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

基本信息

批准号：
10399533
负责人：
Kenneth Hugh Pearce
金额：
$ 58.36万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/10399533
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 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 NF-kappa B Nerve Neurotransmitters PLC gamma1 PLCgamma2 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 leukemia/lymphoma migration novel personalized medicine phospholipase C gamma phospholipase inhibitor response small molecule inhibitor standard of care success therapeutic development therapeutic target tool treatment response

项目摘要

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.

抽象的两种 PLC-gamma 同工酶（PLC-gamma1、-gamma2）对于中继和放大源自众多受体酪氨酸激酶和免疫受体（包括 B 和 T 细胞受体）的信号至关重要。当这种控制失调时，PLC-γ同工酶会导致炎症和免疫性疾病，例如各种白血病和淋巴瘤。例如，PLC-gamma1 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；突变的蛋白质总是具有组成型活性。类似地，PLC-gamma2 的突变、组成型活性形式响应 B 细胞白血病的治疗而出现，并导致难治性疾病。尽管 PLC-γ 同工酶与血液癌之间存在明显的因果关系，但目前还没有专门针对 PLC-γ 同工酶的化学探针或突破性治疗方法。这种缺陷主要源于我们对 PLC-gamma 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。我们最近在原子分辨率下确定了全长 PLC-gamma 同工酶的第一个结构，并用它开发了其调节的变构模型。此外，我们还创建了 XY-69，这是第一个荧光、膜结合的 PIP2 类似物，能够可靠地捕获 PLC-gamma 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抑制剂和变构抑制剂。因此，该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化合物。将追求两个具体目标。在目标 1 中，我们将使用一种新型高通量筛选来鉴定选择性抑制 PLC-gamma 同工酶的类药物化合物。在目标 2 中，将使用强调 T 细胞生物学和趋化性的一系列生化、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，以便进一步优化。最终，这些抑制剂将成为必不可少的、高需求的探针，用于剖析由 PLC-γ 同工酶控制的细胞过程。此外，这些抑制剂将成为开发治疗由 PLC-gamma 同工酶驱动的各种血液癌症的药物的有希望的先导。