌的发生、增殖、迁移和转移 癌症，以及肝细胞癌和神经母细胞瘤。此外，这些基因的表达上调 蛋白质增强了对放射治疗和靶向治疗的抵抗力。几种正构体抑制剂已经被 针对这种酶家族的强制性共底物α-酮戊二酸的结合口袋而开发 (α-KG)，然而α-KG的高细胞浓度阻碍了这些整形外科的细胞有效性 化学探头。我们假设不与这种丰富的细胞竞争的化学探针 在细胞环境中，代谢物具有显著的优势。我们最近的工作已经确定了PhD1结构域， KDM5A内的三个染色质阅读器结构域之一，作为该去甲基酶中的变构调节部位。 在这里，我们建议通过靶向KDM5A的PhD1结构域来开发变构小分子调节剂。 我们将使用高通量筛选来开发PhD1导向的化学探针。具体来说， 将在基于荧光偏振(FP)的结构多样化的高通量屏幕中识别命中 加州大学旧金山分校的小分子发现中心提供了250,000个化合物库。命中数将根据优先顺序进行排序 关于它们的效力和通过化学信息学过滤器，并通过基于FP的正交分析以及 通过基于表面等离子体共振的结合实验。优先命中及其可用导数的选择性 将在一个全面的反筛选中对照相关的染色质阅读器结构域进行评估。使用蛋白质 核磁共振，我们将确定最有效和选择性打击的结合姿势。一系列的活动分析将是 用于评估已鉴定的配体在体外和细胞内的活性和作用方式。拟议的研究 有可能产生Phd1的化学探针，扩大靶向表观遗传的小分子的谱系 阅读器结构域，并实现KDM5A和KDM5B的变构调节。

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.