In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System

细胞内自动闪式氧化 (IC-AutoFox™) 蛋白质足迹系统

• 批准号: 10589128
• 负责人: Scot Randy Weinberger
• 金额: $ 116.54万
• 依托单位: GENNEXT TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589128
• 关键词: Address; Adopted; Adoption; Affect; Antibodies; Antibody Binding Sites; Antineoplastic Agents; Appearance; Award; Behavior; Binding; Biological Assay; Biological Process; Biological Products; Biological Response Modifier Therapy; Cell Line; Cell Separation; Cells; Choline; Collaborations; Comparative Study; Complex; Computer software; Crowding; Cytoplasmic Protein; DNA-Protein Interaction; Dangerousness; Data Analyses; Detection; Detergents; Diffusion; Drug Targeting; Environment; Epitopes; Fluorescence; G-Protein-Coupled Receptors; Goals; Growth Factor; Higher Order Chromatin Structure; Human; Hydroxyl Radical; Immune response; In Vitro; Individual; Kinetics; Label; Laboratories; Lasers; Lead; Letters; Ligands; Link; Lipids; Liquid substance; Marketing; Maryland; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Molecular Conformation; Monoclonal Antibodies; Morphologic artifacts; Muscarinic Acetylcholine Receptor; National Institute of General Medical Sciences; Nature; Nuclear Proteins; Nucleoplasm; Organism; Pathogenicity; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phosphotransferases; Play; Positioning Attribute; Process; Production; Protein Conformation; Protein Footprinting; Proteins; Proteome; Proteomics; Pump; Rattus; Reaction; Reproducibility; Research; Role; Safety; Signal Transduction; Small Business Innovation Research Grant; Structure; Styrenes; Surface; System; Techniques; Technology; Toxin; Ultraviolet Rays; Universities; Work; adverse drug reaction; analytical tool; biopharmaceutical industry; commercialization; computerized data processing; copolymer; dosimetry; drug candidate; experimental study; fluidity; improved; in vivo; innovation; interest; light scattering; maleic acid; member; mimetics; nanodisk; new technology; oxidation; particle; programs; protein complex; protein function; reconstitution; research and development; tool; unilamellar vesicle

The GenNext Phase II SBIR submssion entitled “In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System,” is responsive to the ackowledged need for new and improved tools for higher order structural analysis (HOS) of biopharmaceuticals and membrane protein target studies. An emerging HOS analysis technique is hydroxyl radical foot-printing (HRPF). HRPF involves the irreversible labeling of a protein’s exterior by reaction with hydroxyl radicals with subsequent MS analysis to identify the outer portions of the protein. The most widely used method for generating OH radicals employs a quick burst of UV light, and is appropriately called fast photochemical oxidation of proteins (FPOP).We have developed commercial solutions to perform in vitro FPOP. The practice of applying the results of in vitro structural experiments to authentic in vivo behavior has been brought into question. Macromolecular crowding within a cell limits diffusion, thus altering reaction kinetics, association rates of proteins, and protein-DNA interactions. These effects are not observed while performing in vitro studies. Because of in vitro shortcomings, there has been recent desire to extend the use of FPOP to whole cells in an in vivo manner. The practice of in vivo or in-cell FPOP (IC-FPOP) has been pioneered by Dr. Lisa Jones of the University of Maryland. While showing great promise to address unmet challenges in pharmaceutical research, reproducibility for IC-FPOP is challenged by variability of intracellular background scavenging, cell-to-cell isolation irreproducibility, and the mandatory use of hazardous, complicated, and poorly reproducible excimer lasers. Collaborating with the Jones laboratory, our work will extend our innovative in vitro laser-free FPOP technology to in vivo applications. GenNext Technologies is the only company commercializing products for FPOP HOS analysis. Our goal is to convert the IC-FPOP process from an academic research experiment into a valuable analytical tool. Once simplified and transformed into a robust technique, we envision IC-FPOP to enable cell-based assays to: paratope and epitope the interaction of mAb biopharmaceuticals with their membrane targets; elucidate the dynamics of lead binding to orthosteric or allosteric membrane targets; to reveal secondary messenger signaling cascades of GPCR lead compounds; and to detect the impact of anti-neoplastics upon targets such as kinases and growth factors.

Gennext II期SBIR子序列标题为“内部自动闪光灯” 氧化（IC-AUTOFOX™）蛋白足迹系统，“对 对高阶结构分析（HOS）的新工具（HOS）的新工具的需求 生物药物和膜蛋白靶靶研究。新兴的HOS分析 技术是羟基自由基（HRPF）。 HRPF涉及不可逆的 通过与随后的MS的羟基自由基反应来标记蛋白质的外部 分析以识别蛋白质的外部。最广泛使用的方法 生成OH激进分子的员工快速爆发紫外线，并适当地称为快速 蛋白质的光化学氧化（FPOP）我们已经开发了商业解决方案 在体外进行FPOP。应用体外结构结果的实践 对真实体内行为的实验已受到质疑。 细胞内的大分子拥挤限制了扩散，从而改变了反应动力学， 蛋白质的关联速率和蛋白-DNA相互作用。这些影响不是 在进行体外研究时观察到。由于体外缺点，有 最近希望以体内方式将FPOP扩展到整个细胞。 Lisa博士率先进行了体内或细胞内FPOP（IC-FPOP）的实践 马里兰大学的琼斯。同时表现出未满足的良好承诺 药物研究中的挑战，IC-FPOP的可重复性受到挑战 细胞内背景清除，细胞对细胞隔离性不可重复的可变性， 以及强制使用危险，复杂且繁殖不良的准分子 激光。与琼斯实验室合作，我们的工作将扩大我们的创新性 体外无激光FPOP技术用于体内应用。 Gennext Technologies是 只有公司将产品商业化用于FPOP HOS分析。我们的目标是转换 从学术研究实验到有价值的分析的IC-FPOP过程 工具。一旦简化并转化为强大的技术，我们将IC-FPOP设想为 启用基于细胞的阿萨斯至：副膜和表位mab的相互作用 生物药物及其膜靶；阐明铅结合的动力学 到角膜或变构膜靶标；揭示次级信使信号 GPCR铅化合物的级联；并检测抗塑料对 激酶和生长因子等靶标。