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

项目摘要 染色质修饰蛋白的失调是人类癌症中常见的改变， 这些蛋白质中的大多数涉及肿瘤发生和癌症进展的各个方面，以及 治疗阻力属于KDM 5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的赖氨酸4的甲基化。KDM 5家族的两个成员，KDM 5A和KDM 5 B， 在癌症中频繁扩增和过度表达。这些去甲基化酶的高表达对于 肿瘤发生、增殖、迁移和转移，如乳腺癌、前列腺癌、肺癌、胃癌和结肠癌 癌症以及肝细胞癌和神经母细胞瘤。此外，这些基因的表达增加， 蛋白质促进对放射疗法和靶向疗法的抗性。几种正构抑制剂已经被 开发用于靶向该酶家族α-酮戊二酸的强制性共底物的结合口袋 然而，高细胞浓度的α-KG阻碍了这些正构的细胞有效性。 化学探针我们假设，不与这种丰富的细胞竞争的化学探针， 代谢物在细胞的情况下可以具有显著的优势。我们最近的工作已经确定了PHD 1结构域， KDM 5A内的三个染色质阅读器结构域之一，作为该脱甲基酶中的变构调节位点。 在这里，我们建议通过靶向其PHD 1结构域来开发KDM 5A的变构小分子调节剂。 我们将使用高通量筛选来解决PHD 1定向化学探针的开发。具体地说， 命中将被确定在荧光偏振（FP）为基础的高通量筛选的结构多样的 250，000化合物库可在加州大学旧金山分校的小分子发现中心。点击率将根据 通过化学信息学过滤器，并通过基于正交FP的试验进行验证，以及 通过基于表面等离子体共振的结合测定。优先命中及其可用衍生物的选择性 将在针对相关染色质阅读器结构域的全面反筛选中进行评估。使用蛋白 NMR，我们将确定最有效和选择性命中的结合姿势。将进行一系列活性测定， 用于评估体外和细胞中鉴定的配体的活性和作用模式。拟议研究 有可能产生PHD 1的化学探针，扩大靶向表观遗传的小分子库， 阅读器结构域，并且能够进行KDM 5A和KDM 5 B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.