A high-throughput platform to identify selective allosteric inhibitors of the PLC-y isozymes

用于鉴定 PLC-y 同工酶选择性变构抑制剂的高通量平台

• 批准号: 10185322
• 负责人: Kenneth Hugh Pearce
• 金额: $ 56.97万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10185322
• 关键词: Active Sites; Adult T-Cell Leukemia/Lymphoma; Allosteric Regulation; B-Cell Leukemia; B-Lymphocytes; Binding; Biochemical; Biological Assay; Biophysics; Catalytic Domain; Cell physiology; Cellular Assay; Cellular biology; Chemicals; Chemotaxis; Cleaved cell; Clinical; Collection; Combination Drug Therapy; Cutaneous T-cell lymphoma; Development; Diglycerides; Etiology; Fluorescence; Genetic; Genetic Transcription; Goals; Growth Factor; Hemangiosarcoma; Hematologic Neoplasms; Hormones; Human; Immune System Diseases; Immunologic Receptors; Inflammatory; Inositol; Isoenzymes; Lead; Length; Ligands; Malignant Neoplasms; Mediating; Membrane; Modeling; Monitor; Mutate; Mutation; Nerve; Neurotransmitters; Patient Care; Patients; Peripheral; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase; Phospholipase C; Phospholipids; Phosphorylation; Protein Isoforms; Protein Kinase C; Proteins; Receptor Protein-Tyrosine Kinases; Recurrent disease; Refractory Disease; Regulation; Resolution; Role; Second Messenger Systems; Signal Transduction; Signaling Protein; Stimulus; Structure; T-Cell Lymphoma; T-Cell Receptor; T-Lymphocyte; Therapeutic; Therapeutic Agents; Time; Toxic effect; Ursidae Family; analog; base; design; drug development; extracellular; high throughput screening; human disease; inhibitor/antagonist; leukemia/lymphoma; migration; novel; personalized medicine; phospholipase inhibitor; response; small molecule inhibitor; standard of care; success; therapeutic development; therapeutic target; tool; treatment response

ABSTRACT The two PLC-gamma isozymes (PLC-gamma1, -gamma2) are central to the relay and amplification of signals originating from numerous receptor tyrosine kinases and immune receptors, including the B and T cell receptors. When this control is dysregulated, the PLC-gamma isozymes contribute to inflammatory and immunological diseases as exemplified by various leukemias and lymphomas. For example, PLC-gamma1 is the most frequently (~40%) mutated protein in patients with adult T cell leukemia/lymphoma; the mutated proteins are invariably constitutively active. Similarly, mutated, constitutively active forms of PLC-gamma2 arise in response to treatment of B cell leukemias and lead to refractory disease. Despite the obvious causality between the PLC-gamma isozymes and hematologic cancers, there are no chemical probes or breakthrough therapeutics that specifically target the PLC-gamma isozymes. This deficiency arises mainly from our incomplete understanding of how the PLC-gamma isozymes are regulated as well as inadequate tools to monitor this regulation in a manner amenable to high-throughput screens. We recently determined the first structure of a full-length PLC-gamma isozyme at atomic resolution and used it to develop an allosteric model of its regulation. In addition, we created XY-69, the first fluorescent, membrane-bound analog of PIP2 that reliably captures the allosteric activation of PLC-gamma isozymes in response to either extracellular stimuli or mutation. In preliminary studies, we show that XY-69 can be used to identify both orthosteric and allosteric inhibitors in high-throughput screens. Consequently, the overall objective of this proposal is to identify selective inhibitors of the PLC-gamma isozymes to be used as chemical probes and leads for drug development. Two Specific Aims will be pursued. In Aim 1, we will use a novel high-throughput screen to identify drug-like compounds that selectively inhibit the PLC-gamma isozymes. In Aim 2, these compounds will be profiled and prioritized for further optimization using a battery of biochemical, biophysical and cellular assays that emphasize T cell biology and chemotaxis. Ultimately, these inhibitors will be essential, high-demand probes used to dissect cellular processes controlled by the PLC-gamma isozymes. Moreover, these inhibitors will be promising leads for the development of drugs to treat various hematologic cancers driven by the PLC-gamma isozymes.

摘要 两种PLC-γ同工酶（PLC-γ 1，-γ 2）是源自许多受体酪氨酸激酶和免疫受体（包括B和T细胞受体）的信号传递和放大的中心。当这种控制失调时，PLC-γ同工酶导致炎症和免疫疾病，如各种白血病和淋巴瘤。例如，PLC-γ 1是成人T细胞白血病/淋巴瘤患者中最常见（约40%）的突变蛋白质;突变蛋白质总是具有组成性活性。类似地，PLC-γ 2的突变的组成型活性形式响应于B细胞白血病的治疗而产生，并导致难治性疾病。尽管PLC-γ同工酶和血液癌症之间存在明显的因果关系，但没有特异性靶向PLC-γ同工酶的化学探针或突破性治疗。这种缺陷主要是由于我们对PLC-γ同工酶如何调节的不完全理解以及以适合高通量筛选的方式监测这种调节的工具不足。 我们最近确定了原子分辨率的全长PLC-γ同工酶的第一个结构，并用它来开发其调节的变构模型。此外，我们还创建了XY-69，这是第一个荧光膜结合的PIP 2类似物，可以可靠地捕获PLC-γ同工酶响应细胞外刺激或突变的变构激活。在初步研究中，我们表明XY-69可用于在高通量筛选中鉴定正构和变构抑制剂。因此，本提案的总体目标是确定PLC-γ同工酶的选择性抑制剂，用作药物开发的化学探针和先导物。 将有两个具体目标。在目标1中，我们将使用一种新的高通量筛选来鉴定选择性抑制PLC-γ同工酶的药物样化合物。在目标2中，将使用一系列强调T细胞生物学和趋化性的生化、生物物理和细胞测定来对这些化合物进行分析和优先排序，以进一步优化。最终，这些抑制剂将是必不可少的，高需求的探针，用于解剖PLC-γ同工酶控制的细胞过程。此外，这些抑制剂将是开发药物以治疗由PLC-γ同工酶驱动的各种血液癌症的有希望的线索。