Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

用于探测蛋白激酶高度特异性相互作用的通用纳米传感器

• 批准号: 10719635
• 负责人: LIVIU MOVILEANU
• 金额: $ 42.22万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-23 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719635
• 关键词: Address; Affinity; Algorithmic Analysis; Applications Grants; Basic Science; Binding; Biological Assay; Biosensor; Biotechnology; Caseins; Catalytic Domain; Cell physiology; Competitive Binding; Complex; Cyclic AMP-Dependent Protein Kinases; Dependence; Detection; Development; Devices; Diagnostic; Drug Targeting; Electrophysiology (science); Elements; Engineering; Enzymatic Biochemistry; Enzymes; Epidermal Growth Factor Receptor; Event; FDA approved; Face; Generations; Grain; Growth Factor; Healthcare; Hematologic Neoplasms; Human; Hyperactivity; Integral Membrane Protein; Kinetics; Label; Lead; Ligands; Malignant Neoplasms; Measurement; Measures; Mediating; Medical; Methods; Microelectrodes; Modality; Modeling; Moloney Leukemia Virus; Nanostructures; Noise; Outcome; Peptides; Performance; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Dynamics; Protein Engineering; Protein Isoforms; Protein Kinase; Protein Kinase Interaction; Protein-Serine-Threonine Kinases; Proteins; Receptor Protein-Tyrosine Kinases; Reporter; Reporting; Resolution; Sampling; Serum; Signal Pathway; Signal Transduction; Solid Neoplasm; Specificity; Substrate Interaction; Tail; Technology; Time; antibody mimetics; casein kinase II; cofactor; complex data; design; drug discovery; inhibitor; innovation; inorganic phosphate; leukemia; member; molecular diagnostics; molecular dynamics; nanopore; nanosensors; new technology; overexpression; polypeptide; preservation; prognostic; protein protein interaction; response; screening; sensor; single molecule; small molecule; small molecule inhibitor; synthetic biology; tool

Project Summary Developing novel technologies for identifying and quantifying transient protein-protein interactions is critical in basic research and medical biotechnology. Protein kinases represent a focal group among strategic drug targets for treating numerous hematological malignancies and solid tumors. Yet, creating high-resolution sensors to detect, quantify, and analyze the plasticity of diverse kinome members in a broad dynamic range of interactions remains difficult. This challenge is exacerbated because the kinase superfamily members vary drastically in their complexity. To address this long-standing technological shortcoming, we will formulate, develop, and validate a new class of generalizable and highly specific nanopore sensors (nanosensors) for kinase analytics. The key innovating aspect of this design is fusing a generic protein recognition ligand with a transmembrane protein nanopore. This approach will employ a robust nanostructure made of a single polypeptide entity with no requirement for an additional tail or other exogenous tags. The binding interface of the protein recognition ligand is interchangeable to accommodate the required specificity for a targeted kinase, whereas the nanopore facilitates the generation of a reporting electrical signal. A protein kinase analyte in solution produces a unique electrical signature that varies with its identity and quantity. The reporting signal is mediated by the ligand-kinase assembly at the nanopore tip. In these studies, kinase recognition events will be discriminated at single-molecule precision without the necessity of using complex data analysis algorithms. This engineering strategy substantially broadens the spectrum of applications of these nanosensors to various kinases and their interactions. Our preliminary studies prove the power of this approach by creating a single- molecule nanosensor platform that probes and quantifies structurally and functionally diverse proteins beyond the fundamental limit of sensing inside the nanopore. In addition, such a tactic will enable the detection of competing binding interactions of kinase isoforms against the same recognition ligand. These generalizable nanosensors permit integration into scalable devices, representing versatile elements for small-molecule inhibitor screening and drug discovery pipelines. Further project developments will be aimed at maintaining a high performance of these nanosensors in a complex biofluid. Therefore, they can be utilized using realistic samples, having prospects in molecular diagnostics. The expected immediate outcomes of this project will be the following: (i) the development of high-affinity nanosensors for ultrasensitive analysis of receptor tyrosine kinases (RTKs); (ii) the creation of genetically-encoded nanosensors for probing serine-threonine kinases (STKs); (iii) the detection and analysis of kinases in multiplexed settings and biofluids. These studies will impact healthcare by providing tools and a fundamental framework in biosensor technology, synthetic biology, and single-molecule enzymology.

项目摘要 开发用于识别和量化瞬态蛋白质蛋白相互作用的新型技术至关重要 基础研究和医学生物技术。蛋白激酶代表战略药物中的焦点群 治疗许多血液系统恶性肿瘤和实体瘤的靶标。但是，创造高分辨率 传感器以在广泛的动态范围内检测，量化和分析不同的Kinome成员的可塑性 互动仍然很困难。由于激酶超家族成员有所不同，因此加剧了此挑战 急剧复杂性。为了解决这个长期存在的技术缺点，我们将制定， 开发并验证一类新的可推广和高度特定的纳米孔传感器（纳米传感器） 激酶分析。该设计的关键创新方面是将通用蛋白质识别配体融合在一起 跨膜蛋白纳米孔。这种方法将采用一个由单个制成的强大纳米结构 多肽实体无需额外的尾巴或其他外源标签。绑定界面的 蛋白质识别配体可以互换以适应目标激酶的所需特异性， 而纳米孔促进了报告电信号的产生。蛋白激酶分析物 解决方案会产生独特的电特征，其身份和数量变化。报告信号是 由纳米孔尖端的配体激酶组件介导。在这些研究中，激酶识别事件将是 在单分子精度下进行区分，而无需使用复杂的数据分析算法。 这种工程策略实质上扩大了这些纳米传感器的应用范围 激酶及其相互作用。我们的初步研究通过创建单一的 分子纳米传感器平台，该平台在结构和功能上多样的蛋白质探测和量化 纳米孔内感应的基本限制。此外，这种策略将使检测 激酶同工型与相同识别配体的竞争结合相互作用。这些可推广 纳米传感器允许集成到可扩展的设备中，代表小分子的多功能元素 抑制剂筛查和药物发现管道。进一步的项目发展将旨在维护 这些纳米传感器在复杂的生物流体中的高性能。因此，可以使用逼真的 样品，具有分子诊断的前景。该项目的预期直接结果将是 以下内容：（i）开发高亲和力纳米传感器，用于对受体酪氨酸的超敏分析 激酶（RTK）； （ii）创建遗传编码的纳米传感器，用于探测丝氨酸 - 苏氨酸激酶 （STK）; （iii）多路复用环境和生物流体中激酶的检测和分析。这些研究会 通过提供工具和生物传感器技术，合成生物学的基本框架来影响医疗保健， 和单分子酶学。